The acceleration of human population growth imposes tremendous pressure on natural resources and on the agricultural systems necessary to supply raw materials and foodstuffs ^[1][2][3][4][1,2,3,4]. Modern agriculture employs several chemical compounds to increase productivity and avoid crop losses caused by pests. Among these agrochemicals, pesticides have a primary role ^[5][6][5,6]. Pesticides are chemical substances widely employed for the control of different crop pests in agricultural areas, to avoid decreases in the quality and yield of farm products during food storage, transportation, and commercialization processes. Pesticides also have important applications in the control of vectors for different diseases in livestock and humans ^[7][8][7,8]. Hence, pesticide usage greatly benefits human societies worldwide [9]. However, the intensive application of pesticides is related to adverse effects on the environment [10], biodiversity [11], soil health and fertility [12], food supply, and human health ^[13][14][13,14]. Thus, almost all processes involving pesticides represent a risk to environmental health [8].

Pesticide residues have been identified in all environmental compartments, soil, water, and air [15]. It is estimated that only a small proportion of the total pesticides employed in agricultural areas reaches the targets in crops, with the remaining percentage dispersed through the environment. Pollution caused by pesticides is an important concern in several regions around the world ^[16][17][16,17]. Moreover, in various less-developed countries, many obsolete, unused, or expired pesticides are stored, constituting an environmental risk that endangers environmental and human health ^{[18][19][20][21]}[18,19,20,21]. The release of pesticides into the environment generates different adverse effects such as reducing the overall soil, water, and air quality, threats to wildlife and biodiversity, and the contamination of food destined for livestock and humans, with acute and chronic toxic effects on humans, among the most relevant ^{[17][22][23][24][25][26]}[17,22,23,24,25,26].

Pesticides can reach soils and water bodies because of their production and extensive scale application in agricultural systems. Spills, leaks, wastewater, and inappropriate waste disposal in the pesticide manufacturing industry have been identified as environmental pollution sources ^[27][28][27,28]). However, operations related to pesticide dissolution before application, the filling and cleaning of aspersion equipment and machinery, and accidental spills in crop fields have also been identified as important punctual pesticide pollution sources [29]. The pesticides and their degraded metabolites released from producing factories reach the soil and both surface water and groundwater bodies, threatening aquatic environments and human health [30] through the presence of pesticides in food and drinking water [31]. The adverse impacts of pesticide pollution on the environment and human health make it necessary to implement adequate strategies for the treatment of pesticide residues and the remediation of polluted sites ^[32][33][32,33].

Microbial-mediated bioremediation has been proposed as a cost-competitive, efficient, adaptable, and safe strategy for the treatment of different pollutants [34], including pesticides ^[35][36][37][35,36,37]. Biobeds are one of the microbial-mediated bioremediation approaches proposed for the treatment of pesticide wastes. These systems were developed in Sweden for the control and treatment of pesticide pollution caused by the environmental release of effluents derived from the washing of equipment and machinery employed for pesticide application in crop fields, as well as the inadequate disposal of pesticide residues, accidental spills in the handling and application of pesticides, and the residual water from different pesticide formulation plants and agro-industries ^{[38][39][40][41][42]}[38,39,40,41,42]. This technology has been adopted in many countries and successfully applied for the biodegradation of different pesticide residues ^{[41][42][43][44][45][46]}[41,42,43,44,45,46].

Both small and high-scale agricultural activity involve the use and disposal of pesticides and represent significant pollution sources. Pesticide punctual pollution events are generated because of the activities related to the use of different pesticides in crop fields. The release and dispersion of pesticide residues into the environment represent a high risk for ecosystems and human health on a global scale ^[47][48][47,48].

Three critical points can generate contamination by pesticides during agricultural activity. The first occurs when the pesticide application devices are filled; here, highly concentrated pesticide solutions can be spilled. The second point is when the pesticides are spread in the crop field; after application, pesticides can reach the surrounding environment. The third point is during the handling and washing of the application devices when a high amount of residual water with low-concentration pesticide remnants is generated. Improper handling of these residues causes soil and water pollution by direct disposal, leaching, or runoff processes ^{[31][49][50][51]}[31,49,50,51]. Avoiding pesticide release into the soil and water is a crucial issue in mitigating the environmental pollution associated with agricultural practices, and biobeds have been proposed as a feasible alternative for punctual pesticide pollution mitigation.

