Antioxidant in relation to nutrition, stomata, and yield of lettuce (Lactuca sativa L.) fertilized with seabird guano

Antioxidantes em relação à nutrição, estômatos e produtividade de alface (Lactuca sativa L.) fertilizada com guano de aves marinhas

Abstract

Peru faces a complex economic crisis, exacerbated by persistent food and nutritional insecurity as a result of the pandemic, geopolitical tensions, adverse weather conditions, and other relevant factors. For this reason, an investigation was conducted on antioxidants in relation to nutrition, stomata, and the yield of lettuce fertilized with seabird guano (S.G.). The general objective was to determine the antioxidant capacity in relation to nutrition, stomata, and yield of lettuce fertilized with S.G. The methodology is based on applied research with an experimental approach, as the statistical model of the completely randomized block design was employed, consisting of three blocks and five treatments: T₁ with 0, T₂ with 300, T₃ with 600, T₄ with 900, and T₅ with 1,200 kg ha^-1 of S.G. The doses were applied 15 days after transplanting, and the physical characteristics (plant height, plant weight, commercial yield, and plant diameter) were evaluated. Nutrients (nitrogen, potassium, phosphorus, calcium, magnesium, sulfur, molybdenum, iron, manganese, copper, zinc, boron, chlorides, and sodium), antioxidant capacity, and stomatal density in the leaves were determined. Also, total phosphorus and nitrogen uptake and profitability. The results showed that T₅ stood out in plant height at 25 cm, plant weight at 618.8 g, commercial yield at 61.501 t ha^-1, and diameter at 15.91 cm. In total nitrogen consumption, it stood out with 174.68 kg ha^-1, and in total phosphorus consumption with 180.77 kg ha^-1. In stomatal density, it stood out with 134 stomata/mm² and in profitability with 443.5%. In leaf nutrient concentration, T₂ stood out with calcium, magnesium, sulfur, molybdenum, boron, and sodium, and T₃ excelled in antioxidant capacity with 4,607.8 μmol Trolox/100 g. It is concluded that an adequate antioxidant capacity (4,020.0 μmol Trolox/100 g in T₅) was associated with the highest lettuce yield. Therefore, at higher doses of S.G., stomatal density and nutrients such as nitrogen, phosphorus, copper, and chlorides in the leaves increased, which enhanced the efficiency of gas and nutrient exchange. These characteristics optimized physiological processes and modulated antioxidant pathways, thereby improving yield.

Keywords:
seabird guano; dosage; nutrition; antioxidant; yield; lettuce

Resumo

O Peru enfrenta uma complexa crise econômica, agravada pela persistente insegurança alimentar e nutricional como resultado da pandemia, das tensões geopolíticas, das condições climáticas adversas e de outros fatores relevantes. Por esse motivo, investigou-se sobre antioxidantes em relação à nutrição, estômatos e ao rendimento da alface fertilizada com guano de aves marinhas (G.A.M.). O objetivo geral foi determinar a capacidade antioxidante em relação à nutrição, aos estômatos e ao rendimento de alface fertilizada com G.A.M. A metodologia baseia-se em pesquisa aplicada com abordagem experimental, uma vez que foi empregado o modelo estatístico do delineamento em blocos inteiramente casualizados, que consistiu em três blocos e cinco tratamentos: T₁ com 0, T₂ com 300, T₃ com 600, T₄ com 900 e T₅ com 1.200 kg ha^-1 de G.A.M. As doses foram aplicadas 15 dias após o transplante, e as características físicas (altura da planta, peso da planta, rendimento comercial e diâmetro da planta) foram avaliadas. Foram determinados os nutrientes (nitrogênio, potássio, fósforo, cálcio, magnésio, enxofre, molibdênio, ferro, manganês, cobre, zinco, boro, cloretos e sódio), a capacidade antioxidante e a densidade de estômatos nas folhas. Também a absorção total de fósforo e nitrogênio e a rentabilidade. Os resultados determinaram que a T₅ se destacou em altura da planta com 25 cm, peso da planta com 618.8 g, rendimento comercial com 61.501 t ha^-1 e diâmetro com 15.91 cm. Em consumo total de nitrogênio, destacou-se com 174.68 kg ha^-1 e no consumo total de fósforo com 180.77 kg ha^-1. Na densidade de estômatos, destacou-se com 134 estômatos/mm² e em rentabilidade com 443.5%. Na concentração de nutrientes nas folhas, o T₂ destacou-se com cálcio, magnésio, enxofre, molibdênio, boro e sódio, e o T₃ sobressaiu na capacidade antioxidante com 4,607.8 μmol de Trolox/100 g. Conclui-se que uma capacidade antioxidante adequada (4,020.0 μmol Trolox/100 g em T₅) esteve associada ao maior rendimento da alface. Portanto, quanto maior a dose de G.A.M., maior foi o aumento da densidade estomática e de nutrientes como nitrogênio, fósforo, cobre e cloretos nas folhas, o que favoreceu a eficiência das trocas gasosas e nutricionais. Essas características otimizaram os processos fisiológicos e modularam as vias antioxidantes, o que melhorou a produtividade.

Palavras-chave:
guano de aves marinhas; dosagem; nutrição; antioxidante; produtividade; alface

1. Introduction

Peru is experiencing a multidimensional economic situation linked to food and nutritional security, influenced by the post-pandemic period, the geopolitical context, climate variations, and other factors. This situation has given rise to a severe social and economic crisis that particularly affects the country’s inland areas. In this regard, Hernandez Cornejo et al. (2025) note that poverty has increased significantly in Peru since the COVID-19 pandemic. This fact is corroborated by INEI (2023), which indicate that monetary poverty affected 29.0% of the country’s population, equivalent to 9,780,000 people (5.7% in extreme poverty and 23.3% in non-extreme poverty). This precarious economic situation forms the basis of the nutritional problem, as household food insecurity reflects the multiple ways in which the food crisis affects child growth (Morales-Cahuancama et al., 2025).

In this context, the increase in the price of synthetic fertilizers has raised agricultural production costs, creating a problem with far-reaching repercussions. This situation has indebted the area’s farmers and created uncertainty about potential economic losses. This effect directly impacts food prices, triggering instability in the food supply chain and, as a result, contributing to malnutrition. This problem is explained by the fact that Nacimento (2024) argues that the repercussions of the Russia–Ukraine conflict in Peru demonstrate how rising fertilizer prices severely affect the agricultural production of small farmers, limiting their ability to cover these costs. In fact, Legua Cárdenas et al. (2023) quantify that Peru was harmed by a fertilizer price increase of more than 25% compared to previous years, which raised the production cost of many food products. As confirmation, Zea et al. (2022) state that high prices for chemical fertilizers increase cultivation costs, affecting many producers who cannot cover these expenses, especially during health and food crises.

