Bioremediation of Agricultural Soils Polluted with Pesticides: a Review

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Review Bioremediation of Agricultural Soils Polluted with Pesticides: A Review

Carla Maria Raffa and Fulvia Chiampo *

Department of Applied Science and Technology, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Torino, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-011-090-4685

Abstract: Pesticides are chemical compounds used to eliminate pests; among them, herbicides are compounds particularly toxic to weeds, and this property is exploited to protect the crops from unwanted plants. Pesticides are used to protect and maximize the yield and quality of crops. The excessive use of these chemicals and their persistence in the environment have generated serious problems, namely pollution of soil, water, and, to a lower extent, air, causing harmful effects to the ecosystem and along the food chain. About soil pollution, the residual concentration of pesticides is often over the limits allowed by the regulations. Where this occurs, the challenge is to reduce the amount of these chemicals and obtain agricultural soils suitable for growing ecofriendly crops. The microbial metabolism of indigenous microorganisms can be exploited for degradation since bioremediation is an ecofriendly, cost-effective, rather efficient method compared to the physical and chemical ones. Several biodegradation techniques are available, based on bacterial, fungal, or enzymatic degradation. The removal efficiencies of these processes depend on the type of pollutant and the chemical and physical conditions of the soil. The regulation on the use of pesticides is strictly connected to their environmental impacts. Nowadays, every country can adopt regulations to restrict the consumption of pesticides, prohibit the most harmful ones, and define the admissible concen- Citation: Raffa, C.M.; Chiampo, F. trations in the soil. However, this variability implies that each country has a different perception Bioremediation of Agricultural Soils of the toxicology of these compounds, inducing different market values of the grown crops. This Polluted with Pesticides: A Review. review aims to give a picture of the bioremediation of soils polluted with commercial pesticides, Bioengineering 2021, 8, 92. https:// considering the features that characterize the main and most used ones, namely their classification doi.org/10.3390/bioengineering8070092 and their toxicity, together with some elements of legislation into force around the world.

Academic Editors: Bruna Matturro and Marco Zeppilli Keywords: pesticides; bioremediation; agricultural soil; environmental pollution; sustainable agriculture; toxicity; health effects Received: 6 May 2021 Accepted: 23 June 2021 Published: 2 July 2021 1. Introduction Publisher’s Note: MDPI stays neutral Soil pollution is a worldwide problem that draws its origins from anthropologic with regard to jurisdictional claims in and natural sources. Urbanization, industrialization, and food-demand increases have published maps and institutional afﬁl- required the use of compounds, substances, and chemical agents, which, over the years, iations. have brought on the dispersion and accumulation of pollutants in the environment. The common pollutants present in the soil are heavy metals, polycyclic aromatic hydrocarbons (PAHs), or pesticides [1]. Pesticides are chemical compounds used to eliminate pests. They are chemical or Copyright: © 2021 by the authors. biological agents, that weaken, incapacitate, and kill pests. Based on the types of targeted Licensee MDPI, Basel, Switzerland. pests, the pesticides can be divided into several groups, namely insecticides, herbicides, This article is an open access article rodenticides, bactericides, fungicides, and larvicides. distributed under the terms and During the 19th and 20th centuries, the extracts from plants, namely pyrethrins, were conditions of the Creative Commons used as insecticides, fungicides, and herbicides. The increase in pesticide use happened Attribution (CC BY) license (https:// with synthetic chemistry during the 1930s. In this period, inorganic chemicals such as creativecommons.org/licenses/by/ arsenic and sulfur compounds were applied for crop protection. The arsenic poison was 4.0/).

Bioengineering 2021, 8, 92. https://doi.org/10.3390/bioengineering8070092 https://www.mdpi.com/journal/bioengineering Bioengineering 2021, 8, x FOR PEER REVIEW 2 of 29

Bioengineering 2021, 8, x FOR PEER REVIEW 2 of 29

Bioengineering 2021, 8, 92 2 of 29

with synthetic chemistry during the 1930s. In this period, inorganic chemicals such as ar- withsenic synthetic and sulfur chemistry compounds during were the 1930s.applied In fothisr crop period, protection. inorganic The chemicals arsenic suchpoison as ar-was senicfatalfatal toand to insects, insects, sulfur while whilecompounds the the sulfur sulfur were was was applied used used as asfo a ra fungicide. cropfungicide. protection. At At the the beginningThe beginning arsenic of ofpoison the the Second Second was fatalWorldWorld to War,insects, War, numerous numerous while the pesticides pesticides sulfur was werewere used synthesized, as a fungicide. mainly mainly At theorganic organic beginning chemicals, chemicals, of the such suchSecond as as di- Worlddichlorodiphenyltrichloroethanechlorodiphenyltrichloroethane War, numerous pesticides (DDT), were (DDT), synthesized, aldrin, aldrin, and and mainlydieldrin dieldrin organic used used as chemicals, as insecticides, insecticides, such while whileas di- 2- chlorodiphenyltrichloroethane2-methyl-4-chlorophenoxyaceticmethyl-4-chlorophenoxyacetic (DDT), acid acid (MCPA) aldrin, and and 2,4-dichlorophenoxyaceticdieldrin2,4-dichlorophenoxyacetic used as insecticides, acidacid while (2,4-D) (2,4-D) 2- methyl-4-chlorophenoxyaceticwerewere used used as as herbicides herbicides [2 [2].]. acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were AfterusedAfter as 1945, 1945, herbicides there there was was[2]. a a rapid rapid development development of of the the agrochemical agrochemical ﬁeld, field, characterized characterized byby the theAfter introduction introduction 1945, there of ofwas many many a rapid insecticides, insecticides, development fungicides, fungicides, of the herbicides, herbicides,agrochemical and and field, other other characterized chemicals, chemicals, to to bycontrolcontrol the introduction pests pests and and ensure ensureof many the the insecticides, yields yields of of agricultural agricu fungicides,ltural production. production. herbicides, Moreover, andMore otherover, pesticideschemicals, pesticides are toare controlappliedapplied pests in in aquaculture, aquaculture,and ensure horticulture, the horticulture, yields of and agricu and for variousforltural various production. general general household More householdover, applications. pesticides applications. They are appliedareThey also are usedin alsoaquaculture, to used control to control vector-borne horticulture, vector-borne diseases and for diseases (e.g., various malaria (e.g., general mala andria dengue)household and dengue) [3]. applications. [3]. They FromareFrom also 1990 1990 used toto to2018, control there there vector-borne have have been been register diseases registereded (e.g.,amounts amounts mala ofria used of and used pesticides dengue) pesticides [3]. by all by coun- all countriestriesFrom in the in1990 theworld, to world, 2018, especially there especially have in Asiabeen in Asia andregister and America. America.ed amounts The The world of world used average pesticides average quantity quantity by all coun-has has in- increased from 1.55 kg·ha−1 in 1990 to 2.63 kg·ha−1 in 2018, as shown in Figure1. Looking triescreased in the from world, 1.55 especiallykg·ha−1 in 1990in Asia to 2.63and kg·haAmerica.−1 in 2018,The worldas shown average in Figure quantity 1. Looking has in- at at the types, fungicides− and1 bactericides are used−1 more than the others (Figure2). creasedthe types, from fungicides 1.55 kg·ha and in bactericides 1990 to 2.63 ar kg·hae used in more 2018, than as shown the others in Figure (Figure 1. 2).Looking at the types, fungicides and bactericides are used more than the others (Figure 2).

Figure 1. Pesticides use per area of cropland (data from [4]). Figure 1. Pesticides use per area of cropland (data from [4]). Figure 1. Pesticides use per area of cropland (data from [4]).