Biobeds are biological systems used to treat pesticide residues derived from the operations related to the application of pesticides in crop fields. Their use helps minimize pesticide delivery into the environment, as well as protecting soil and water from pesticide pollution ^[52][53][52,53]. Biobed technology was developed in Sweden in the 1990s by Torstenson and Castillo (1997) [54], as a low-cost and efficient alternative to mitigate pesticide pollution from specific punctual sources. In the original proposal, biobeds had a simple design. The system was a trench packed with a mixture of 50% wheat straw, 25% soil, and 25% peat (biomixture), covered with a grass lid. The biomixture has the function of absorbing pesticide residues and serving as a support for the development of different microorganisms, mainly bacteria, and fungi, needed for pesticide degradation in the system (Figure 1).

Biobeds were fast adopted in Swedish agriculture to treat effluents with different pesticide residues [55]. After that, biobed systems were integrated into several countries of the European Union, and subsequently, experimental devices were established in several countries around the world [56]. In 2016, the number of installed biobed systems in the European Union was around 9000, located mainly in France (4500), Sweden (750), and the United Kingdom (450), while in the context of Latin America, 1500 biobed systems were installed in Guatemala, and additional experimental scale biobeds are located in Africa, Asia, and North America [57]. The biobed design has been modified according to the climatic characteristics of the regions in which it is located and the availability of the materials for the biomixture, including different lignocellulosic materials instead of wheat straw, or compost instead of peat, as some examples (Figure 2).

The biobeds’ pesticide treatment capacity is related to the design, surface, scale, and biomixture composition. The water-holding capacity of the biomixture is related to the components that integrate it. For example, Henriksen et al. (2003) [58] determined an absorption between 1.6 and 5.2 L·kg⁻¹ of biomixture (wheat straw, soil, and peat; 2:1:1) for the herbicides isoproturon and mecoprop. Foog et al. (2004) [59] determined the maximum water-holding in the system for efficient pesticide dissipation was 1121 L/m² for a biobed (1.5 m deep) packed with a biomixture composed of wheat straw, soil, compost (2:1:1). Recently, Lescano et al. (2022) [41] treated 200 L of wastewater with the presence of different pesticides in a pilot biobed system (1000 L capacity) packed with a biomixture of soil and millet stubble (1:1).

On the other hand, the climatic conditions of each region can affect the efficiency of pesticide dissipation in these systems. The most important climatic factors that modify the effectiveness of pesticide dissipation in a biobed are the environmental temperature and precipitation/moisture. At low environmental temperatures, the degradation efficiency is reduced; in some regions, low temperatures can freeze the system [60]. On the other hand, high levels of precipitation can generate an imbalance in the water content of the system so that the efficiency and speed of dissipation of pesticides are reduced and can generate pesticide leaching events [60]. Finally, climatic conditions affect the integrity of the biomixture; in temperate climates, the biomixture should be replaced every five to eight years [55], but in areas with tropical climates, the biomixture should be replaced in six months to two years ^[52][61][52,61]. These facts must be taken in count for the biobeds systems’ implementation and adequate pesticide residues treatment.