In light of this situation, it is necessary to innovate sustainable alternatives, such as the use of S.G., which is generated in excess in coastal areas and, especially, on the country’s islands. In this way, when used as a nutritional source, it can provide a higher concentration of nitrogen, phosphorus, potassium, and other essential micronutrients for the plant compared to other fertilizers. This, in turn, improves nutrient availability for greater uptake by the plant, optimizing biochemical reactions and strengthening its resistance to nutritional, environmental, and pest stress, resulting in better yield and fruit quality. In this regard, Agro Rural (2023a) proposes marketing 25,000 tons of S.G. as an organic alternative to address the reduction in fertilized areas caused by high fertilizer prices and thereby support producers’ livelihoods. This statement is based on data from Agro Rural (2025b), which indicate that S.G. contains 10–14% nitrogen, 10–12% phosphorus, 2–3% potassium, 10–15% calcium, 0.8–1.5% magnesium, 1.5–2% sulfur, 600–760 ppm iron, 170–23 ppm zinc, 48–50 ppm manganese, 187–197 ppm boron, and 76–80 ppm molybdenum. Likewise, Van Rees et al. (2025) note that S.G. improves vegetation productivity at the start of the season and increases sediment retention, although they caution that the magnitude of these effects varies depending on seasonal conditions and the local environmental context.

It is crucial to mention that G.S. provides the soil with nutrients and beneficial microorganisms that improve the availability and uptake of essential elements by the plant. This nutritional improvement optimizes key biochemical pathways, including those associated with the biosynthesis of flavonoids and other bioactive compounds with antioxidant capacity. These compounds contribute to defense mechanisms by neutralizing reactive oxygen species (ROS). Consequently, the defense mechanism against nutritional, environmental, and pest stress is strengthened, resulting in an increase in lettuce yield. In this context, Appoo et al. (2024) state that the nutrients from G.S. enrich mangrove plants, enhance the trophic networks of their invertebrates, and are exported to nearby coastal habitats by tides, demonstrating their ecosystem-wide impact. In contrast, Slamet et al. (2017) observed that compost had a lesser effect on leaf area, leaf area index, and photosynthetic rate, while the number of leaves, total chlorophyll, and antioxidant content in lettuce were not affected by the different organic nitrogen fertilizers. Complementarily, El Sebai et al. (2023) state that the use of organic fertilizer improved plant productivity by increasing soil fertility, reducing nutrient losses, and enhancing water retention capacity.

It is worth mentioning that G.S., recognized for its nutritional richness and diversity of native microorganisms, promotes the availability and absorption of essential elements for the plant. These nutrients enter the roots in ionic form and participate in vital biochemical processes, including the activation of antioxidant enzymes such as peroxidase (POD) and superoxide dismutase (SOD), as well as other metabolic mediators. These enzymes catalyze the degradation of ROS such as hydrogen peroxide and other oxidative compounds, thereby helping to maintain the organism’s oxidative balance and prevent cellular damage. Consequently, antioxidant capacity reinforces the plant’s defense mechanisms against physiological stress caused by environmental, nutritional, and pest factors, thereby improving fruit yield and quality. In this regard, Irin and Hasanuzzaman (2024) mention that vermicompost, biochar, and manure increase the activity of antioxidant enzymes, stabilize ionic balance, mitigate osmotic and oxidative stress, and modulate gene expression, collectively promoting plant growth and productivity. Likewise, Rao et al. (2025) analyze that the main enzymatic antioxidants, such as SOD, catalase (CAT), reductases, and POD, as well as non-enzymatic antioxidants, such as ascorbic acid, glutathione, polyphenols, and flavonoids, play a decisive role in ROS detoxification.

For this reason, the relationship between antioxidants, nutrition, stomata, and the yield of lettuce fertilized with G.S. was investigated. The overall objective was to determine the antioxidant capacity in relation to nutrition, stomata, and the yield of lettuce fertilized with G.S. To this end, a completely randomized block design statistical model with three blocks and five treatments, including the control and the standard dose, was employed. The G.S. doses were applied fifteen days after transplanting, and subsequently the physical, chemical, and biological characteristics of the lettuce were evaluated.

2. Methodology

2.1. Location of the experimental area

The experiment was carried out in the Tres Piedras sector, in the Supe Puerto district, Barranca province, Lima. The coordinates are 10°47'19.916" South latitude and 77°44'8.539" West longitude, at an altitude of 75 meters above sea level. Under current weather conditions, the temperature ranges from 19 to 23°C, and the relative humidity from 70 to 75%.

2.2. Research type

It is based on applied research with an experimental approach, since the physical characteristics of the lettuce sample were continuously evaluated and the corresponding data were obtained. After statistical analysis of these data, the appropriate dose of G. S. was determined, which will serve as a recommendation for farmers in the area.

2.3. Population

The population refers to lettuce plants grown at an altitude of 50 to 150 meters above sea level, under the agroecological conditions of the area. Therefore, the values obtained in the experiment were validated.

2.4. Sample

A 50% sample of the plants in each plot was taken and marked in the central rows to avoid edge effects. The plants were evaluated based on their physical characteristics, such as height, weight, yield, and equatorial diameter.

2.5. Study factor

To determine the application rate of G.S. per hectare, the soil analysis results and the amounts applied by local farmers—ranging from 400 to 800 kg ha^-1 for lettuce—were considered. Additionally, the research by Chávez-Centeno (2015) was considered, which evaluated treatments ranging from 250 to 1,000 kg ha^-1 of G.S. combined with phosphate rock and demonstrated that applying 1,000 kg ha^-1 of G.S. along with 500 kg ha^-1 of phosphate rock produced the highest yield. Based on this background, a standard dose of 600 kg ha^-1 of G.S. was established (see Table 1). It should be noted that this dose was applied only once and that all other agricultural practices remained the same across all treatments.

Table 1
G.S. doses for lettuce cultivation by treatment.

Treatment	G.S. dose (kg ha-1)
T1	0
T2	300
T3	600
T4	900
T5	1,200

2.6. Quantification of nitrogen in the soil

2.6.1. Determination of the weight of the arable layer

To determine the amount of nitrogen, the weight of the soil’s arable layer mentioned by Castañeda Chirre et al. (2022) was calculated.

