Introduction

In the cheese industry, 100 L of processed milk yields between 85 and 90 L of whey with retention of up to 55% of milk nutrients (^{Utama et al., 2017}; ^{Montalvo-Salinas et al., 2018}). Globally, between 180-190 million tonnes of whey are generated annually (^{Guerrero-Rodríguez et al., 2012}; ^{Mieles-Cedeño et al., 2018}). In Mexico, more than 2.4 million tonnes of whey are generated annually, but only 50% is processed (^{Mazorra-Manzano and Moreno-Hernández, 2019}), the rest is discarded into water bodies or in the soil and causes serious environmental damage (^{Dainka et al., 2019}).

^{González (2012}) reports a biological oxygen demand (BOD) of 40 g L^-1 and a chemical oxygen demand (COD) of 60 g L^-1 with a biodegradability index (BOD5/COD) between 0.4 and 0.8, which exceed the limits allowed by the Official Mexican Standard NOM-001-ECOL-1996, on the maximum permissible limits of pollutants in discharges of wastewater for agricultural use, which is 0.2 g L^-1 (^{DOF, 1997}). ^{Ramírez (2012}) equates the polluting force of 5 L of whey to that of sewage produced in one day by a person.

To take advantage of whey lactose, proteins, and microelements (^{Chandrapala et al., 2015}; ^{Andrade et al., 2017}), several studies have been carried out to apply it in human and animal food, production of bioplastics and fertilizers (^{Quille et al., 2021}), and for agricultural irrigation (^{Araújo et al., 2017}; ^{Krause et al., 2017}; ^{Araújo et al., 2020}). Where cheese factories with low technification predominate (^{Faría et al., 2002}; ^{Villegas-Soto et al., 2018}), the use of sweet whey in agricultural applications represents a good option.

When whey is dumped into agricultural soil, it physically and chemically affects its structure (^{Carvalho et al., 2013}) and therefore decreases agricultural yield (^{Parra, 2009}; ^{Araújo et al., 2013}). However, other studies have shown that it improves soil aggregation (^{Kelling, 1981}) and soil fertility (^{Robbins and Lehrsch, 1998}; ^{Jones et al., 1993}).

Bioassays performed on BOD and COD are used to estimate the toxicity or potential biological impact that exposure to industrial effluents has on the physiological function of living organisms (^{Schultz et al., 2002}). These tests determined the concentration that inhibits the germination of 50% of the exposed organisms, which is known as the mean lethal concentration (LC₅₀), as well as the concentration at which a sublethal effect (SLC) is produced, characterized by growth retardation (^{Uc-Peraza and Delgado-Blas, 2012}).

In addition to tests carried out in vitro, ^{Teacă and Bodĭrlău (2008}) propose bioassays for phytotoxicity in hydroponic systems. Therefore, the objective was to evaluate the toxicity of different concentrations of whey in the irrigation of alfalfa (Medicago sativa L.) and corn (Zea mays L.) in the stages of germination and development of seedlings.

Materials and methods

The experiments were conducted in the Laboratory of Microbial Molecular Ecology of the Microbiological Sciences Research Center (CICM, for its acronym in Spanish) of the Meritorious Autonomous University of Puebla (ICUAP-BUAP, for its acronym in Spanish). Preparation and analysis of whey. Sweet whey was obtained from the production of fresh cheese in the cheese factory ‘Lácteos Galeazzi’ in Chipilo Puebla, which is characterized by its low acidity, neutral pH, and absence of salts. It was immediately refrigerated at 4 °C in 1 L Schott bottles until use. Its characterization was carried out by measuring, in triplicate, its pH with a Hanna 2010 potentiometer, and its acidity by titration expressed in degrees Dornic (°D) (^{AOAC, 1995}).

Results and discussion

Physicochemical analysis. Two types of whey were found as a by-product of cheese making: sweet and salty. The first, which comes mainly from the production of fresh cheese, presented a pH close to neutral (6.8 ±0.3) and low acidity (19 ±0.5 °D) because it had no added salts and had low concentrations of calcium chloride. The second, which comes from the production of quesillo, cottage cheese, and ranch cheese, had a pH of 5.41 ±0.3 and acidity of 38 ±0.6 °Dornic. These results are similar to those reported by ^{Panesar et al. (2010}) for sweet whey and by ^{Guerrero-Rodríguez et al. (2012}) for acid whey.

It is important to consider that the characteristics of whey vary depending on the type of cheese that is processed and that its agricultural use will depend mainly on whether it is free of added salt. On the other hand, whey, although it has a low acidity at the time it is generated, tends to acidify rapidly naturally due to the development of its microbial flora: the lactic acid generated in this process can have a beneficial effect in agricultural processes such as in the chelation and solubilization of phosphates (^{Paredes-Mendoza and Espinosa-Victoria, 2010}) but it will be a priority to take care of the development of pathogenic microorganisms with controlled fermentation processes, for example with the technology of beneficial and/or efficient microorganisms (^{Morocho and Leiva, 2019}).