4. Key Factors in Biobeds’ Effectiveness

4.1. Biomixture

The composition of the biomixture is a key factor for the efficiency of pesticide degradation in biobed systems; so, each component (wheat straw, soil, and peat) plays an important role [62]. For example, wheat straw is a lignocellulosic substrate that acts as an adsorbent for pesticides in the system, serves as physical support for the development of microbial communities, provides essential nutrients for the growth of fungi and bacteria, and stimulates the production of ligninolytic enzymes, such as laccases and peroxidases, reported to be highly efficient in the degradation of different pesticides. The soil supplies microorganisms to the system and stimulates the microbial activity that mediates the degradation of the pesticides, while peat is a porous material that increases pesticide retention in the biobed system, regulates the moisture, and reduces the pH, factors that favor pesticide dissipation ^{[63][64][65][66]}[63,64,65,66]. In the biomixture, wheat straw can be replaced by other lignocellulosic substrates, depending on the availability of these materials in a particular country where biobed systems are applied. For example, Karanasios et al. (2010) [67] reported the use of different low-cost lignocellulosic materials, such as sunflower crop residues, olive leaves, grape stalks, orange peels, corn cobs, and spent mushroom substrate for the degradation of mixtures of pesticides in biobed systems. In this study, the alternative substrates favored the retention of pesticides in the system, and comparable pesticide half-life values, concerning those observed in the biobeds with the presence of wheat straw, were documented. In another study, Diez et al. (2013) [68] complemented the biomixture composition with the addition of lignocellulosic materials, such as pine sawdust (25%) and barley husk (25%), for the degradation of the pesticides carbendazim, isoproturon, and chlorpyrifos. The systems that contained wheat straw/barley husk (25%/25%) showed higher degradation percentages for carbendazim and chlorpyrifos after 90 days compared to the systems with only wheat straw (50%) and wheat straw/pine sawdust (25%/25%). In a similar study, Urrutia et al. (2013) [66] evaluated the addition of lignocellulosic materials such as barley husk, oat husk, and sawdust to biobed biomixtures for the treatment of the pesticides atrazine, chlorpyrifos, and isoproturon. Among the three lignocellulosic materials, oat husk was the best substitute for wheat straw, with similar pesticide degradation rates compared to the biomixture that included just wheat straw. In contrast, barley husk and sawdust can be added to the biomixtures in combination with wheat straw but not as the sole lignocellulosic material in the biomixture composition. Gongora-Echeverría et al. (2017) [69] evaluated the suitability of wheat straw substitution in biobed systems, employing different materials of high availability in southeastern Mexico such as compost, sisal fibers, corn stoves, and seaweed in combination with soil for the treatment of a pesticide mixture composed of 2,4-dichloro phenoxy acetic acid (2,4-D, 1.08 mg/cm³ of mixture), atrazine (2.5 mg/cm³ of mixture), carbofuran (0.23 mg/cm³ of mixture), diazinon (0.34 mg/cm³ of mixture), and glyphosate (0.36 mg/cm³ of mixture), mimicking the composition of pesticide effluents generated by farmers in Yucatan, Mexico. In all evaluated biomixtures, the five pesticides’ dissipation was over 99% after 41 days. Peat is an important component in biobed biomixtures; however, in some regions, this material has low availability or high cost. So, in biomixtures, peat has been substituted by alternative material or just eliminated from the biomixture composition ^[70][71][70,71]. Among the alternative materials to peat for biobed mixtures, compost ^{[72][73][74][75][76]}[72,73,74,75,76] or vermicompost ^{[43][77][78][79]}[43,77,78,79] are notable for being the most reported. Various agro-industrial wastes have been employed in the biomixture composition in biobed systems to treat fungicides, herbicides, and insecticides from different chemical families. They include spent coffee grounds [80], coir [80], cotton crop residues ^[81][82][81,82], garden wastes ^[83][84][83,84], livestock manure ^[80][85][80,85], olive leaves ^[68][86][87][68,86,87], pine bark [80], and sewage sludge [88]. Spent mushroom substrates and biochar have also been incorporated into the biomixture composition as complementary materials in pesticide dissipation ^{[38][39][42][70][89][90][91]}[38,39,42,70,89,90,91].