Figure 2. Pesticides use from 1990 to 2016 (data from [4]). FigureFigure 2. PesticidesPesticides use use from from 1990 to 2016 (data from [4]). [4]). There has been no decrease even over the years, and directives have been imple- mentedThereThere in hashas many beenbeen countries no no decrease decrease around even even the over overworld the the years, to years,reduce and directivesand the usedirectives of have pesticides, beenhave implementedbeen for example, imple- mentedinthe many Regulation in countries many countries(EC) around 1107/2009 around the world[5] the of world tothe reduce European to reduce the useUnion the of use or pesticides, ofthe pesticides, Stockholm for example,for Convention example, the theRegulation[6], Regulation which (EC)focuses (EC) 1107/2009 on1107/2009 eliminating [5] [5] of the ofor the Europeanreducing European of Union persistent Union or theor organic the Stockholm Stockholm pollutants Convention Convention (POPs). [6 To], [6],which which focuses focuses on eliminatingon eliminating or reducing or reducing of persistent of persistent organic organic pollutants pollutants (POPs). (POPs). To this To purpose, the governments have to take measures to eliminate or reduce the release of POPs into the environment. Bioengineering 2021, 8, 92 3 of 29

When pesticides are used, a part of them remains in the soil, and the accumulation affects the microorganisms living there. Human exposure can occur through the ingestion of pesticide-contaminated water and food, the inhalation of pesticide-contaminated air, and directly from occupational, agricultural, and household use. The pesticides can enter the human body by dermal, oral, eye, and respiratory pathways [7]. The toxicity of pesticides depends on the electronic properties and the structure of the molecule, dosage, and exposure times [8,9]. For these reasons, the residual pesticide concentration present in the soil must be reduced, and effective remediation techniques must be used to do this. An ecofriendly, cost-effective, rather efﬁcient method is bioremediation, which is an alternative to more expensive and toxic approaches, such as chemical and physical methods. In biodegradation, the removal can be achieved by exploiting the microbial activity of microorganisms. The microorganisms, primarily bacteria [10], or fungi [11] transform pesticides into less complex compounds, CO2, water, oxides, or mineral salts, which can be used as carbon, mineral, and energy source. In these reactions, the enzymes have an important role since they act as catalysts [12]. Several techniques are available for the biodegradation of pesticides, which could develop in aerobic or anaerobic conditions based on types of microorganisms. Moreover, the bioremediation techniques can be divided into three categories depending on where the remediation treatment is done, namely in situ, ex situ, or on-site. In the in situ approach, the treatment is involved in the contaminated zone, and usually, the process is aerobic. The main in situ techniques are natural attenuation, bioaugmentation, biostimulation, bioventing, and biosparging. In the ex situ methods, the contaminated soil is removed from polluted sites and transported to other places for treatment. Bioreac- tors, composting, landfarming, and biopiles are ex situ treatments. The on-site approach consists of the treatment of polluted soil on the surrounding site, to say the soil is removed from its original position but cleaned up in the neighborhood without any impact due to its transport. In the literature, several reviews on pesticides have been published in the past years. Each of them is mainly focused on one topic. However, by this approach, the knowledge of the pesticide sector and its problems is lacking. Table1 reports a shortlist of these publications.

Table 1. Reviews on pesticides.

Topic References Pesticide diffusion in the environment [13,14] Toxic effects on living organisms [13,15,16] Legislation [14] Physical techniques for pesticide degradation [17,18] Chemical techniques for pesticide degradation [16–18] Biological techniques for pesticide degradation [17–22] Microorganisms capable of degrading pesticides [13,19,21–23] Enzymatic degradation [24] Economic analysis [17] Degradation of organochlorine pesticides [14,16] Degradation of herbicides. [13] Monitoring of pesticide clean-up [20]

The current review aims to give an overview of the bioremediation of soils polluted with commercial pesticides, considering the features that characterize the main and most used ones, namely, their classiﬁcation and their toxicological issues, together with some elements of legislation into force around the world. Bioengineering 2021, 8, x FOR PEER REVIEW 4 of 29

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BioengineeringBioengineering2021, 8, 922021, 8, x FOR2. Classification PEER REVIEW of Pesticides 4 of 29 4 of 29