LC₅₀ in seed germination. At concentrations of 0 to 40% whey, germination is maintained at almost 100%, however, with an LC₅₀-5, germination fell to 53% in alfalfa seeds (Figure 1), a value obtained graphically by interpolation, and that coincides when substituting the value y= 50 (50% of the population that does not survive) in the polynomial equation. The decrease in germination from 40% whey concentration is due to the high concentration of NaCl, which raises osmotic pressure and caused an adverse physiological effect.

Figure 1 Toxic effect of sweet whey at different concentrations on the germination of alfalfa seeds at five days. Corrected values that were considered with 83.5% germination of the control. 

^{Dantas et al. (2005}) found similar results in germination of alfalfa seeds exposed to salt stress. ^{Laynez-Garsaball et al. (2008}) observed a decrease in germination due to the increase in osmotic potential exerted by NaCl, while ^{Porta et al. (1999}) demonstrated that the total inhibition of germination in high salt concentrations is due to the accumulation of chloride, which decreases water absorption and affects the germination rate.

For their part, ^{Mahdavi and Sanavy (2007}) report that ions present in whey can also cause hardening of cell walls and affect seedling growth. LC₅₀ was not obtained in corn, because in all treatments there was contamination by filamentous fungi. This may be due to the natural microbial load of corn, which is favored by the contribution of nutrients from whey.

SLC in sprout development. Sweet whey showed a sublethal effect at concentrations greater than 12% in the development of alfalfa sprouts at 10 days of exposure, observing significant differences between the total length of sprouts and hypocotyls compared to the drinking water control (p< 0.05) (Figure 2 a and b), although no effects were observed on total biomass (data not presented). In similar works, ^{Navarro et al. (2006}) report the inhibition in the growth of hypocotyls of chicory, lettuce, and endive seeds exposed to neutralized effluents from citrus and wine industries diluted to 10³.

Figure 2 Sublethal effect of sweet whey on total length (a) and length of the hypocotyl; and (b) 10-day-old sprouts of alfalfa. Different letters between columns indicate a significant difference (t-Student p< 0.05). 

If this value is compared with that obtained in the present study, in which inhibition of hypocotyl development was observed at a concentration of 12% (10¹), then it can be affirmed that sweet whey has low toxicity. On the other hand, SLC values were not obtained for corn because, as in the previous experiment, the seeds were contaminated with filamentous fungi in all treatments.

LC₅₀ in seedling emergence. The lethal concentration of sweet whey in corn seedlings was obtained at a concentration of 40%, and similarly in alfalfa seedlings, although 100% lethality was not observed in alfalfa at high concentrations of whey, the survival of 50% of the population is evidence that this species is more tolerant to sweet whey than corn (Figure 3). Various authors such as ^{Wang (1987}); ^{Navarro et al. (2006}), in works carried out on lettuce, found differences in tolerance to the toxicity of different substances in different species, with lettuce being one of the most used species in this type of study due to its high sensitivity.

Figure 3 Toxic effect of sweet whey at different concentrations on the emergence of seedlings of corn (Zea mays L.) and alfalfa (Medicago sativa L.). 

Alfalfa tolerance can be explained because it is a seed more resistant to low pH, as reported by ^{Köpp et al. (2011}), who found that alfalfa can grow optimally between pH 5 and 6 but can tolerate even lower pH values, while the optimal pH of corn growth is 6 to 7.2 and exhibits growth inhibition at pH values lower than 5 (^{Aldrich and Long, 1994}).

SLC in seedling development. No differences were observed in variations in total length in all concentrations studied (p< 0.5) so it is assumed that sweet whey has no sublethal effect at a concentration lower than 10%, in corn or alfalfa seedlings; however, a beneficial effect was observed at a concentration of 5 ±2.6% for alfalfa length (p< 0.05) (Figure 4a and 4b).

Figure 4 Sublethal effect of sweet whey on length (a) and biomass; and (b) of 60-day-old alfalfa seedlings. Different letters between columns indicate a significant difference (t-Student p< 0.05). 

The differences observed between experiments with seeds and seedlings indicate that the phytotoxicity is lower for alfalfa than for corn and that in both species, whey is more toxic in the early stages of development. On the other hand, it is necessary to carry out more research to evaluate what is the optimal concentration in other stages of the development of the plants studied, as well as the use of whey under field conditions.

Conclusions

According to the chemical characterization carried out, it was found that the main wheys generated in Chipilo Puebla are sweet and acid-salty. The first is the one with the greatest potential for biotechnological use since it has low toxicity (LC₅₀ of 55%), although it was determined that at a concentration of 12%, it has a negative effect on the growth of 10-day-old alfalfa sprouts, which indicates that sweet whey has low toxicity, with high probabilities to be used safely in the agricultural irrigation of species such as alfalfa and corn.

Its use is recommended at concentrations lower than 12% in order to maximize its use and at concentrations less than 4% as a plant growth promoter. The use of agro-industrial by-products represents an area of research with great potential.

The use of whey to make products of commercial interest, particularly as a raw material to produce biofuels, metabolites, and biofertilizers by biotechnological transformation, is gaining increasing attention. It is suggested to continue studying its use as a nutrient medium to promote the growth of microorganisms of agricultural interest such as fungi and bacteria that fix nitrogen, solubilize phosphorus, and are antagonistic to phytopathogens and compare its effect on corn and alfalfa compared to untreated whey.