Formula of the arable layer (Equation 1)

$[WAL]=\left(DA.\right)\left(SD\right)\left(Ha\right)$ (1)

Where:

WAL: Weight of the arable layer (2,800 t ha^-1)

DA: Apparent density (1.4 g cm^-3)

SD: Soil depth (0.20 m)

Ha: Hectare (10,000 m²)

2.6.2. Quantification of organic carbon

Then, the Van Bemmelen formula was applied: [C. Org.] = (O.M. × 0.58) (Vela et al., 2012).

Formula of organic carbon (Equation 2).

$[C.org.]=\left(O.M.\right)\left(0.58\right)$ (2)

Where:

C. org.: Organic carbon (0.8352%)

O.M.: organic matter (1.44%) (see Table 2) (INIA, 2024a)

Table 2
Soil analysis of the experimental area.

N° Lab.	E.C. 1:2:5 dS/m	pH 1:2:5	O.M. (%)	N (%)	P (ppm)	K (ppm)	CaCO3 (%)	Cation exchange capacity (mEq/100 g of soil)				C.E.C.
								Ca	Mg	Na	K
080292-24	0.0344	7.83	1.44	0.07	16.70	155.08	0.01	7.17	1.81	0.97	0.40	10.34

• Source: INIA (2024a). Note: C.E.C. (Cation Exchange Capacity).

2.6.3. Quantification of the C/N ratio

Next, the C/N ratio formula was applied. For this, the organic carbon data was used.

Carbon-to-nitrogen ratio formula (Equation 3)

$[C./N]=\frac{C.org.}{N}$ (3)

Where:

C/N: Carbon-to-nitrogen ratio (11.93)

C. org.: Organic carbon (0.8352%)

N: Nitrogen (0.07%) (see Table 2) (INIA, 2024a)

2.6.4. Conversion of nitrogen to available nitrogen

After obtaining a C/N ratio of 11.93, it was considered to be within acceptable limits, allowing the available nitrogen to be set at 140 ppm (see Table 3).

Table 3
Conversion factor from total nitrogen to available nitrogen.

Margin C/N ratio	Conversion factor from total nitrogen (N.T.) in percent to nitrogen in ppm
Greater than 12	11.2
From 10 to 12	140
Less than 10	225

• Source: Kass (1998).

After determining the C/N ratio, the value was taken as an indicator within the established range (see Table 3), yielding 140 ppm of available nitrogen (N.A.). Next, the N.A. formula was applied, which is 140 ppm multiplied by 0.07% N (see Table 2) (INIA, 2024a), resulting in 9.8 ppm N.A. Subsequently, this value was projected using the weight of the arable layer, which is 2,800 t ha^-1, yielding 27.44 kg ha^-1 of soil-available nitrogen (ASN).

2.6.5. Determination of the standard nitrogen dose

To determine the standard nitrogen dose, the recommended amount of 120 kg ha^-1 was used (see Table 4) (INIA, 2024b). The soil nitrogen content of 27.44 kg ha^-1 of NDS was subtracted from this amount, resulting in 92.56 kg ha^-1 of nitrogen.

Table 4
Macronutrient recommendations for lettuce cultivation.

Nutrients (kg ha-1)
N	P	K
120	40	40

• Source: INIA (2024b).

Next, the nitrogen concentration in the G.S. was measured, which amounts to 12.27% (see Table 1) (INIA). This proportion indicates that there are 12.27 kg of nitrogen per 100 kg of fertilizer. This concentration was projected for doses of 0, 300, 600, 900, and 1,200 kg ha^-1 of G. S., as indicated in Table 1. The resulting amounts of nitrogen applied were 0, 36.81, 73.62, 110.43, and 147.24 kg ha^-1, respectively.

Finally, the data obtained from the INIA recommendation in Huaral, after subtracting the nitrogen already present in the soil, yielded 92.56 kg ha^-1. When projecting the fertilizer doses in nitrogen, 0, 36.81, 73.62, 110.43, and 147.24 kg ha^-1 were obtained, respectively. These results were compared, and it was determined that the nitrogen rate of 73.62 kg ha^-1 was the closest match between the two, so the standard dose is 600 kg ha^-1 of G.S. in T₃.

2.7. Calculation of phosphorus in the soil

To determine the amount of phosphorus in the soil, the following steps were carried out:

2.7.1. Determination of the weight of the soil’s arable layer

To determine the amount of phosphorus in the soil, the weight of the arable layer exposed by Castañeda E. et al. (2022) was considered.

Formula for the arable layer (Equation 4).

$[WAL]=\left(DA.\right)\left(SD\right)\left(Ha\right)$ (4)

Where:

WAL: Weight of the arable layer (2,800 t ha^-1 of soil)

DA: Apparent density (1.4 g cm^-3)

SD: Soil depth (0.20 m)

Ha: hectare (10,000 m²)

2.7.2. Calculation of phosphorus (P₂) in the soil

Next, the amount of P₂ in the soil was determined. To do this, the amount of P₂ obtained through soil analysis was projected based on the weight of the topsoil, following the procedure described by Bello and Pino (2000).

Formula to calculate P₂ in soil (Equation 5).

$\left[TW{P}_2\right]=\frac{\left[WAL\right]\left[W{P}_{2}inS.\right]}{kg}$ (5)

Where:

TW P₂: Total weight of P₂ (46.76 kg ha^-1 of P₂)

WAL: Weight of the arable layer (2,800 t ha^-1 of soil)

W P₂ in S.: P₂ content in the soil (16.70 ppm of P₂)

2.7.3. Determination of the weight of Diphosphorus Pentoxide (P₂O₅)

Then, the weight of P₂O₅ in the soil was determined. To this end, the amount of P₂ in the soil was taken and projected using the moles of P₂ and P₂O₅; this procedure is supported by Carrasco and Aguirre (2023).

Formula to determine the weight of P₂O₅ (Equation 6)

$\left[W.P_2{O}_5\right]=\frac{\left[nmoles{P}_2{O}_5\right]\left[TW{P}_2\right]}{nmoles{P}_2}$ (6)

Where:

W. P₂O₅: Weight of P₂O₅ (107.09 kg ha^-1 of P₂O₅)

n moles P₂O₅: Number of moles of P₂O₅ (142 moles of P₂O₅)

TW P₂: Total weight of P₂ (46.76 kg ha^-1 of P₂)

n moles P₂: Number of moles of P₂ (62 moles of P₂)

2.8. Statistical processing

2.8.1. Statistical analysis of the physical characteristics of lettuce

1. Statistical analysis

   • Analysis of variance

Once the data on plant height, weight, yield, and equatorial diameter of the lettuce were obtained, they were processed using an analysis of variance. In this way, it was determined whether there was a dose effect among the treatments (F-calculated > F-tabulated) or not. In other words, it was determined whether the application of G.S. influenced the crop’s development, yield, and quality. It is also noted that the tabulated F-values were obtained from the Fisher table with a 5% error.