2. Classification of Pesticides 2. ClassificationThe pesticides of Pesticidescan be classified by different criteria such as chemical classes, func- 2. ClassificationThe pesticides of Pesticidescan be classified by different criteria such as chemical classes, func- tionalThe groups, pesticides mode can of action,be classified and toxicity. by different criteria such as chemical classes, func- tional2. Classificationgroups,2. Classification mode of of Pesticides action, of Pesticides and toxicity. tionalThe groups, pesticides mode can of action, be classified and toxicity. by different criteria such as chemical classes, func- tional2.1. Classification groups,The pesticides Themode by pesticides Origin of can action, be classifiedcan and be toxicity.classified by different by criteriadifferent such criteria as chemical such as classes, chemical functional classes, func- 2.1. Classification by Origin 2.1. groups,ClassificationThe tionalpesticides mode groups, by of action,Origincan mode be and classified of toxicity. action, by and their toxicity. origin, namely chemical pesticides and bi- 2.1.opesticides. ClassificationThe pesticides Chemical by Origincan pesticides be classified are bygenerally their origin, organic namely compounds chemical that pesticides can have andnatural bi- 2.1.The Classification pesticides2.1. Classification bycan Origin be by classified Origin by their origin, namely chemical pesticides and biopesticides.sourcesThe orpesticides byChemical chemical can pesticides besynthesis classified are [7]. generallyby Biopes their origin, ticidesorganic namelyare compounds natural chemical substances that pesticides can have that naturalandcontrol biopesticides.The pesticidesChemical canpesticides be classified are generally by their origin, organic namely compounds chemical pesticidesthat can have and biopes-natural sourcesopesticides.pests by or nontoxic by Chemical Thechemical mechanismspesticides pesticides synthesis can [25]. beare [7]. classified generally Biopes byticides organic their areorigin, compounds natural namely substances thatchemical can havepesticidesthat controlnatural and bi- sourcesticides. or Chemicalby chemical pesticides synthesis are generally [7]. Biopes organicticides compounds are natural that cansubstances have natural that sources control pestssources by ornontoxicopesticides. by chemical mechanisms Chemical synthesis [25].pesticides [7]. Biopes are generallyticides are organic natural compounds substances that that can havecontrol natural pestsor by by nontoxic chemicalsources ormechanisms synthesis by chemical [7]. [25]. Biopesticidessynthesis [7]. are Biopes naturalticides substances are natural that substances control pests that by control pests2.1.1.nontoxic byClassification nontoxic mechanisms mechanisms by Chemical [25]. [25]. Composition 2.1.1. Classificationpests by nontoxic by Chemical mechanisms Composition [25]. 2.1.1. WithClassification this classification, by Chemical four Composition main groups can be identified: organochlorines, organ- 2.1.1.2.1.1.With Classification Classification this classification, by byChemical Chemical four Compositionmain Composition groups can be identified: organochlorines, organophosphates,With 2.1.1.this carbamates, classification,Classification and byfour pyrethrinsChemical main groups Composition and pyrethroidscan be identified: (Figure organochlorines, 3 and Table 2). Theorgan- in- ophosphates,formationWithWith thison this carbamates, theclassification, classification, chemical and and four four pyrethrins physical main groupsgroups characte and canpyrethroids can beristics be identified: identified: of (Figurepesticides organochlorines, organochlorines, 3 and is very Table useful organophos- 2). The organ- in in-de- ophosphates, carbamates,With this classification, and pyrethrins four mainand pyrethroids groups can be (Figure identified: 3 and organochlorines, Table 2). The in- organ- formationophosphates,phates, on carbamates, thecarbamates, chemical and pyrethrins andand pyrethrins physical and pyrethroidscharacte and pyrethroidsristics (Figure of 3pesticides (Figure and Table 3 and2is). very The Table informationuseful 2). The in de- in- formationterminingophosphates, onthe the mode chemical of carbamates, application, and physical and precaution pyrethrins charactes that risticsand needpyrethroids of pesticidesto be taken (Figure is during very 3 and useful the Table applica- in 2). de- The in- terminingon the chemicalthe mode and of physicalapplication, characteristics precaution ofs pesticidesthat need is to very be taken useful during in determining the applica- the terminingformationtion, and formation the theon application themode chemical on of theapplication, rates chemical and [26]. physical andprecaution physical charactes thatcharacteristics need ofristics topesticides be of taken pesticides is during very is useful verythe applica- useful in de- in de- tion,terminingmode and the of the application, application mode of application, precautionsrates [26]. precaution that need tos that be taken need during to be taken the application, during the and applica- the tion,application and thetermining application rates [the26]. mode rates of [26]. application, precautions that need to be taken during the application, and thetion, application and the application rates [26]. rates [26]. E.g.: DDT, DDD, Organochlorines E.g.: DDT, DDD, Organochlorines E.g.:dieldrin, DDT, aldrinDDD, Organochlorines E.g.:dieldrin, DDT,E.g.: aldrin DDD, DDT, DDD, OrganochlorinesOrganochlorines dieldrin,E.g.: HETP, aldrin Organophosphates dieldrin,E.g.:parathion, HETP,dieldrin, aldrin aldrin E.g.: HETP, Organophosphates chlorpyrifosparathion, Organophosphates E.g.:parathion, HETP,E.g.: HETP, Chemical pesticides Organophosphates chlorpyrifosparathion, Chemical pesticides Organophosphates chlorpyrifosparathion, Chemical pesticides E.g.:chlorpyrifos furadan,chlorpyrifos Chemical pesticides Carbamates sevin,E.g.: fenobucarbfuradan, Chemical pesticides Carbamates E.g.: furadan, Carbamates sevin, fenobucarbE.g.: furadan, CarbamatesCarbamates sevin,E.g.: fenobucarb furadan, Pyrethrin and sevin,E.g.: fenobucarbcyfluthrin,sevin, fenobucarb Pyrethrin and bifenthrin,E.g.: cyfluthrin, allethrin Pyrethrinpyrethroids and E.g.: cyfluthrin, PyrethrinpyrethroidsPyrethrin and and bifenthrin,E.g.: cyfluthrin,E.g.: allethrin cyfluthrin, pyrethroids bifenthrin,bifenthrin, allethrin allethrin pyrethroids bifenthrin, allethrin Figure 3. Classification of the chemical pesticides. DDT:pyrethroids dichlorodiphenyltrichloroethane; DDD: dichlorodiphenyldi- Figure 3. Classification of the chemicalchloroethane; pesticides. HETP: DDT: dichhexaethyllorodiphenyltrichloroethane; tetraphosphate. DDD: dichlorodiphenyldi- Figure 3. Classification of the chemical pesticides. DDT: dichlorodiphenyltrichloroethane; DDD: dichlorodiphenyldi- Figure 3. ClassificationFigure ofchloroethane; 3.theClassification chemical HETP: pesticides. of the hexaethyl chemical DDT: dich tetraphosphate. pesticides.lorodiphenyltrichloroethane; DDT: dichlorodiphenyltrichloroethane; DDD: dichlorodiphenyldi- DDD: Figure 3. Classification of the chemicalchloroethane; pesticides.chloroethane; HETP: DDT: hexaethyldich HETP:lorodiphenyltrichloroethane; hexaethyl tetraphosphate. tetraphosphate. DDD: dichlorodiphenyldi- Tabledichlorodiphenyldichloroethane; 2. Chemicalchloroethane; composition HETP: hexaethyl of the HETP: pesticides. tetraphosphate. hexaethyl tetraphosphate. Table 2. Chemical composition of the pesticides. Table 2. ChemicalTableGroup 2. compositionChemical composition of the pesticides. of the pesticides. Chemical Structure TableTable 2. Chemical 2. Chemical composition composition of of the the pesticides. pesticides. Group Group ChemicalChemical Structure Structure OrganochlorinesGroup Chemical Structure GroupGroup Chemical Chemical Structure Organochlorines OrganochlorinesOrganochlorines Organochlorines Organochlorines Organophasphates OrganophasphatesOrganophasphates OrganophasphatesOrganophasphates Organophasphates

Carbamates CarbamatesCarbamatesCarbamates Carbamates Carbamates

PyrethrinsPyrethrins and and pyrethroids pyrethroids Pyrethrins and pyrethroids Pyrethrins and pyrethroids Pyrethrins and pyrethroids Pyrethrins and pyrethroids

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Organochlorines Organochlorine pesticides (OCs) are organic compounds, namely hydrocarbon chains bonded with at least one covalently bonded atom of chlorine (Table2). These compounds are widely used in agriculture, especially as insecticides to control a broad range of insects. The most common organochlorines are dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethane (DDD), dicofol, dieldrin, lindane, aldrin, chlordane, endosulfan, isodrin, isobenzan, toxaphene, and chloropropylate [27]. These compounds are lipophilic and are difﬁcult to decompose, thus tending to bioaccumulate in tissues and remaining in the environment. For their high persistence in the environment, OCs belong to the class of persistent organic pollutants (POPs). They may cause damage to living beings, causing mutagenic effects, histopathological effects, enzyme-inducing and/or enzyme-inhibiting, carcinogenicity, and teratogenicity [28]. For human health, organochlorine exposure may increase the risk of breast cancer [29,30].

Organophosphates Organophosphates (OPs) are synthetic pesticides, which include phosphoric acid esters or thiophosphoric acid esters. The general structure is reported in Table2. Hexaethyl tetraphosphate (HETP) was the first one synthesized and used as agricultural insecticides. OPs are acutely toxic for insects and other animals, including birds, amphibians, and mammals. The cause of their toxicity is due to inhibition of the acetylcholinesterase (AChE) in the central and peripheral nervous system [31,32]. The inhibition of this enzyme causes muscarinic and nicotinic effects. Muscarinic symptoms are linked to the assumption system: • for inhalation, the symptoms may be cough, expectoration of frothy secretions, chest tightness, and wheeze, pulmonary edema; • for ingestion hypersalivation, nausea, vomiting, abdominal cramps, diarrhea, and tenesmus; • for eye, miosis, blurred vision, and eye pain. Nicotinic effects are profuse sweating, fasciculation, progressive flaccidity, and weak- ness of proximal muscle groups, such as the neck flexors, then the extraocular muscles and muscles of respiration [33].

Carbamates Carbamates are compounds derived from carbamic acid. Their chemical structure is characterized by an amino group bonded with an ester group, as shown in Table2. Typically, R1 and R2 are organic radicals or hydrogen. If R2 is hydrogen, it is possible to understand the target considering the functional group R1 [34]. It is possible to have:

• insecticides, when R1 is a methyl group; • herbicides, when R1 is an aromatic group; • fungicides, when R1 is a benzimidazole moiety. Carbamates are also biocides for industry and household products for the control of household pests [35]. The common carbamate pesticides are aldicarb, carbofuran, fenoxycarb, carbaryl, ethienocarb, and fenobucarb [36]. As the organophosphates, carbamates are inhibitors of acetylcholinesterase activity, and therefore, their toxicity acts on the neurological system [37]. Exposure to carbamate pesticides increases the risk of non-Hodgkin’s lymphoma in humans since they inhibit and induce apoptosis of human natural killer (NK) cells and cytotoxic T lymphocytes that provide defense against tumors [38].

