**1. Introduction**

Pests and diseases have always had repercussions directly on losses of crops and livestock products and indirectly over the income decreases due to insufficient harvests of commercial crops. Chemical pesticides are used in an excessive way and without prior technical assistance to pest distribute control, which instead of solving the problem, has produced strong damage to agricultural and livestock productivity, as well as important environmental effects with implications to public health [1]. Pesticides are chemical substances designed to prevent, delay, repel or fight any pests [2].

fetal development and adverse effects on testicular function (semen, sperm, and sperm motility decrease) due to the mimetization or antagonism of reproductive hormones [18, 19]. Thus, DDT metabolites and DDT are considered endocrine disruptors, with estrogenic properties related to several types of estrogen-dependent cancers, such as breast cancer; therefore, the use of this pesticide was prohibited on the decade of the 1970s in most countries [20]. Nevertheless, pesticides remain in the environment (persistence); therefore, the pesticides have been widely distributed, and their traces can be detected in all areas of the environment (air, water, and soil) [21, 22]; therefore, current tendency is focused on natural sources for biological pesticide control. On the other hand, the indiscriminate and uncontrolled application of synthetic pesticide besides to accumulate residues in the environment and, in some cases, in living beings, has caused resistance in some pest. The first case reported of DDT resistance occurred in 1947, and since then, it has increased alarmingly, and it has been estimated that there are currently around 489 species of pest resistant to 400 different pesticides in the world [23]. The irrational use of synthetic pesticide has produced genotypic and phenotypic changes in many species, generating resistance to the action of most of them, including inorganics, DDT, cyclodienes, organophosphates, carbamates, pyrethroids, juvenile hormone analogues, avermectins, neonicotinoids, and antimicrobial [22–24]. Though the use of pesticides has offered significant economic benefits by enhancing the production and yield of food and fibers and the prevention of vector-borne diseases, evidence suggests that their use has adversely affected the health of human populations and the environment [21]. Because of this problem, several researchers are focused their studies on the identification of new natural sources containing active metabolites that could be used on the

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The development of essential oils (EOs) as plant protection products is especially suited to organic farming as well as to integrated pest management. They are natural in origin and biodegradable, have diverse physiological targets within insects, and, consequently, may delay the evolution of insect resistance [27]. EOs act as fumigants, pesticides, repellents, and antifeeds that could affect some biological parameters such as grown rate, biological cycle, and reproduction [26]. One of the most widely analyzed oils is neem (*Azadirachta indica*) which has a toxic effect over several pests and it is a potential alternative to the synthetic pesticide [28]. Neem oil active compounds are azadirachtin, salannin, nimbin, and their respective analogues; being the azadirachtin the most abundant compound [29]. Azadirachtin acts on the immature stages of the insects avoiding their molt or maturation from larva to pupa and generating mutations in the development of different essential parts for their survival, because it affects their ability to oviposit in mature stage and hatch during the larval stage [30]. Its effect is reinforced by the action from the rest of limonoides, such as salannin and nimbin, which have repellent and antifeeder effects over many insects [31]. Although the concentration of azadirachtin is sufficient and its location well established in the seed, its extraction presents some problems, because it is soluble in polar organic solvents, but slightly soluble in water,

besides is photosensitive and thermolabile, which conditions its activity in the oil.

Among the methods used for the extraction of azadirachtin, it could be mentioned the cold extrusion in a mechanical press, maceration, and percolation with the use of organic solvents. Each of the proposed methods stimulates the extraction of azadirachtin but in different proportions. Various researchers have suggested that the high variability in the extraction

control of pest [25, 26].

In [3], it was mentioned that Mexico ranked fifth in the world in the use of 1,1-bis(4-chlorophenyl)- 2,2,2-trichloroethane (DDT) in agricultural programs and fourth place for its use in public health. As many as 69,545 ton of DDT were used in health campaigns for the control of malaria and agricultural activities, from 1957 onward, DDT was applied every 6 months indoors and outdoors with a coverage of 2 g/m2 , and almost 1000 ton DDT/y were used in agricultural areas [4]. Indeed, DDT was used in Mexico until the year 2000, and DDT and its metabolites have been found in the environment [5] as well as in human tissues [5, 6], breast milk [7], raw cow's milk and bovine meat [8, 9]. DDT is a very stable organochlorine pesticide that is almost completely metabolized, but small percentage remains as o,p´-DDT, while the most of its concentration is transformed into p,p´-DDE, which is characterized for its poor solubility in water and high affinity for lipids. It is considered a pollutant of high persistence due to its half-life of up to 15 years in the environment [10]. This pesticide persistence is responsible of the wild flora and fauna deterioration, as well as the contamination of soil, water table, continental, and coastal waters. Besides, pesticides can be incorporate into pasture, vegetables, and edible animals, which when consumed, act as transporters facilitating its accumulation in living organisms [11, 12].