1. Duncan’s test

Next, the physical characteristic data were processed using Duncan’s test at a 5% error level, which allowed us to determine which treatment stood out from the others. This test also allowed the means to be classified and grouped using letters. In this way, it was determined whether there was homogeneity or statistically significant differences when the letters were different.

2.8.2. Statistics on total nitrogen consumption

To determine the total nitrogen consumption related to lettuce yield, we used the nitrogen value from the fertilizer analysis, which is 12.27% (see Table 5) (INIA, 2024c). This value was projected for the G.S. doses of 0, 300, 600, 900, and 1,200 kg ha^-1, yielding the following results: 0, 36.81, 73.62, 100.43, and 147.24 kg ha^-1 of nitrogen. Then, 27.44 kg ha^-1 of soil nitrogen was added to each value, resulting in a total nitrogen uptake of 27.44, 64.25, 101.06, 137.87, and 174.68 kg ha^-1. These amounts, along with the total antioxidant capacity and lettuce yield, were arranged in ascending order in a table, allowing for analysis of the results (see Table 6).

Table 5
Chemical analysis of G.S.

N° Lab.	ID Sample	pH	EC (mS cm-1)	Humidity (%)	O.M. (%)	N (%)	P2O5 (%)	K2O (%)	CaO (%)	MgO (%)	C/N
AO205-DO-24	Fertilizer	7.72	0.19	5.97	59.94	12.27	6.14	2.82	6.56	0.66	11.42

• Source: INIA (2024c).

Table 6
Total nitrogen consumption for lettuce yield.

Treatment	G.S. dose (kg ha-1)	* Nitrogen in G.S. (kg ha-1)	Soil nitrogen (kg ha-1)	Total nitrogen (kg ha-1)	Total antioxidant capacity (μmol Trolox/100 g sample)	Commercial yield (t ha-1)
T1	0	0	27.44	27.44	3,644.7	45.325
T2	300	36.81	27.44	64.25	3,760.5	52.754
T3	600	73.62	27.44	101.06	4,607.8	55.846
T4	900	110.43	27.44	137.87	4,262.5	57.720
T5	1,200	147.24	27.44	174.68	4,020.0	61.501

• Note: *G.S. contains 12.27% nitrogen (see Table 5) (INIA, 2024c).
• This amount was projected for an application rate of 1,200 kg ha^-1, resulting in 147.24 kg ha^-1 of nitrogen. This procedure was carried out for all treatments.

2.8.3. Total P₂O₅ consumption statistics

It consisted of using the 6.14% P₂O₅ content of the fertilizer (see Table 5) (INIA, 2024c), and this concentration was projected for the G.S. doses specified in Table 1, resulting in applications of 0, 18.42, 36.84, 55.26, and 73.68 kg ha^-1 of P₂O₅. To each of these amounts, the available soil P₂O₅, estimated at 107.09 kg ha^-1, was added, resulting in total P₂O₅ uptake of 107.09, 125.51, 143.93, 162.35, and 180.77 kg ha^-1. Finally, these data, along with total antioxidant capacity and yield, were tabulated and sorted from lowest to highest in a table, allowing for analysis of the results (see Table 7).

Table 7
Total P₂O₅ consumption for lettuce yield.

Treatment	G.S. dose (kg ha-1)	* P2O5 in G.S. (kg ha-1)	P2O5 in soil (kg ha-1)	Total P2O5 (kg ha-1)	Total antioxidant capacity (μmol Trolox/100 g sample)	Commercial yield (t ha-1)
T1	0	0	107.09	107.09	3,644.7	45.325
T2	300	18.42	107.09	125.51	3,760.5	52.754
T3	600	36.84	107.09	143.93	4,607.8	55.846
T4	900	55.26	107.09	162.35	4,262.5	57.720
T5	1,200	73.68	107.09	180.77	4,020.0	61.501

• Note: *G.S. contains 6.14% phosphorus (see Table 5) (INIA, 2024c).
• This amount was projected for an application rate of 1,200 kg ha^-1, resulting in 73.68 kg ha^-1 of phosphorus. This procedure was carried out for all treatments.

2.8.4. Stomatal density statistic in lettuce leaves

To quantify stomatal density, lettuce leaves were observed using a Quanta scanning electron microscope. Next, the micrograph was printed on an A4 sheet at a 200 µm scale and projected onto the total area of the micrograph, yielding 0.454179 mm². In this area, stomata were counted and divided by the area, thus obtaining the stomatal density. This procedure was performed for all treatments, and the results were placed in a table sorted from lowest to highest, along with the antioxidant capacity and yield (see Table 8). This allowed us to analyze how stomata influence yield.

Table 8
Stomatal density in lettuce leaves in relation to yield.

	T1	T2	T3	T4	T5
Stomata (number of stomata/0.454179 mm2)	30	33	36	39	61
Stomatal density (stomata/mm2)	66.1	72.7	79.3	85.9	134.3
Total antioxidant capacity (μmol Trolox/100 g)	3,644.7	3,760.5	4,607.8	4,262.5	4,020.0
Commercial yield (t ha-1)	45.325	52.754	55.846	57.720	61.501

• Note: The projected lens area is 0.454179 mm².

Formula for calculating stomatal density (Equation 7).

$SD=\frac{SN}{LA}$ (7)

Where:

SD: Stomatal density

SN: Number of stomata

LA: Lens area (0.454179 mm²)

2.9. Data collection technique

2.9.1. Data collection techniques and instruments for lettuce

Regarding data collection techniques, meticulous observations and measurements were used in all evaluations. To evaluate the physical characteristics, precision instruments such as a digital scale and a tape measure were used. To evaluate nutrient concentrations, laboratory materials from AGQ Peru S.A.C. were used, while materials from the Nutritional Research Institute (IIN) were used to evaluate antioxidants. Additionally, a scanning electron microscope was used to quantify the number of stomata on the leaves.