Pyrethrins and Pyrethroids Pyrethrins are natural insecticides, in which active principle comes from the ﬂowers of Tanacetum cinerariaefolium, also called Chrysanthemum cinerariaefolium or Pyrethrum cinerariaefolium. Their active constituents are esters of 2,2-dimethyl-3-(2-methyl-l-propenyl)-l- Bioengineering 2021, 8, x FOR PEER REVIEW 6 of 29

Pyrethrins and Pyrethroids Pyrethrins are natural insecticides, in which active principle comes from the flowers Bioengineering 2021, 8, 92 6 of 29 of Tanacetum cinerariaefolium, also called Chrysanthemum cinerariaefolium or Pyrethrum cinerariaefolium. Their active constituents are esters of 2,2-dimethyl-3-(2-methyl-l-propenyl)- l-cyclopropanecarboxylic acid (chrysanthemic acid) and of 3-(2-methoxycarbonyl-l-pro- cyclopropanecarboxylicpenyl)-2,2-dimethyl-l-cyclopropanecarboxylic acid (chrysanthemic acid) acid and (pyrethric of 3-(2-methoxycarbonyl-l-propenyl)- acid). Six types were iden- 2,2-dimethyl-l-cyclopropanecarboxylictified, as shown in Table 2. acid (pyrethric acid). Six types were identiﬁed, as shownPyrethroids in Table2. are synthetic compounds that are obtained by modifying the chrysan- themicPyrethroids acid moiety are of synthetic pyrethrin compounds I and esterifying that are the obtained alcohols. by They modifying can be thedivided chrysan- into themic[39]: acid moiety of pyrethrin I and esterifying the alcohols. They can be divided into [39]: • First-generation pyrethroids: esters of chrysanthemic acid and oneone alcohol,alcohol, havinghaving aa furanfuran ringring andand terminalterminal sideside chainchain moieties.moieties. • Second-generation pyrethroids: they have 3-phenoxybenzyl3-phenoxybenzyl alcoholsalcohols derivativesderivatives inin thethe alcoholalcohol moietymoiety andand havehave some some of of the the terminal terminal side side chain chain moieties moieties replaced replaced with with a dichlorovinyla dichlorovinyl or or dibromovinyl dibromovinyl substitute substitute and and aromatic aromatic rings. rings. Pyrethroids areare synthesizedsynthesized toto increaseincrease the the insecticidal insecticidal power power and and decrease decrease the the sensi- sen- tivitysitivity to to air air and and light, light, compared compared to to the the pyrethrins. pyrethrins. Generally, inin thethe air,air, thethe pyrethrinspyrethrins andand manymany pyrethroidspyrethroids areare rapidlyrapidly degradeddegraded byby sunlight, while they remain for a long time in the soil as they bind strongly to it [40]. [40]. Pyrethrins and pyrethroids may be foundfound on leaves,leaves, fruits,fruits, andand vegetablesvegetables sincesince theythey areare sprayed directly ontoonto cropscrops andand plantsplants [[41].41]. The pyrethrins and pyrethroids disruptdisrupt thethe sodiumsodium channelschannels inin thethe axonsaxons damagingdamaging the neurologicneurologic systemsystem [ 42[42].]. They They are are toxic toxic for for insects insects but but less less harmful harmful to humans. to humans. However, How- itever, was it notedwas noted that thethat exposure the exposure of these of these pesticides pesticides can can have have respiratory respiratory effects effects such such as coughas cough or upperor upper respiratory respiratory irritation irritation after after inhalation inhalation of of dust dust or or aerosol aerosol droplets; droplets; neuro- neu- logicalrological effects effects such such as as headache headache or or dizziness; dizziness; gastrointestinal gastrointestinal effects effects such such as as nauseanausea andand vomiting; andand irritationirritation and/orand/or dermal dermal effect effectss [43]. [43]. Pyrethroids may cause cardiovascular problems [44]. problems [44]. 2.1.2. Biopesticides 2.1.2. Biopesticides Biopesticides are pesticides derived from nature (animals, plants, microorganisms, and minerals).Biopesticides They are can pesticides be divided derived into threefrom majornature classes (animals, (Figure plants,4) based microorganisms, on the type ofand active minerals). ingredient They used, can be namely, divided biochemical, into three major plant-incorporated classes (Figure protectants 4) based on (PIPs), the type and microbialof active ingredient pesticides. used, namely, biochemical, plant-incorporated protectants (PIPs), and microbial pesticides.

Biochemical pesticides

Plant incorporated Biopesticides protectants (PIPs)

Microbial pesticides

Figure 4. ClassiﬁcationClassification of biopesticides.

Biochemical Pesticides They are naturalnatural compoundscompounds thatthat controlcontrol pestspests byby nontoxicnontoxic mechanisms.mechanisms. TheyThey cancan be extracted from natural sources oror synthesizedsynthesized to have thethe samesame structurestructure andand functionfunction as the natural ones [45]. Semiochemicals are chemical compounds emitted by plants or animals. Pheromones, allomones, kairomones, and attractants are examples of these compounds. They are connected to the vital functions, such as feeding, mating, and egg-laying (ovipositing) of the pests [46]. Therefore, acting on their concentration can be exploited to inﬂuence the pest life cycle. Bioengineering 2021, 8, x FOR PEER REVIEW 7 of 29

Bioengineering 2021, 8, x FOR PEER REVIEW 7 of 29

as the natural ones [45]. Semiochemicals are chemical compounds emitted by plants or animals.as the natural Pheromones, ones [45]. allomones, Semiochemicals kairomones, are chemical and attractants compounds are examples emitted of by these plants com- or pounds.animals. TheyPheromones, are connected allomones, to the kairomones,vital function ands, such attractants as feeding, are mating,examples and of egg-layingthese com- (ovipositing)pounds. They of are the connected pests [46]. to Therefore, the vital function acting ons, such their as concentration feeding, mating, can andbe exploited egg-laying to influence the pest life cycle. Bioengineering 2021, 8, 92 (ovipositing) of the pests [46]. Therefore, acting on their concentration can7 of be 29 exploited to influence the pest life cycle. Plant-Incorporated Protectants Plant-IncorporatedThe plant-incorporated Protectants protectants (PIPs) can be produced by the plants themselves Plant-IncorporatedwhenThe the Protectants plant-incorporatedpest feeds on them. protectantsTo force their (PIPs) production, can be produced the plants by can the be plants genetically themselves mod- The plant-incorporatedifiedwhen introducing the pest feeds protectants the on gene them. acting (PIPs) To force on can a theirspecific be produced production, pesticidal by thethe protein plantsplants into themselvescan the be geneticallygenetic material mod- when the pestofified the feeds introducing plant on itself. them. theIn To thisgene force way, acting their the on production,plant a specific can synthesize pesticidal the plants the protein can toxic be intocompounds genetically the genetic for materialselected modified introducingpestsof the [47]. plant the itself. gene In actingthis way, on athe specific plant can pesticidal synthesize protein the toxic into compounds the genetic for selected material of thepests plant [47]. itself. In this way, the plant can synthesize the toxic compounds for selected pestsMicrobial [47]. Pesticides MicrobialMicrobial Pesticides pesticides include living organisms, such as bacteria, fungi, algae, and vi- Microbial Pesticides ruses,Microbial that control pesticides the pests. include They living suppress organisms, pests either such producing as bacteria, toxic fungi, metabolites algae, and that vi- Microbialcauseruses, pesticides damagethat control includeand diseasesthe livingpests. or organisms,They preventing suppress suchth epests establishment as bacteria,either producing fungi, of othe algae,r toxicmicroorganisms andmetabolites [48].that viruses, thatcause control damage the pests. and They diseases suppress or preventing pests either the producing establishment toxic of metabolites other microorganisms that [48]. cause damage2.2. and Classification diseases or by preventing Targets the establishment of other microorganisms [48]. 2.2. Classification by Targets 2.2. Classification byPesticides Targets can be classified by the roles that they play and the types of pests that they attack. The main classes are insecticides, herbicides, rodenticides, bactericides, and fungi- Pesticides canPesticides be classified can be by classified the roles by that the theyroles playthat they and play the types and the of types pests of that pests that they cides. they attack. Theattack. main The classes main classes are insecticides, are insecticides, herbicides, herbicides, rodenticides, rodenticides, bactericides, bactericides, and and fungi- Based on their chemical structure, they may interact in a different way with pests and fungicides. cides. with different toxicity. Based on theirBased chemical on their structure, chemical they structure, may interact they may in a differentinteract in way a different with pests way and with pests and with differentwith toxicity. different toxicity. 2.2.1. Insecticides 2.2.1. Insecticides2.2.1.Insecticides Insecticides are chemical and biological compounds that attack and kill insects. Lar- Insecticidesvicides areInsecticides are chemical specific are and insecticides chemical biological and that compounds biological target the that compounds larval attack life and stage that kill ofattack insects. an insect. and Larvi- kill insects. Lar- cides are specificvicidesThese insecticides are compoundsspecific that insecticides target are used the larvalthat in agriculture,target life stage the larval ofhorticulture, an life insect. stage forestry, of an insect. and gardening but These compoundsare alsoThese used arecompounds usedto control in agriculture, are vectors, used in horticulture,such agriculture, as mosquitoes forestry, horticulture, andand gardeningticks, forestry, which butand are are gardening involved but in also used to controlspreadingare also vectors, used human to such control and as animal mosquitoes vectors, diseases, such and ticks,suchas mosquitoes as which dengue are involvedand[49] andticks, inmalaria which spreading [50]. are involved in human and animalspreadingThe diseases, most-used human such and insecticides as animal dengue diseases, [belong49] and suchto malaria the as classes dengue [50]. of [49] organophosphates, and malaria [50]. pyrethroids, The most-usedand carbamates.The insecticides most-used They belonginsecticides act on to the the belongnervous classes to ofsyst the organophosphates,em classes of the of victims, organophosphates, pyrethroids,causing spasms, pyrethroids, respira- and carbamates.toryand failure,carbamates. They act and/or on They the death. nervous act Inon Table the system nervous 3, several of thesyst insecticides victims,em of the causing victims,used in spasms, agriculturalcausing respi- spasms, soil are respira- sum- ratory failure,marized.tory and/or failure, death. and/or In death. Table3 In, several Table 3, insecticides several insecticides used in agriculturalused in agricultural soil are soil are sum- summarized.marized. Table 3. Classification of the most common insecticides used in agricultural soil. Table 3. Classification of the most common insecticides used in agricultural soil. Name PesticideTable 3. Class Classification of Chemical the most Structure common insecticides used in agricultural Mode ofsoil. Action NameName Pesticide Pesticide Class Class Chemical Chemical StructureStructure Mode ofMode Action of Action