In the state of Chiapas, México, [13] found high levels of total DDT in outdoor soil samples that ranged from 0.002 to 27 mg kg−<sup>1</sup> , while the levels found by [4] in Chihuahua, México, ranged from 0.001 to 0.788 mg kg−<sup>1</sup> . Taking into account the guideline for total DDT in residential soil of 0.7 mg kg−<sup>1</sup> in Canada [14], the soil samples from Chiapas had levels higher than the guideline. The high levels of OCs observed may be due to ongoing usage as well as the emission of old residues from soil. Soils are an important sink and source for persistent organic pollutants to the atmosphere. In many of the cases, the contamination of soil with pesticides is due to its incorrect storage, either by leakage of corroded tanks containing liquid pesticides or by aerial dissemination of powder pesticide. However, when pesticides infiltrate the soil, their dissemination depends on the nature of the pesticide, as well as the composition, moisture, pH, and temperature [12, 15]. Because of that, a small portion of spilled pesticides can generate a high soil contamination. Moreover, soil pesticides infiltration can cause their introduction and distribution in the food chain, accumulating successively on each ecological niche until reaching lethal doses for some constituent organisms of the chain, or until reaching high levels of the trophic network [16].

This problem is aggravated due to the excessive use of pesticides in the agricultural sector, the absence of technological remediation and the lack of safety interval (waiting period) for the harvest of agricultural products, which has significant impacts on public health [17]. Some of the toxic effects of DDT and its metabolite identified in mammalian have been alterations in fetal development and adverse effects on testicular function (semen, sperm, and sperm motility decrease) due to the mimetization or antagonism of reproductive hormones [18, 19]. Thus, DDT metabolites and DDT are considered endocrine disruptors, with estrogenic properties related to several types of estrogen-dependent cancers, such as breast cancer; therefore, the use of this pesticide was prohibited on the decade of the 1970s in most countries [20]. Nevertheless, pesticides remain in the environment (persistence); therefore, the pesticides have been widely distributed, and their traces can be detected in all areas of the environment (air, water, and soil) [21, 22]; therefore, current tendency is focused on natural sources for biological pesticide control. On the other hand, the indiscriminate and uncontrolled application of synthetic pesticide besides to accumulate residues in the environment and, in some cases, in living beings, has caused resistance in some pest. The first case reported of DDT resistance occurred in 1947, and since then, it has increased alarmingly, and it has been estimated that there are currently around 489 species of pest resistant to 400 different pesticides in the world [23]. The irrational use of synthetic pesticide has produced genotypic and phenotypic changes in many species, generating resistance to the action of most of them, including inorganics, DDT, cyclodienes, organophosphates, carbamates, pyrethroids, juvenile hormone analogues, avermectins, neonicotinoids, and antimicrobial [22–24].

**1. Introduction**

delay, repel or fight any pests [2].

110 Soil Contamination and Alternatives for Sustainable Development

ranged from 0.002 to 27 mg kg−<sup>1</sup>

from 0.001 to 0.788 mg kg−<sup>1</sup>

of 0.7 mg kg−<sup>1</sup>

ers facilitating its accumulation in living organisms [11, 12].

with a coverage of 2 g/m2

Pests and diseases have always had repercussions directly on losses of crops and livestock products and indirectly over the income decreases due to insufficient harvests of commercial crops. Chemical pesticides are used in an excessive way and without prior technical assistance to pest distribute control, which instead of solving the problem, has produced strong damage to agricultural and livestock productivity, as well as important environmental effects with implications to public health [1]. Pesticides are chemical substances designed to prevent,

In [3], it was mentioned that Mexico ranked fifth in the world in the use of 1,1-bis(4-chlorophenyl)- 2,2,2-trichloroethane (DDT) in agricultural programs and fourth place for its use in public health. As many as 69,545 ton of DDT were used in health campaigns for the control of malaria and agricultural activities, from 1957 onward, DDT was applied every 6 months indoors and outdoors