2.9.2. Techniques for determining soil nutrients

To analyze the elements present, a representative 1 kg soil sample was taken to INIA-Huaral, where methods and techniques were employed. To determine the pH of the soil and residues, method 9045D was used. For organic matter, the Walkley–Black AS-07 method was used, and the nitrogen content was calculated from the organic matter data. To determine the amount of phosphorus, the Olsen method was used, and for potassium, the calcium carbonate determination techniques (AS-29 acid neutralization method). Finally, to measure calcium, magnesium, sodium, and potassium, the soil’s cation exchange capacity and exchangeable bases were determined using ammonium acetate.

2.9.3. Techniques for determining antioxidant capacity

To measure the antioxidant capacity of lettuce leaves, the ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) method was used, which involves scavenging the radical cation formed by this compound. The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was also used, which is based on scavenging a free radical (Aguilar Felices et al., 2021). Trolox, carbonates, ethanol, and distilled water were used as the antioxidant standard, along with various instruments, primarily the UV-Vis spectrophotometer (Mejía-Reyes et al., 2021). The equation used to calculate inhibition is referenced in studies such as Cuapio-Rodriguez et al. (2024).

Percentage inhibition formula (Equation 8).

$\text{\% Inh}.=\frac{\left(ABTS{}_{t=0}-ABT{S}_{t=6}\right)}{ABTS{}_{t=0}}*100$ (8)

Where:

% Inh.: Inhibition percentage

ABTS _(t=0): Absorbance obtained at time zero.

ABTS _(t=6): Absorbance after 6 minutes.

Subsequently, the equation corresponding to the TEAC-DPPH method was applied to determine the antioxidant capacity, according to the procedure described by Paucar Luna et al. (2021).

Antioxidant capacity formula (Equation 9):

$C.A.\left(\mu \text{mol TROLOX eq}./\text{g}\right)=Y\frac{\left(W+V\right)}{W}*Fd$ (9)

Where:

C. A.: Antioxidant capacity

Y: μmol (Micromole)

W: Sample weight (g)

V: Solvent volume (mL)

Fd: Dilution factor

2.9.4. Procedures

G.S., an organic fertilizer sourced from the guano islands of Peru, rich in macronutrients (nitrogen, phosphorus, and potassium) as well as various micronutrients, was used. This fertilizer was applied to the lettuce crop, which promoted its development.

The land preparation was carried out in the usual manner, that is, as the farmers in the Supe Puerto district of Barranca Province (Lima) do. Afterward, soil samples were taken using a zigzag method at a depth of 0.25 m. They were piled onto a blanket, mixed, and a 1 kg sample was taken from them. This sample was taken to the INIA-Huaral laboratory, where the soil’s chemical characteristics were determined.

On April 27, 2025, the lettuce seedbed was established, using certified seeds with a high germination rate, varietal purity, and no damage. The seeds were broadcast sown, and two weeks later the seedlings were thinned to prevent nutritional competition. The corresponding cultural practices were also carried out, and pests were controlled. After 30 days, they were removed for transplanting.

The transplant was carried out on May 27 and consisted of removing the seedlings from the seedbed at an average height of 12 cm. Additionally, the roots were disinfected in a solution containing 10 g of benomyl per liter of water. Afterward, they were transplanted with a spacing of 0.25 m between plants across the entire area. It is worth mentioning that the plot measured 2.5 m in length and 1.2 m in width, comprising two twin rows, and a 0.5 m alley was left between blocks.

On June 10, 2025, fifteen days after transplanting, the G.S. doses were applied. During that time, the physical characteristics of the lettuce were evaluated until harvest on July 31, 2025, i.e., 66 days after transplanting. Once the data were obtained, they were processed using statistical analysis.

On the same day, 200 g leaf samples were taken from each treatment—without repeating any block—and sent to the AGQ Peru S.A.C. laboratory to determine nutrient concentrations. Samples were also taken to the IIN to determine the antioxidant concentration and to the scanning electron microscope to quantify the stomata.

Finally, the cost and yield per plot were determined, and the production cost and commercial yield were calculated. The latter was multiplied by the unit price, thus obtaining the total revenue. Subsequently, that amount was subtracted from the production cost, and the result was divided by the production cost to obtain the profitability. This procedure was carried out for all five treatments. Below is the formula for profitability.

Profitability formula (Equation 10)

$P.=\frac{U}{C.p.}*100$ (10)

Where:

P. : Profitability

U : Utility

C.p. : Cost of production.

3. Results

3.1. Soil analysis of the experimental area

After analyzing the soil at INIA Huaral, it was determined that the soil pH is slightly alkaline, while organic matter and nitrogen levels are low. Furthermore, phosphorus and potassium levels are within the appropriate ranges (Ángeles et al., 2021). Electrical conductivity (EC) indicates that the soil has slight salinity. The concentrations of calcium and potassium are medium, while that of manganese is high. Sodium indicates that the soil is moderately sodic, according to Agrolab’s ranges (Agrolab, 2005) (see Table 2) (INIA, 2024a). Therefore, the analysis indicates that the soil is suitable for growing lettuce, although it is necessary to apply organic matter, such as G.S.

After the soil analysis, the nutritional recommendation for lettuce cultivation was determined (see Table 4) (INIA, 2024b), establishing a nitrogen requirement of 120 kg ha^-1. This value highlights the importance of applying an appropriate concentration of G.S., an aspect that was considered when defining the standard fertilizer dose.

3.2. Chemical analysis of G.S.

Regarding the G.S. analysis presented in Table 5 (INIA, 2024c), it shows a high concentration of organic matter, nitrogen, and phosphorus, indicating that it is a considerable nutritional source. Likewise, the carbon-to-nitrogen ratio is within the appropriate ranges, indicating that it is a suitable, mature fertilizer. This characterization coincides with Schnug et al. (2018), who reported that G.S. in Peru contains 7–8% H₂O, 45% organic carbon, 8–15% nitrogen, 8–15% P₂O₅, and 2–4% K₂O. Additionally, Román et al. (2013) state that the ideal ranges for mature compost are between 3 and 6 months, with a C/N ratio of 10:1 to 15:1 and moisture content of 30% to 40%. Therefore, the compost is suitable for lettuce.

The micronutrient analysis of G.S. is presented in Table 9 (INIA, 2024d). It shows iron, zinc, copper, and manganese concentrations lower than those of other fertilizers, such as humus derived from plant residues. According to Cruz Nieto et al. (2025a), humus contains 486 ppm of iron, 68.50 ppm of zinc, 15.20 ppm of copper, and 186 ppm of manganese. However, despite its lower micronutrient content, G.S. optimized physiological processes and positively modulated antioxidant activity, thereby increasing lettuce yield and quality.

Table 9
Micronutrient analysis of G.S.