Interaction with sodium ion channels in neurons, caus- Interaction with sodium ion channels in DDT Organochlorine ing their inactivation, which leads to spasms and even- Interactionneurons, with causing sodium their ion inactivation, channels in neurons, caus- DDT Organochlorine DDT Organochlorine ingwhich their inactivation, leads to spasms tualwhich anddeath. leads eventual to spasms and even- death.tual death.

Interference with nerve signaling by Bioengineering 2021, 8, x FOR PEER REVIEW Interference with nerve signaling by inhibition of8 the of 29 Cyﬂuthrin Cyfluthrin PyrethroidPyrethroid inhibition of the membrane sodium Interferencemembrane with nerve sodium sign channelaling by systems. inhibition of the Cyfluthrin Pyrethroid channel systems. membrane sodium channel systems.

Interruption of functioning of the Interruption of functioning of the nervous system, inter- TeﬂuthrinTefluthrin PyrethroidPyrethroid nervous system, interfering with sodiumfering with channels. sodium channels.

Inhibition of cholinesterase prevents the breakdown of Aldicarb Carbamate acetylcholine in the synapse. It leads to respiratory failure.

Interference with nerve signaling by inhibition of the λ-cyhalothrin Pyrethroid membrane sodium channel systems.

Interference with sodium channels disrupts the function Permethrin Pyrethroid of neurons and causes muscles to spasm, culminating in paralysis and death.

Inactivation of acetylcholinesterase by phosphorylation Terbufos Organophosphate of the hydroxyl group of serine present at the active site of the enzyme.

Disruption of nervous systems by inactivation of acetyl- Chlorpyrifos Organophosphate cholinesterase.

Disruption of nervous systems by inactivation of acetyl- Methyl-parathion Organophosphate cholinesterase.

Disruption of nervous systems by inactivation of acetyl- Dimethoate Organophosphate cholinesterase.

Disruption of nervous systems by inactivation of acetyl- Carbofuran Carbamate cholinesterase.

2.2.2. Herbicides Herbicides are used to control and remove undesirable plants and weeds. These compounds are mainly applied in agricultural soils, before or during farming to maximize crop productivity. Herbicides are also used in forest management and in suburban and urban areas. Bioengineering 2021, 8, x FOR PEER REVIEW 8 of 29

Bioengineering 2021, 8, x FOR PEER REVIEW 8 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 8 of 29 Bioengineering Bioengineering 2021 2021, ,8 8, ,x x FOR FOR PEER PEER REVIEW REVIEW 88 of of 29 29 Bioengineering 2021, 8, x FOR PEER REVIEW 8 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 8 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 8 of 29

Interruption of functioning of the nervous system, inter- Tefluthrin Pyrethroid Interruption offering functioning with sodium of the channels. nervous system, inter- Tefluthrin Pyrethroid Interruption offering functioning with sodium of the nervouschannels. system, inter- Bioengineering 2021, 8Tefluthrin, 92 Pyrethroid InterruptionInterruption of of functioning functioning of of the the nervous nervous8 of 29 system, system, inter- inter- TefluthrinTefluthrin PyrethroidPyrethroid Interruption offering functioning with sodium of the channels. nervous system, inter- feringfering with with sodium sodium channels. channels. Tefluthrin Pyrethroid Interruption offering functioning with sodium of the channels.nervous system, inter- Tefluthrin Pyrethroid InterruptionInhibition of of cholinesterase functioning of prevents the nervous the breakdown system, inter- of

fering with sodium channels. Tefluthrin Pyrethroid

Aldicarb Carbamate Table 3. Cont. acetylcholineInhibition of feringcholinesterasein the withsynapse. sodium prevents It leads channels. tothe respiratory breakdown fail- of Inhibition of cholinesterase prevents the breakdown of Aldicarb Carbamate acetylcholine in the synapse.ure. It leads to respiratory fail- InhibitionInhibition of of cholinesterase cholinesterase prevents prevents the the breakdown breakdown of of NameAldicarb PesticideCarbamate Class Chemical Structure acetylcholineInhibition of Modecholinesterasein the synapse. of Actionure. prevents It leads to the respiratory breakdown fail- of Aldicarb Carbamate acetylcholine in the synapse. It leads to respiratory fail- Aldicarb Carbamate acetylcholine in the synapse.ure. It leads to respiratory fail-

Inhibition of cholinesterase prevents the breakdown of Aldicarb Carbamate acetylcholine in the synapse.ure. It leads to respiratory fail-

ure. Aldicarb Carbamate acetylcholineInhibitionInhibition of cholinesterase ofin cholinesterasethe synapse.ure. prevents It preventsleads tothe respiratory breakdown fail- of AldicarbAldicarb CarbamateCarbamate acetylcholineInterferencethe breakdown within the of nerve synapse. acetylcholine signure. alingIt leads inby theto inhibition respiratory of thefail- λ-cyhalothrin Pyrethroid Interferencesynapse.membrane It leadswith nerve to sodium respiratory signure. channelaling failure. by systems. inhibition of the λ-cyhalothrin Pyrethroid Interferencemembrane with nerve sodium sign alingchannel by inhibitionsystems. of the λ-cyhalothrin Pyrethroid InterferenceInterference with with nerve nerve sign signalingaling by by inhibition inhibition of of the the λλ-cyhalothrin-cyhalothrin PyrethroidPyrethroid Interferencemembrane with nerve sodium sign channelaling by systems. inhibition of the λ-cyhalothrin Pyrethroid Interferencemembranemembrane with sodium sodium nerve signaling channel channel systems. bysystems. Interference with nerve signaling by inhibition of the λ membrane sodium channel systems. -cyhalothrinλ-cyhalothrin PyrethroidPyrethroid inhibition of the membrane sodium InterferenceInterferencemembrane with with sodium nerve sodium signcha channelnnelsaling disruptsby systems. inhibition the function of the

λ-cyhalothrin Pyrethroid channel systems.