DDT was used in Mexico until the year 2000, and DDT and its metabolites have been found in the environment [5] as well as in human tissues [5, 6], breast milk [7], raw cow's milk and bovine meat [8, 9]. DDT is a very stable organochlorine pesticide that is almost completely metabolized, but small percentage remains as o,p´-DDT, while the most of its concentration is transformed into p,p´-DDE, which is characterized for its poor solubility in water and high affinity for lipids. It is considered a pollutant of high persistence due to its half-life of up to 15 years in the environment [10]. This pesticide persistence is responsible of the wild flora and fauna deterioration, as well as the contamination of soil, water table, continental, and coastal waters. Besides, pesticides can be incorporate into pasture, vegetables, and edible animals, which when consumed, act as transport-

In the state of Chiapas, México, [13] found high levels of total DDT in outdoor soil samples that

line. The high levels of OCs observed may be due to ongoing usage as well as the emission of old residues from soil. Soils are an important sink and source for persistent organic pollutants to the atmosphere. In many of the cases, the contamination of soil with pesticides is due to its incorrect storage, either by leakage of corroded tanks containing liquid pesticides or by aerial dissemination of powder pesticide. However, when pesticides infiltrate the soil, their dissemination depends on the nature of the pesticide, as well as the composition, moisture, pH, and temperature [12, 15]. Because of that, a small portion of spilled pesticides can generate a high soil contamination. Moreover, soil pesticides infiltration can cause their introduction and distribution in the food chain, accumulating successively on each ecological niche until reaching lethal doses for some constituent organisms of the chain, or until reaching high levels of the trophic network [16].

This problem is aggravated due to the excessive use of pesticides in the agricultural sector, the absence of technological remediation and the lack of safety interval (waiting period) for the harvest of agricultural products, which has significant impacts on public health [17]. Some of the toxic effects of DDT and its metabolite identified in mammalian have been alterations in

, and almost 1000 ton DDT/y were used in agricultural areas [4]. Indeed,

, while the levels found by [4] in Chihuahua, México, ranged

. Taking into account the guideline for total DDT in residential soil

in Canada [14], the soil samples from Chiapas had levels higher than the guide-

Though the use of pesticides has offered significant economic benefits by enhancing the production and yield of food and fibers and the prevention of vector-borne diseases, evidence suggests that their use has adversely affected the health of human populations and the environment [21]. Because of this problem, several researchers are focused their studies on the identification of new natural sources containing active metabolites that could be used on the control of pest [25, 26].

The development of essential oils (EOs) as plant protection products is especially suited to organic farming as well as to integrated pest management. They are natural in origin and biodegradable, have diverse physiological targets within insects, and, consequently, may delay the evolution of insect resistance [27]. EOs act as fumigants, pesticides, repellents, and antifeeds that could affect some biological parameters such as grown rate, biological cycle, and reproduction [26]. One of the most widely analyzed oils is neem (*Azadirachta indica*) which has a toxic effect over several pests and it is a potential alternative to the synthetic pesticide [28]. Neem oil active compounds are azadirachtin, salannin, nimbin, and their respective analogues; being the azadirachtin the most abundant compound [29]. Azadirachtin acts on the immature stages of the insects avoiding their molt or maturation from larva to pupa and generating mutations in the development of different essential parts for their survival, because it affects their ability to oviposit in mature stage and hatch during the larval stage [30]. Its effect is reinforced by the action from the rest of limonoides, such as salannin and nimbin, which have repellent and antifeeder effects over many insects [31]. Although the concentration of azadirachtin is sufficient and its location well established in the seed, its extraction presents some problems, because it is soluble in polar organic solvents, but slightly soluble in water, besides is photosensitive and thermolabile, which conditions its activity in the oil.