4.2. Microorganisms

In the biobed systems, the microbiota colonizing the biomixture are responsible for the pesticide degradation. Microorganisms such as bacteria and fungi may use pesticide molecules such as carbon, nitrogen, phosphorous, and energy sources for their growth. The efficient pesticide degradation by microorganisms is related to their great genetic plasticity, the production of diverse pesticide-degrading enzymes, fast growth, and adaptability to living in polluted environments [89]. The materials that integrate the biomixture retain the pesticide molecules in the biobeds and serve as a habitat for the development of different soil autochthonous microorganisms [92]. In biobeds, the presence of lignocellulosic materials reduces the pH in the system generating an environment that favors the growth and development of lignin-degrading fungi, such as different species of white-rot fungi [45]. White-rot fungi are organisms broadly reported in the biodegradation of several organic pollutants, including pesticides from different chemical families ^{[93][94][95][96][97][98]}[93,94,95,96,97,98]. Fungi can produce extracellular enzymes, such as peroxidases, laccases, and the cytochrome P450 complex, implicated in pesticide degradation ^{[99][100][101]}[99,100,101]. On the other hand, the presence of peat in the biomixture also favors the development of white-rot fungi in biobeds; however, in biomixtures without peat, the pesticide degradation is mediated mainly by the bacterial community [45]. Bacteria may act in synergy with fungi to enhance the pesticide and derived metabolites degradation; so, they can also produce different pesticide-degrading enzymes, such as dehalogenases, hydrolases, oxidoreductases, oxygenases, and esterases ^{[102][103][104][105][106]}[102,103,104,105,106]. In biobed systems, the biomixture supports the development of broad microbial diversity, and recent studies have evaluated such microbial complexity. For example, through a metagenomics approach, Bergsveinson et al. (2018) [107] assessed the bacterial and fungal diversity in four biobed systems employed for treating pesticide rinsates with differential composition and pesticide concentrations. As a result of the study, around 440 bacterial genera and an average of 285 fungal genera were identified in each biobed system. In a similar study, Góngora-Echeverría et al. (2018) [108] identified several archaea (23), bacteria (598), and fungi (64) species in lab-scale biobed systems with the presence of a mixture of commercial pesticide formulates (2,4-D, atrazine, carbofuran, diazinon, and glyphosate). In addition, Russell et al. (2021) [109] evaluated the bacterial diversity in a two-cell biobed system. After the treatment of pesticide residues, in cell one, 81 bacterial species from 58 genera were identified, while in cell two, 36 bacterial species from 33 genera were identified. The most representative bacterial genera in both biobed cells were Afipia, Sphingopyxis, and Pseudomonas. The development of a great diversity of microorganisms in the biobed systems is crucial for the efficient treatment of pesticide residues. However, the metabolic activities of the indigenous microbiota do not always guarantee total pesticide degradation. Due to this, bioaugmentation strategies have been employed to enhance pesticide biodegradation efficiency in biobed systems. This strategy is based on the addition of selected endogenous or exogenous microorganisms, such as specific fungi and bacterial strains, or the use of characterized or non-characterized microbial consortia ^[45][52][45,52]. The key characteristics for selecting microorganisms for a bioaugmentation strategy include pesticide resistance, high pesticide degradation efficiency, fast growth, and simple culture in lab conditions ^[110][111][110,111]. Examples of microorganisms used in biobed bioaugmentation strategies include uncharacterized microbial consortia, archaea species, bacteria of different phyla such as Actinobacteria (Streptomyces spp.), Bacteroidetes, Firmicutes, and mainly Proteobacteria (Achromobacter ssp., Bordetella ssp., Pseudomonas ssp., and Variovorax ssp.), and white-rot fungi from different classes (Aphelidiomycetes and Pezizomycetes) and species (Trametes versicolor and Stereum hirsutum).

4.3. Physicochemical Parameters

Pesticide dissipation effectiveness in biobed systems is strongly related to the biomixture composition and the metabolic activity of the different microorganisms; however, other key parameters include the pre-incubation time, moisture, temperature, and pesticide concentration in the system. Fernández-Alberti et al. (2012) [112] evaluated the effect of the biomixture pre-incubation time and moisture on chlorpyrifos (insecticide) degradation. In the study, the biomixture was composed of wheat straw, peat, and soil (2:1:1), pre-incubation took place (25 ± 1 °C) over 0, 15, and 30 days, and three water-holding-capacity percentages (WHC 40%, 60%, and 80%) were evaluated. The best condition for chlorpyrifos degradation (>70%) was 15 days pre-incubation and 60% WHC. Pre-incubation favors the microbial community proliferation in the biomixture, while at a high moisture (60% WHC), the ligninolytic enzyme activity in the biomixture increases. In a similar study, Tortella et al. (2012) [113] evaluated the effect of the biomixture maturity and concentration on the chlorpyrifos degradation; the biomixture (wheat straw, peat and soil, 2:1:1) was pre-incubated over 0, 15, and 30 days; after that time, three chlorpyrifos concentrations (200, 320, and 480 mg·kg⁻¹) were added to the biomixture. The biomixture’s maturity did not affect the chlorpyrifos degradation; all the biomixtures showed degradation percentages above 50%. However, increasing the chlorpyrifos concentration reduced the degradation efficiency and the hydrolytic and phenoloxidase activities in the systems. More recently, Kumari et al. (2019) [114] evaluated the effect of pre-incubation, pesticide concentration, and moisture on the degradation process of azoxystrobin (fungicide) and imidacloprid (insecticide) in biobed systems, employing biomixtures that included rice straw/corn cobs, peat, and compost (2:1:1). Ten days of biomixture pre-incubation before pesticide application reduced by 5–9 times the degradation rate of the insecticide imidacloprid, while the increase in the WHC from 60% to 80% had a positive effect on the degradation rates of both pesticides, reducing their half-life time. However, increases in the concentration of the pesticides from 30 to 100 mg·kg⁻¹ reduced the degradation rates of both pesticides. Cordova-Méndez et al. (2021) [46] evaluated the effect of moisture and temperature on the dissipation of five pesticides, two insecticides: carbofuran and diazinon, and three herbicides: atrazine, 2,4-D, and glyphosate; five temperatures (5, 15, 25, 35, and 45 °C) and five water holding capacity percentages (20%, 40%, 60%, 80%, and 100%) were evaluated. The increasing temperature positively affected the dissipation of the five pesticides evaluated; the highest dissipation percentages were observed at temperatures of 35 and 45 °C. However, the increase in the water-holding percentages did not show a significant improvement in pesticide dissipation. The observed increase in pesticide dissipation was related to higher microbial activity at higher temperatures. According to the reviewed studies, for efficient pesticide treatment in biobed systems, physicochemical parameters, such as the preincubation time, moisture, temperature, and pesticide concentration in the system, must be optimized.