Permethrin Pyrethroid ofInterference neuronsmembrane and with causes sodium sodium muscles cha channelnnels to spasm, disrupts systems. culminating the function in

Permethrin Pyrethroid Interferenceof neurons andwith causes sodiumparalysis muscles cha andnnels todeath. spasm,disrupts culminating the function in Interference Interference with with sodium sodium cha channelsnnels disrupts disrupts the the function function Permethrin Pyrethroid ofInterference neuronsInterference and with causes withsodiumparalysis sodiummuscles cha andnnels channelsto death. spasm, disrupts culminating the function in PermethrinPermethrin PyrethroidPyrethroid of of neurons neurons and and causes causes muscles muscles to to spasm, spasm, culminating culminating in in Interferencedisrupts thewith function paralysissodium of cha and neuronsnnels death. disrupts and the function PermethrinPermethrin PyrethroidPyrethroid of neurons and causes muscles to spasm, culminating in causes musclesparalysisparalysis to spasm, and and culminating death. death. Inactivation of acetylcholinesterase by phosphorylation Permethrin Pyrethroid ofInterference neurons and with causes sodiumparalysis muscles cha andnnels to death. spasm, disrupts culminating the function in in paralysis and death. PermethrinTerbufos OrganophosphatePyrethroid ofInactivation theneurons hydroxyl and of acetylcholinesterasecausesgroupparalysis ofmuscles serine and todeath.present spasm, by phosphorylationat culminating the active site in Terbufos Organophosphate Inactivationof Inactivationthe hydroxyl of acetylcholinesterase of group acetylcholinesteraseparalysisof ofthe serine enzyme. and death.present by byphosphorylation at the active site InactivationInactivation of of acetylcholinesterase acetylcholinesterase by by phosphorylation phosphorylation Terbufos Organophosphate ofInactivation phosphorylationthe hydroxyl of acetylcholinesterasegroup ofof theof the serine hydroxyl enzyme. presentgroup by phosphorylationat the active site TerbufosTerbufos OrganophosphateOrganophosphate of the hydroxyl group of serine present at the active site Terbufos Organophosphate of the hydroxyl group of serine present at the active site Terbufos Organophosphate ofInactivation theof hydroxyl serine of present acetylcholinesterase groupof at theof the serine enzyme. active present site by of phosphorylation at the active site Inactivation of acetylcholinesteraseofof the the enzyme. enzyme. by phosphorylation Terbufos Organophosphate of the hydroxyl thegroup enzyme. of serine present at the active site of the enzyme. Terbufos Organophosphate Disruptionof the hydroxyl of nervous groupof systemsofthe serine enzyme. by present inactivation at the active of acetyl- site

Chlorpyrifos Organophosphate Disruption of nervouscholinesterase.of systemsthe enzyme. by inactivation of acetyl- Chlorpyrifos Organophosphate Disruption of nervous systems by inactivation of acetyl- Chlorpyrifos Organophosphate DisruptionDisruption of nervous of nervouscholinesterase. systems systems by inactivation by of acetyl- ChlorpyrifosChlorpyrifos OrganophosphateOrganophosphate Disruption of nervouscholinesterase. systems by inactivation of acetyl- Chlorpyrifos Organophosphate Disruptioninactivation of nervous of acetylcholinesterase.cholinesterase. systems by inactivation of acetyl- Chlorpyrifos Organophosphate cholinesterase. Disruption of nervouscholinesterase. systems by inactivation of acetyl- Chlorpyrifos Organophosphate Disruption of nervous systems by inactivation of acetyl-

Chlorpyrifos Organophosphate cholinesterase.

Disruption of nervous systems by inactivation of acetyl- Methyl-parathion Organophosphate cholinesterase. Disruption of nervouscholinesterase. systems by inactivation of acetyl- Methyl-parathion Organophosphate DisruptionDisruption of nervous of nervouscholinesterase. systems systems by inactivation by of acetyl- Methyl-parathionMethyl-parathion Organophosphate Organophosphate DisruptionDisruption of of nervous nervous systems systems by by inactivation inactivation of of acetyl- acetyl- Methyl-parathionMethyl-parathion OrganophosphateOrganophosphate Disruptioninactivation of nervous of acetylcholinesterase.cholinesterase. systems by inactivation of acetyl- Methyl-parathion Organophosphate cholinesterase.cholinesterase.

Disruption of nervous systems by inactivation of acetyl- Methyl-parathion Organophosphate cholinesterase.

Disruption of nervouscholinesterase. systems by inactivation of acetyl-

Methyl-parathion Organophosphate Disruption of nervouscholinesterase. systems by inactivation of acetyl-

Dimethoate Organophosphate DisruptionDisruption of nervous of nervouscholinesterase. systems systems by inactivation by of acetyl- DimethoateDimethoate OrganophosphateOrganophosphate Disruptioninactivation of nervous of acetylcholinesterase.cholinesterase. systems by inactivation of acetyl- Dimethoate Organophosphate DisruptionDisruption of of nervous nervous systems systems by by inactivation inactivation of of acetyl- acetyl- DimethoateDimethoate OrganophosphateOrganophosphate Disruption of nervouscholinesterase. systems by inactivation of acetyl- Dimethoate Organophosphate cholinesterase.cholinesterase. Disruption of nervouscholinesterase. systems by inactivation of acetyl- Dimethoate Organophosphate Disruption of nervous systems by inactivation of acetyl-

cholinesterase. Dimethoate Organophosphate

DisruptionDisruption of nervous of nervouscholinesterase. systems systems by inactivation by of acetyl- CarbofuranCarbofuran CarbamateCarbamate Disruptioninactivation of nervous of acetylcholinesterase.cholinesterase. systems by inactivation of acetyl- Carbofuran Carbamate Disruption of nervouscholinesterase. systems by inactivation of acetyl- Carbofuran Carbamate DisruptionDisruption of of nervous nervous systems systems by by inactivation inactivation of of acetyl- acetyl- CarbofuranCarbofuran CarbamateCarbamate Disruption of nervouscholinesterase. systems by inactivation of acetylcholinesterase.cholinesterase. Carbofuran Carbamate Disruption of nervous systems by inactivation of acetyl- Carbofuran Carbamate cholinesterase.