Elements	Iron	Zinc	Copper	Manganese
Amount (mg kg−1)	210.37	2.52	3.45	2.81

• Source: INIA (2024d).

3.3. Physical characteristics of lettuce

After processing the data on plant height and weight, commercial yield, and equatorial diameter of the lettuce by means of an analysis of variance, it was determined that the G.S. dose had no effect; that is, no statistically significant differences were found among the treatments. Furthermore, the highest dose (T₅) stands out compared to the other treatments (see Table 10 and Figure 1). This result is interpreted as indicating that the application of G.S. did not affect the development, yield, or quality of the lettuce, meaning that the treatments were statistically homogeneous. However, T₅ showed higher values.

Table 10
Physical characteristics of the lettuce crop by treatment.

Treatment	G.S. dose (kg ha-1)	Plant height (cm)	Plant weight (g)	Yield Commercial (t ha-1)	Plant diameter (cm)
T5	1,200	25.01 a	618.79 a	61.501 a	15.91 a
T4	900	24.05 ab	594.18 a	57.720 a	15.64 ab
T3	600	23.66 ab	573.00 a	55.846 a	15.44 ab
T2	300	23.05 ab	543.00 a	52.754 a	15.02 ab
T1	0	22.67 b	508.17 a	45.325 a	14.14 b
Significance		N.S.	N.S.	N.S.	N.S.
Coefficient of variation (%)		4.75	19.58	16.14	5.50

• Note: Significant (S), not significant (N.S.). Identical letters, as assigned by Duncan, indicate that they are statistically homogeneous.

Figure 1
Comparison of lettuce development according to G.S. doses.

3.4. Total nitrogen consumption for yield

According to the evaluation of total nitrogen uptake, T₅, with 1,200 kg ha^-1 of G.S., stood out with 174.68 kg ha^-1 of nitrogen, which influenced the considerable increase in total antioxidant capacity to 4,020 μmol Trolox/100 g of sample and a higher lettuce yield (see Table 6). This result is interpreted as indicating that higher nitrogen uptake is associated with a considerable increase in total antioxidant capacity, which in turn leads to higher yield.

3.5. Total phosphorus consumption for yield

Regarding total phosphorus uptake, the highest dose (180.77 kg ha^-1 of P₂O₅) was applied in T₅. This led to a considerable increase in antioxidant capacity (reaching 4,020 μmol Trolox/100 g of sample) and the highest lettuce yield (see Table 7). Based on these results, it is interpreted that a higher application of G. S. is associated with increased P₂O₅ uptake, which in turn is linked to a considerable increase in antioxidant capacity and higher yield.

3.6. Nutrient concentration in lettuce leaves

After analyzing the nutrient content of the lettuce leaves, it was determined that T₂ exhibited higher concentrations of most elements, such as calcium, magnesium, sulfur, molybdenum, boron, and sodium. However, these nutrient concentrations did not influence the highest yield, as T₅, with nitrogen, phosphorus, copper, and chlorides, exhibited the highest lettuce yield response (see Table 11) (AGQ Perú S.A.C., 2025). This result is interpreted as indicating that at the highest G.S. dose (T₅), with a higher concentration of a reduced number of elements (N, P, Cu, and Cl), a superior yield response was obtained.

Table 11
Nutrient concentrations in lettuce leaves by treatment.

Elements	T1	T2	T3	T4	T5	Normal range according to AGQ Peru S.A.C. (2025)
Macronutrients (%)
Total Nitrogen	2.20	2.70	2.50	2.30	3.10	4.00 – 5.00
Potassium	3.51	4.74	4.75	4.91	4.16	6.00 – 7.00
Phosphorus	0.379	0.305	0.368	0.330	0.436	0.40 – 0.60
Calcium	0.70	0.76	0.49	0.67	0.63	2.30 – 3.50
Magnesium	0.189	0.209	0.152	0.172	0.178	0.50 – 0.80
Sulfur	0.24	0.28	0.24	0.27	0.24	0.20 – 0.60
Micronutrients (mg kg-1)
Molybdenum	0.20	0.30	< 0.10	0.30	0.20	0.20 – 1.00
Iron	137	129	84.0	89.4	124	50.0 – 100
Manganese	24.8	20.1	17.4	17.6	21.2	15.0 – 250
Copper	5.47	5.08	4.73	3.49	6.15	8.00 – 25.0
Zinc	34.3	33.6	29.7	26.5	33.7	25.0 – 250
Boron	27.3	31.9	20.4	27.1	25.7	25.0 – 60.0
Phytotoxic elements (%)
Chlorides	0.391	0.584	0.587	0.529	0.589	0.40 – 0.60
Sodium	0.172	0.279	0.228	0.231	0.189	0.20 – 0.30

• Source: AGQ Peru S.A.C. (2025).

3.7. Total antioxidant capacity in leaves

Regarding the evaluation of total antioxidant capacity in lettuce leaves, T₃ stood out with 4,607.8 μmol Trolox/100 g of sample; however, this value had no impact on yield. T₅, despite having 4,020.8 μmol Trolox/100 g of sample, achieved a higher yield: 61.501 t ha^-1 of lettuce (see Table 12) (IIN, 2025). Therefore, this result indicates that an adequate concentration of antioxidant capacity was associated with higher lettuce yield.

Table 12
Total antioxidant capacity in lettuce leaves by treatment.

Treatment	G.S. dose (kg ha-1)	Result (μmol Trolox/100 g sample)	Commercial yield (t ha-1)
T1	0	3,644.7	45.325
T2	300	3,760.5	52.754
T3	600	4,607.8	55.846
T4	900	4,262.5	57.720
T5	1,200	4,020.0	61.501

• Source: IIN (2025).

3.8. Stomatal density in lettuce leaves

Once the stomata on the lettuce leaves were evaluated, it was determined that T₅ stood out with 134 stomata/mm², which contributed to a considerable antioxidant capacity of 4,020.0 μmol Trolox/100 g. This resulted in higher lettuce yield (see Table 8 and Figure 2). The interpretation suggests that a higher stomatal density, which differs by more than 50.8% from the control, is associated with a considerable concentration of antioxidant capacity, which in turn led to higher yield.

Figure 2
Micrograph of stomata in lettuce leaves by treatment.

3.9. Economic analysis of lettuce

Regarding the economic analysis, Table 13 presents the profitability per treatment. It is evident that with a higher dose of G.S. (T₅), a 443.5% increase was achieved, exceeding T₁ (365.3%) by 17.63%. This result is interpreted as indicating that a higher dose of G.S. yields a gain equivalent to 4.4 times that of the other treatments, demonstrating a significant economic benefit and supporting its recommendation for local lettuce farmers.