Among the methods used for the extraction of azadirachtin, it could be mentioned the cold extrusion in a mechanical press, maceration, and percolation with the use of organic solvents. Each of the proposed methods stimulates the extraction of azadirachtin but in different proportions. Various researchers have suggested that the high variability in the extraction of azadirachtin from neem depends on several factors as age of the tree, region of its production, stage of fruit development, availability of the internal portion of the seed, storage conditions of the seed, methods and solvents used for its extraction, and the particle size [32–35]. However, one aspect that has not been taken into account is the physical barrier exerted by the cell wall of the seed that directly affects the availability and extraction of azadirachtin and other acaricidal compounds. This problem has been overcome with great success in the plant extract industry through the application of cellulases or preparations with multiple enzymatic activities (cellulases, hemicellulases, and pectinases) [34, 36, 37].

cellulose structures and the azadirachtin release was evaluated. Neem seed hydrolysis was indirectly determined by the quantification of reducing sugar released during the enzymatic reaction. Total protein was estimated with [40] bovine serum albumin (BSA) as standard, and enzymatic activity (EA) for each enzymatic preparation was defined as changes in absorbance

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.

The optimal conditions of pH and temperature were established based on the enzymatic hydrolysis of the neem seed and the release of azadirachtin. To determine the optimum pH of the enzymatic preparations, 1 g of the dehydrated seed was homogenized with 19 mL of phosphate buffer (3.0, 3.5, 4.0, 4.5, 5.0, and 5.5) and was conditioned at 50°C for 5 min and after 1 mL of the corresponding enzyme preparation was added. The enzymatic reaction was carried out for 2 h and was stopped by immersion in ice water. Subsequently, 1 mL of the sample was taken for reducing sugar determination and 1 mL for azadirachtin quantification by HPLC [41]. Once the optimum pH of each enzyme preparation was identified, the effect of the temperature at 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70°C was evaluated. Additionally, one extract without enzyme added was prepared as control, and the concentrations of reducing sugars released were subtracted from the values obtained in each of the enzymatic treatments. Furthermore, enzyme:neem seed (dry base) ratio was evaluated under optimal conditions of pH and temperature, and for this, 2 g of neem seeds (wet base) were used and 0.5, 1.0, 2.0, and

To determine kinetics of azadirachtin release, 100 g of neem seed (wet base) was homog-

3 min using an Ultra Turray homogenizer, T-25 basic (IKA®, Wilmington, NC). Five extracts were elaborated, one for each enzyme preparation and one without enzyme (control), and were incubated at optimum temperatures for each one. An aliquot was taken at 0, 2, 4, 6, 12, 18, and 24 h for the determination of azadirachtin. All samples were analyzed by triplicate.

Azadirachtin quantification was carried out by the HPLC technique proposed by [41], using a binary HPLC system (Waters 1525) and a photodiode detector (Waters 2996). The analytical separation was carried out with a Nova-Pak C18 column of 4 μm (3.9 × 150 mm) SUM (Waters®

analyzing by HPLC. The samples were filtered through acrodiscs (Millipore) of 0.22 μm, and

The optimal conditions of pH, temperature, and the enzyme:substrate ratio were analyzed by means of analysis of variance (ANOVA) at a level of significance of p < 0.05, and Tukey's tests

), and azadirachtin was read at 217 nm in a retention time of 3.1 min.

Milford, MA). Neem samples were centrifuged and diluted with acetonitrile (1:1, v v−<sup>1</sup>

20 μl was injected into the column. The flow rate was set at 1 mL min−<sup>1</sup>

) ratio for

) before

, the mobile phase was ace-

enized with phosphate buffer at the optimum pH previously identify (1:10, w v−<sup>1</sup>

**2.3. Optimum conditions of pH, temperature, and enzyme concentration**

in 0.001 of reducing sugars mg protein−<sup>1</sup> min−<sup>1</sup>

4 mL of each of the enzyme preparations were tested.

**2.4. Kinetics of azadirachtin release**

**2.5. Determination of azadirachtin**

tonitrile: water (40:60, v v−<sup>1</sup>

**2.6. Statistical analysis**

The assisted extraction by cellulolytic enzymes has proven to be a viable and feasible tool to obtain bioactive metabolites from plants, due to its effect on lignocellulosic structures of its cell wall, which increase the yield of oils, pigments, flavorings, and aromas extracted in comparison with traditional extraction methods [38]. Due to the advantages of the use of cellulolytic enzymes for the production of bioactive metabolites from plants and the inherent need to develop sustainable and environmentally friendly alternatives that avoid the resistance phenomenon, the present study had the purpose of evaluating the use of food-grade enzyme preparation on the hydrolysis of the neem seed to obtain extracts with higher concentrations of azadirachtin, but without the use of solvents.