Disruption of nervous systems by inactivation of acetyl- Carbofuran 2.2.2. HerbicidesCarbamate2.2.2. Herbicides cholinesterase. 2.2.2. Herbicides cholinesterase. Herbicides2.2.2. areHerbicidesHerbicides used to are control used andto control remove and undesirable remove undesirable plants and plants weeds. and weeds. These These com- 2.2.2.pounds2.2.2. HerbicidesHerbicides Herbicides are mainly are usedapplied to control in agricultural and remove soils, undesirable before or plantsduring and farming weeds. to These maximize com- compounds2.2.2. are mainlyHerbicides Herbicides applied are inused agricultural to control soils,and remove before or undesirable during farming plants to and maximize weeds. These com- croppounds Herbicidesproductivity. are mainly are Herbicides usedapplied to control in are agricultural alsoand removeused soils,in undesirableforest before management or plantsduring and andfarming weeds. in suburban to These maximize com- and crop productivity.pounds2.2.2.Herbicides Herbicides Herbicides are mainly are areused applied also to control used in agricultural in and forest remove management soils, undesirable before and or plants during in suburban and farming weeds. and to These maximize com- urbanpounds2.2.2.cropHerbicides productivity.Herbicides areas. are mainly are Herbicides usedapplied to control in areagricultural andalso removeused soils, in undesirableforest before management or plantsduring and farmingand weeds. in suburban to These maximize com- and urban areas.poundscrop Herbicidesproductivity. are mainly are Herbicides appliedused to controlin areagricultural also and usedremove soils, in forestundesirable before management or duringplants and farmingand weeds. in suburban to Thesemaximize com- and The modespoundscropurban of productivity. actionareas. are mainly depend Herbicides applied on the chemicalin areagricultural also composition, used soils, in forest before and management usually,or during they farmingand involve in suburban to maximize and cropurbanpounds productivity.Herbicides areas. are mainly are Herbicides usedapplied to control in are agricultural also and used remove soils,in forest undesirable before management or plantsduring and andfarming weeds. in suburban to These maximize com-and a plant enzymecropurban or productivity. areas. a biological Herbicides system. In thisare also way, used the regularin forest plant management growth and and devel- in suburban and urbancroppounds productivity. areas. are mainly Herbicides applied in are agricultural also used soils,in forest before management or during andfarming in suburban to maximize and opments areurban injured areas. or disrupted, causing eventual plant death [51]. In Table4, different urbancrop productivity. areas. Herbicides are also used in forest management and in suburban and herbicides usedurban in agricultureareas. soil are reported. Bioengineering 2021, 8, x FOR PEER REVIEW 9 of 29 BioengineeringBioengineeringBioengineering 2021 20212021,, , 8 88,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 999 of ofof 29 2929 Bioengineering 2021, 8, x FOR PEER REVIEW 9 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 9 of 29

TheTheThe modes modesmodes of ofof action actionaction depend dependdepend on onon the thethe chemical chemicalchemical composition, composition,composition, and andand usually, usually,usually, they theythey involve involveinvolve a plantTheThe enzyme modes modes ofor of actiona action biological depend depend system. on on the the In chemical chemicalthis way, composition, composition,the regular plantand and usually, growthusually, theyand they develop-involve involve aa plantplantThe enzymeenzyme modes oror of aa action biologicalbiological depend system.system. on the InIn thchemicalthisis way,way, thecomposition,the regularregular plantplant and growth growthusually, andand they develop-develop- involve aments aplant plantThe enzymeare enzyme modes injured or orof a aoractionbiological biological disrupted, depend system. system. causing on the In In th chemicaltheventuisis way, way,al thecomposition, plantthe regular regular death plant plant [51].and growth usually,growthIn Table and andthey 4, develop- differentdevelop- involve Bioengineering 2021 8 mentsmentsa plant areare enzyme injuredinjured or orora biological disrupted,disrupted, system. causingcausing In eventueventuthis way,alal plantplantthe regular deathdeath plant [51].[51]. IngrowthIn TableTable and 4,4, different differentdevelop- , , 92 mentsherbicidesaments plant are areenzyme injured usedinjured orin or aagricultureor biologicaldisrupted, disrupted, soilsystem. causing causing are reported.In eventu theventuis way, alal plant theplant regular death death plant[51]. [51]. Ingrowth In Table Table and 4, 94, ofdifferent develop-different 29 herbicidesherbicidesments are usedusedinjured inin agriculture agricultureor disrupted, soilsoil causing areare reported.reported. eventu al plant death [51]. In Table 4, different herbicidesmentsherbicides are used injuredused in in agriculture oragriculture disrupted, soil soil causingare are reported. reported. eventu al plant death [51]. In Table 4, different Table 4.herbicides Classification used of in the agriculture most common soil herbicides are reported. used in agricultural soil. TableTable 4.4. ClassificationClassification ofof thethe mostmost commoncommon herbicidesherbicides usedused inin agriculturalagricultural soil.soil. TableTable 4. 4. Classification Classification of of the the most most common common herbicides herbicides used used in in agricultural agricultural soil. soil. Name Table 4. PesticideTableClassiﬁcation 4. Classification Class of the most of the common most Chemical common herbicides Structure herbicides used in used agricultural in agricultural soil. Modesoil. of Action NameName Pesticide PesticideTable 4. ClassificationClassClass of the most Chemical Chemical common StructureStructure herbicides used in agricultural Mode Modesoil. ofof ActionAction NameName Pesticide Pesticide Class Class Chemical Chemical Structure Structure Mode Mode of of Action Action Name Pesticide Class Chemical Structure Mode of Action Name Pesticide Class Chemical Structure Disruption Mode of shikimic of Action acid pathway DisruptionDisruption ofof shikimicshikimic acidacid pathwaypathway Glyphosate Organophosphate throughDisruptionDisruptionDisruption inhibition of of of shikimic shikimic shikimic of the enzymeacid acid acid pathway pathway 5-enolpy- GlyphosateGlyphosate OrganophosphateOrganophosphate throughthroughDisruption inhibitioninhibition of shikimic ofof thethe enzymeenzyme acid pathway 5-enolpy-5-enolpy- GlyphosateGlyphosate OrganophosphateOrganophosphate throughthroughpathwayruvylshikimate-3-phosphateDisruption inhibition inhibition through of shikimicof inhibition of the the enzyme enzyme acid of pathwaysynthase. the 5-enolpy- 5-enolpy- Glyphosate Organophosphate throughruvylshikimate-3-phosphateruvylshikimate-3-phosphateruvylshikimate-3-phosphate inhibition of the enzyme synthase. synthase.synthase. 5-enolpy- Glyphosate Organophosphate enzyme 5-enolpyruvylshikimate-3- Glyphosate Organophosphate throughruvylshikimate-3-phosphate inhibition of the enzyme synthase. 5-enolpy-

ruvylshikimate-3-phosphatephosphate synthase.

synthase. Inhibition of the photosynthetic pathway, Atrazine Organochlorine InhibitionInhibitionInhibition of ofof the thethe photosynthetic photosyntheticphotosynthetic pathway, pathway,pathway, AtrazineAtrazineAtrazine OrganochlorineOrganochlorineOrganochlorine InhibitionInhibitionspecifically of of the the thephotosynthetic photosynthetic photosystem pathway, II. Atrazine Organochlorine Inhibitionspecificallyspecificallyspecifically of the the thethephotosynthetic photosystem photosystemphotosystem pathway, II. II.II. AtrazineAtrazine OrganochlorineOrganochlorine Inhibitionpathway,specifically of the speciﬁcally photosynthetic the photosystem the pathway, II. Atrazine Organochlorine specifically the photosystem II. specifically the photosystem II. photosystem II.

Imitation of plant growth hormone auxin ImitationImitation ofof plantplant growthgrowth hormonehormone auxinauxin ImitationImitationandImitation uncontrolled of of of plant plant plant growth cell growth division hormone hormone in vascular auxin auxin 2,4-D Organochlorine andauxinandandImitation uncontrolled uncontrolleduncontrolled and uncontrolledof plant cell cellcell growth division divisiondivision cell hormone division in inin vascular vascularvascular auxin 2,4-D2,4-D2,4-D OrganochlorineOrganochlorineOrganochlorine tissue,and uncontrolled leading to uncontrolled cell division growthin vascular and 2,4-D2,4-D OrganochlorineOrganochlorine tissue,tissue,tissue,andin uncontrolled vascularleading leadingleading to toto tissue, uncontrolled uncontrolleduncontrolled cell leading division to growth growthgrowth in vascular and andand 2,4-D Organochlorine tissue, leadingeventually to uncontrolled death of plants. growth and uncontrolledtissue, eventuallyleadingeventuallyeventually growth to uncontrolled death deathdeath and of of eventuallyof plants. plants.plants. growth and tissue, leadingeventually to uncontrolled death of plants. growth and

eventuallydeath of plants. death of plants. eventually death of plants.