Table 13
Economic profitability analysis by treatment.

Treatment	G.S. dose (kg ha-1)	Commercial yield (kg ha-1)	Unit value ($)	Total income ($)	Production cost ($)	Utility ($)	Profitability (%)
T1	0	4,5325	0.2907	13,175.9	2,831.67	10,344.2	365.3
T2	300	5,2754	0.2907	15,335.5	2,972.60	12,362.9	415.9
T3	600	5,5846	0.2907	16,234.3	3,078.30	13,156.0	427.4
T4	900	5,7720	0.2907	16,779.1	3,184.00	13,595.1	427.0
T5	1,200	6,1501	0.2907	17,878.2	3,289.69	14,588.5	443.5

• Note: The price of $1.00 is equal to S/. 3.44 soles.

4. Discussion

4.1. Physical characteristics of lettuce

When analyzing the data on the physical characteristics of the lettuce, it was determined that there was no dose effect and that T₅ stood out among the treatments. In terms of yield, this treatment stood out with 61.501 t ha^-1, representing a 26.30% difference compared to the control (see Table 10). This result is interpreted as indicating that applying the highest dose of G.S. promotes microbial activity in the soil, thereby enhancing the availability of nutrients for plant uptake. This nutritional improvement optimizes photosynthesis, carbohydrate formation and translocation to reserves and other physiological processes, and the modulation of important biochemical pathways, such as the production of flavonoids and bioactive compounds with antioxidant properties. This acts as a defense mechanism against biotic and abiotic stress. Consequently, it improves lettuce yield and quality. The above is supported by Justel‐Díez et al. (2023), who note that seabird colonies can modulate the functioning of microbial communities, especially at the local level, subsequently affecting higher trophic levels. Likewise, Nikolaidou et al. (2021) state that inoculation with beneficial microorganisms, such as the arbuscular mycorrhizal fungus Rhizophagus irregularis and the growth-promoting rhizobacterium Bacillus subtilis, influenced the growth and development of lettuce and represents a sustainable solution, although further studies on their effects are needed. For their part, Liu et al. (2024) mention that the application of organic fertilizer improves leaf water status, nutrient uptake and homeostasis, chlorophyll synthesis, osmolite, hormone, and secondary metabolite production, antioxidant activity, and gene expression, leading to greater tolerance to drought, salinity, heat, and heavy metals.

4.2. Total nitrogen consumption for yield

It was determined that T₅, with 1,200 kg ha^-1 of G.S., stood out with 174.68 kg ha^-1 of nitrogen, equivalent to 84.29% compared to the control (see Table 6). This result indicates that incorporating the highest dose of G.S. into the soil provides nutrients and promotes the proliferation of beneficial microorganisms, including Azotobacter and Azospirillum, as well as other nitrogen-fixing bacteria, thereby increasing nitrogen availability for the plant. Its absorption enhances physiological processes such as photosynthesis, carbohydrate synthesis, and their translocation to storage organs. Biochemical activity, such as the biosynthesis of bioactive compounds with antioxidant capacity, is enhanced. These biochemical processes strengthen the plant against stress, thereby improving lettuce yield and quality. According to Giri et al. (2025), biofertilizers incorporate microorganisms such as Azotobacter, Azospirillum, cyanobacteria, mycorrhizae, Rhizobium, and aquatic ferns like Azolla, with Azospirillum being one of the most widely used nitrogen-fixing bacteria due to its ability to convert atmospheric nitrogen (N₂) into ammonia and promote plant growth. For their part, Hindersah et al. (2020) highlight that Azotobacter improves crop production through nitrogen fixation and the synthesis of phytohormones, as it produces IAA (indole-3-acetic acid), CKs (cytokinins), and GAs (gibberellins), which stimulate plant growth.

4.3. Total phosphorus consumption for yield

It was determined that the T₅ dose at 180.77 kg ha^-1 differed by 40.75% from the control (see Table 7). This is due to the higher G.S. dose; it incorporated beneficial microorganisms and nutrients that improved phosphorus availability. In line with this result, Healing et al. (2024) mention that seabirds connect habitats by transporting nutrients from pelagic zones to terrestrial and marine ecosystems via their guano, which is rich in nitrogen and phosphorus. In turn, Etesami et al. (2021) report that arbuscular mycorrhizal fungi supply phosphorus and other nutrients to their host plants in exchange for photosynthates, expanding the soil volume available for mineral uptake through hyphae that extend from the roots. Once absorbed, phosphorus participates in the synthesis of energy compounds such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) and in processes such as respiration, photosynthesis, and carbohydrate metabolism. Additionally, it participates in protein phosphorylation, regulating cell signaling, gene expression, and adaptation to environmental conditions. Consequently, this signaling activates antioxidants such as SOD, CAT, and ascorbate peroxidase (APX), which neutralize ROS, maintain cellular homeostasis, and enhance lettuce tolerance to abiotic and biotic stress. For their part, Khan et al. (2023) highlight that phosphorus plays a crucial role in various stages of the photosynthetic process, including ATP synthesis and the formation of ribulose-1,5-bisphosphate (RuBP), a vital molecule in the Calvin cycle. Likewise, Zhang et al. (2014) note that inorganic phosphorus (Pi) deficiency is detected in the root system, where hormones act as signaling molecules in developmental reprogramming; root-to-shoot signals are transmitted via the xylem and phloem, with microRNAs (micro ribonucleic acid), mRNAs (messenger ribonucleic acid), and sucrose coordinating responses throughout the plant. Finally, Hernández and Munné-Bosch (2015) indicated that, to counteract the effects of ROS and control their levels for signaling purposes, plants have developed a network of antioxidant defenses.