4.4. Analysis of the Biobeds’ Treated Effluents

According to the biobed design, the systems are isolated from the soil through an impermeable layer, and it has been proposed that treated effluents ending from biobed systems could be reused for crop irrigation [115]. However, leachate analysis in biobed systems is essential to guarantee the efficiency of the treatment process and avoid releasing pesticides into the environment, causing soil and water pollution. In this sense, Henriksen et al. (2003) [58] evaluated the dissipation of the herbicides mecoprop and isoproturon in a biobed system. To determine the pesticide dissipation, the concentration of both herbicides in the leachate was assessed after a year, the concentrations of isoproturon and mecoprop were of 1.4% and 13%, respectively, of the initial dose (8 g), and the presence of a higher concentration of mecoprop in the biobed leachate was associated with its lower retention in the biobed biomixture. The excessive effluent load in the biobed systems could be a limiting factor for pesticide retention and dissipation efficiency, causing the release of leachates with pesticide concentration above the limits established in the regulations. Foog et al. (2004) [59] evaluated the effect of reducing the effluent loads in a biobed system (1.5 m deep) over the concentration of pesticides in leachates. They observed that a reduction from 1175 to 688 L/m² of biomixture decreased the pesticide concentration in leachate from <0.32% to <0.006%, while a decrease to 202 L/m² reduced the pesticide concentration to <0.0001%; according to their results, the maximum water holding in the system for efficient pesticide dissipation was 1121 L/m². In another study, Spliid et al. (2006) [116] evaluated the presence of pesticides in leachates from a biobed system. The concentrations of 21 pesticides (5 g each in the mixture) were assessed through LC-MS/MS; after 593 days of treatment, only the herbicide bentazone showed a significant presence in leachates (14% of the original dose), ten pesticides were not detected in leachates, and the ten remaining pesticides showed reductions below 2% of the initial dose. The authors concluded that the biobeds were effective in retaining and degrading pesticides, generating effluents with lower pesticide concentrations. More recently, Karas et al. (2015) [38] evaluated the risk associated with the environmental release of the biobed-depurated wastewater, the leachates containing fungicides from pilot biobed systems. The leachates included traces of fungicides (diphenylamine, imazalil, ortho-phenylphenol, and thiabendazole); acute effects were evaluated in aquatic organisms, such as the crustacean Daphnia magna and the fish Oncorhynchus mykiss, while chronic effects were assessed in the fish Oncorhynchus mykiss, the algae Pseudokirchneriella subcapitata, and sediment-dwelling invertebrates such as Chironomus sp. The biobed-depurated effluents with diphenylamine, imazalil, and ortho-phenylphenol did not show either an acute or chronic exposure risk in any bioindicator organism; only the effluents with thiabendazole showed an acute exposure risk for Daphnia magna and a chronic exposure risk for Oncorhynchus mykiss. In the same study, the treatment of fungicides using bioaugmentation with fungicide-degrader bacteria generated effluents that showed no acute or chronic exposure risk for the organisms evaluated. The authors conclude that biobed-treated effluents do not represent an environmental risk and can be safely disposed.