4.4. Nutrient concentration in lettuce leaves

When analyzing the nutrient concentrations in the leaves, it was determined that calcium, magnesium, sulfur, molybdenum, boron, and sodium were most prominent in T₂. However, T₅, which contained nitrogen, phosphorus, copper, and chlorides, was associated with the highest yield (see Table 11) (AGQ Perú S.A.C., 2025). This result indicates that applying high doses of G.S. added nutrients to the soil, improving their availability. By absorbing them, the plant increased the concentration of nitrogen, phosphorus, copper, and chlorides in its leaves, thereby enhancing the efficiency of energy compound formation such as ATP and ADP. Consequently, transpiration, photosynthesis, carbohydrate synthesis, and related processes improved. Copper played a vital role in optimizing the formation of antioxidant enzyme compounds that served as a defense mechanism against biotic and abiotic stress, thereby promoting higher lettuce yields. In line with these findings, Tian et al. (2018) mention that nitrogen and phosphorus concentrations in leaves limit photosynthetic and metabolic processes, plant growth, and productivity. Additionally, Fathi (2022) states that nitrogen is one of the main components of chlorophyll; since chlorophyll and nitrogen content are linked, nitrogen concentration determines the photosynthetic rate and plant production. Vey et al. (2025) highlight that phosphorus is essential for plants, as it participates in cellular energy flow and in the composition of various macromolecules. Similarly, Barra et al. (2019) state that phosphorus is part of the structure of nucleotides and phospholipids, such as ATP and ADP, and is involved in photosynthesis, respiration, energy storage and transfer, as well as cell division and growth. Finally, Xu et al. (2024) indicate that copper is an essential nutrient for plant growth and development, acting as an enzymatic cofactor in numerous biochemical pathways and playing a key role in photosynthesis, respiration, ethylene detection, and antioxidant systems.

4.5. Total antioxidant capacity in leaves

Regarding the evaluation of antioxidant capacity, T₃ exhibited 4,607.8 μmol Trolox/100 g, while T₅, with 4,020.8 μmol Trolox/100 g of sample, achieved the highest yield, at 61.501 t ha^-1 of lettuce (see Table 12) (IIN, 2025). This result suggests that the T₅ dose favored the accumulation of bioactive compounds in the leaves, which was associated with adequate antioxidant capacity and, consequently, with increased yield. This is explained by the fact that this antioxidant capacity efficiently activated POD and SOD enzymes, which balance ROS, and stimulated defense mechanisms against abiotic and biotic stress, resulting in higher yield. In line with this, Aina et al. (2019) reported that applying organic fertilizer resulted in better fruit quality, with higher concentrations of bioactive compounds such as phenols, flavonoids, and lycopene, as well as antioxidant activity associated with a greater percentage of ROS scavenging. Complementarily, Rao et al. (2025) highlight that the main enzymatic antioxidants, such as SOD, CAT, APX, and glutathione reductase (GR), constitute the first line of defense by neutralizing ROS, while non-enzymatic compounds such as ascorbate, glutathione, and polyphenols, including flavonoids, reinforce this protection in plants by scavenging free radicals.

4.6. Stomatal density in lettuce leaves

Regarding stomatal density in the leaves, T₅ stood out with 134 stomata/mm², which was associated with a considerable antioxidant capacity of 4,020.0 μmol Trolox/100 g (see Table 8). This result indicates that the highest dose of G.S. increased stomatal density and enhanced nutrient levels (nitrogen, phosphorus, copper, and chlorides), optimizing gas exchange and nutrient assimilation. These characteristics favored transpiration efficiency, carbohydrate synthesis and accumulation, and physiological processes that increased antioxidant capacity. In an integrated manner, these conditions acted as defense mechanisms against abiotic and biotic stress. Consequently, stomatal density was considered a relevant indicator, as it was associated with high antioxidant capacity that led to increased lettuce yield. Fernández et al. (2015) mention that stomata are pores surrounded by two guard cells that regulate their opening and closing; present in high densities on leaves, they control gas exchange and transpiration in the plant. Meanwhile, Nguyen et al. (2023) highlight that stomata regulate the balance between CO₂ uptake for photosynthesis and water loss through transpiration, constituting a key mechanism for improving crop yields and optimizing water-use efficiency in the face of climate change. Accordingly, Gálvez Torres et al. (2021) determined that, in the variation of stomatal number, T₄ with 8 t ha^-1 of sugarcane compost reached a density of 161 stomata/mm², resulting in a considerable yield of 15,738 t ha^-1 of beet. Finally, Cruz Nieto et al. (2025b) mention that stomatal density is established as an indicator that optimizes photosynthesis, evapotranspiration, carbohydrate translocation, and antioxidant capacity, strengthening the plant against environmental stress and, consequently, increasing yield.

4.7. Economic analysis of lettuce

Regarding the economic analysis of the lettuce, T₅ showed a 443.5% profitability compared to the other treatments (see Table 13). This result indicates that applying the highest dose of G.S. yielded a profit 4.4 times greater than that of the other treatments, demonstrating the profitability of this fertilizer. Therefore, the dose is considered advisable for farmers in the Supe Puerto district, as it promotes sustainable cultivation.

5. Conclusion

It was determined that T₃, with 600 kg ha^-1 of G.S., stood out for its antioxidant capacity of 4,607.8 μmol Trolox/100 g, while T₅, with 4,020.0 μmol Trolox/100 g, achieved the highest yield at 61.501 t ha^-1 of lettuce. Therefore, at higher fertilizer doses, the concentrations of nitrogen, phosphorus, copper, and chlorides increased, as did stomatal density in the leaves, which optimized transpiration, photosynthesis, carbohydrate synthesis, and other physiological processes, while also boosting antioxidant levels—all of which collectively contributed to the crop’s vigor and yield.

Additionally, it was determined that T₂ excelled in most nutrients, such as calcium, magnesium, sulfur, molybdenum, boron, and sodium, while T₅ stood out in nitrogen, phosphorus, copper, and chlorides in the leaves, achieving the highest lettuce yield. Therefore, with a higher fertilizer dose, nutrient concentrations were reduced and the efficiency of ATP and ADP synthesis was increased, which optimized transpiration, photosynthesis, carbohydrate synthesis, related physiological processes, and the modulation of antioxidant pathways. This strengthened resistance to abiotic and biotic stress and improved yield.

Finally, it was determined that T₅ stood out in stomatal density with 134 stomata/mm², which was associated with considerable antioxidant capacity and resulted in the highest lettuce yield. Therefore, stomatal density was considered an indicator since, together with nutrients such as nitrogen, phosphorus, copper, and chlorides in the leaves, it promoted gas exchange and nutritional uptake efficiency. These factors optimized transpiration, photosynthesis, carbohydrate synthesis, and physiological processes and modulated the activity of antioxidant pathways. This strengthened the plant’s resistance to abiotic and biotic stress, resulting in higher yield.

• Data Availability Statement
  I mention to you that the entire data set supporting the results of this study was published in the article itself.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Data availability

I mention to you that the entire data set supporting the results of this study was published in the article itself.

Publication Dates

• Publication in this collection
  13 Mar 2026
• Date of issue
  2026

History

• Received
  09 Dec 2025
• Accepted
  16 Jan 2026