Imitation of plant growth hormone auxin ImitationImitationImitation of ofofof plant plantplantplant growth growthgrowth hormone hormonehormone auxin auxinauxin auxinandImitation uncontrolled and uncontrolled of plant cell growth division cell divisionhormone in vascular auxin Dicamba Organochlorine andandandImitation uncontrolled uncontrolleduncontrolled of plant cell cellcell growth division divisiondivision hormone in inin vascular vascularvascular auxin DicambaDicambaDicambaDicamba OrganochlorineOrganochlorineOrganochlorineOrganochlorine tissue,andin uncontrolled vascular leading tissue,to uncontrolled cell leading division to growthin vascular and Dicamba Organochlorine tissue,tissue,tissue,and uncontrolled leading leadingleading to toto uncontrolled uncontrolleduncontrolled cell division growth growthgrowth in vascular and andand Dicamba Organochlorine uncontrolledtissue, leadingeventually growth to uncontrolled death and eventuallyof plants. growth and tissue, eventuallyleadingeventuallyeventually to uncontrolled death deathdeath of ofof plants. plants.plants. growth and eventuallydeath of plants. death of plants.

eventually death of plants.

InhibitionInhibition of of cell cell mitosis, mitosis, acting acting on on the meri- Trifluralin Organophosphate InhibitionInhibitionInhibition of ofof cell cellcell mitosis, mitosis,mitosis, acting actingacting on onon the thethe meri- meri-meri- TriﬂuralinTrifluralinTrifluralinTrifluralin OrganophosphateOrganophosphateOrganophosphateOrganophosphate Inhibitionstemsthe and meristems of tissues cell mitosis, andof underground tissues acting of on theorgans. meri- Trifluralin Organophosphate stemsInhibitionstemsstems and andand of tissues tissuestissues cell mitosis, of ofof underground undergroundunderground acting on organs.organs.theorgans. meri- Trifluralin Organophosphate stems undergroundand tissues of organs.underground organs. stems and tissues of underground organs.

InhibitionInhibition of elongases of elongases and and the the geranylgera- InhibitionInhibition ofof elongaseselongases andand thethe geranylgera-geranylgera- Metolachlor Organochlorine InhibitionInhibitionnylgeranylgeranyl pyrophosphate of of elongases elongases pyrophosphate cycl and andases, the the importantgeranylgera- geranylgera- in MetolachlorMetolachlorMetolachlor OrganochlorineOrganochlorineOrganochlorine Inhibitionnylnyl pyrophosphatepyrophosphate of elongases cyclcycl andases,ases, the importantimportant geranylgera- inin MetolachlorMetolachlor OrganochlorineOrganochlorine Inhibitionnylcyclases,nylthe pyrophosphate pyrophosphate synthesis important of elongases of long-chain cycl in cycl theandases,ases, synthesis the important fattyimportant geranylgera- acids. in in thethe synthesissynthesis ofof long-chainlong-chain fattyfatty acids.acids. Metolachlor Organochlorine nylthethe pyrophosphatesynthesisof synthesis long-chain of of long-chain long-chain fatty cycl acids.ases, fatty importantfatty acids. acids. in the synthesis of long-chain fatty acids.

Inhibition of the photosynthetic Inhibition of the photosynthetic pathway, CyanazineCyanazine OrganochlorineOrganochlorine InhibitionInhibitionInhibitionpathway, of ofof the thethe speciﬁcally photosynthetic photosyntheticphotosynthetic the pathway, pathway,pathway, CyanazineCyanazineCyanazine OrganochlorineOrganochlorineOrganochlorine Inhibitionspecifically of the thephotosynthetic photosystem pathway, II. Cyanazine Organochlorine Inhibitionspecificallyspecificallyspecificallyphotosystem of the the thethephotosynthetic photosystem photosystemphotosystem II. pathway, II. II.II. Cyanazine Organochlorine specifically the photosystem II. specifically the photosystem II.

specifically the photosystem II.

2.2.3. Rodenticides 2.2.3.2.2.3.2.2.3. Rodenticides2.2.3. Rodenticides RodenticidesRodenticides 2.2.3.Rodenticides Rodenticides act to kill rodents, such as rats, mice, squirrels, and nutria; all these ro- Rodenticides2.2.3.RodenticidesRodenticides Rodenticides act to actact kill toto rodents, killkill rodents,rodents, such suchsuch as rats, asas rara mice,ts,ts, mice,mice, squirrels, squirrels,squirrels, and andand nutria; nutria;nutria; all allall these thesethese ro-ro- dentsRodenticidesRodenticides can cause damage act act to to kill killto rodents,crop, rodents, transmit such such as asdisease, ra rats,ts, mice, mice, and squirrels, causesquirrels, ecological and and nutria; nutria; damage. all all these these Rodent ro- ro- rodentsdentsdents can Rodenticides can causecan causecause damage damagedamage act to to crop, tokillto crop,crop, transmitrodents, transmittransmit such disease, disease,disease,as ra andts, causemice, andand cause causesquirrels, ecological ecologicalecological and damage. nutria; damage.damage. Rodent all these RodentRodent ro- dentsinfestationsdents Rodenticidescan can cause cause arise damage damage in act a towide tokill to crop, crop,varietyrodents, transmit transmit of such situations: disease, asdisease, rats, inmice,and and agriculture cause squirrels,cause ecological ecological soils, and nutria;inside damage. damage. andall theseRodent aroundRodent ro- infestationsinfestationsinfestationsdents arisecan cause inarisearise a wide damage inin aa varietywidewide to varietyvarietycrop, of situations: transmit ofof situations:situations: disease, in agriculture inin and agricultureagriculture cause soils, ecological soils, insidesoils, insideinside and damage. around andand around aroundRodent infestationsbuildings,dentsinfestations can cause inarise arisesewers, damagein in a ainwide wide waste to variety crop,variety dumps, transmit of of situations:and/or situations: disease, in open in in andagriculture agriculture areas. cause ecological soils, soils, inside inside damage. and and around Rodentaround buildings,buildings,buildings,buildings,infestations in sewers, in inin sewers, arisesewers,sewers, in wastein in inain wide waste wastewaste dumps, variety dumps, dumps,dumps, and/or of and/or and/orand/or situations: in open in inin open openopen areas. in agricultureareas. areas.areas. soils, inside and around Mostbuildings, rodenticides in sewers, act asin anticoagulantswaste dumps, and/or that interfere in open with areas. blood clotting and cause buildings, in sewers, in waste dumps, and/or in open areas. death due to excessive bleeding. The rodenticide products are baits in block or paste form [52]. The most common rodenticides are summarized in Table5. Bioengineering 2021, 8, x FOR PEER REVIEW 10 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 10 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 10 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 10 of 29 Bioengineering 2021, 8, x FOR PEER REVIEW 10 of 29

Most rodenticides act as anticoagulants that interfere with blood clotting and cause Most rodenticides act as anticoagulants that interfere with blood clotting and cause deathMost due torodenticides excessive bleeding. act as anticoagulants The rodentici thatde products interfere are with baits blood in block clotting or paste and cause form deathMost due rodenticidesto excessive bleeding.act as anticoagulants The rodentici thatde productsinterfere arewith baits blood in blockclotting or pasteand cause form [52].death TheMost due most torodenticides excessive common bleeding. actrodenticides as anticoagulants The rodenticiare summarized thatde products interfere in Table are with baits5. blood in block clotting or paste and causeform Bioengineering 2021, 8, 92 death[52]. The due most to excessive common bleeding. rodenticides The rodenticiare summarizedde products in Table are baits 5. in block or10 paste of 29 form [52].death The due most to excessive common bleeding. rodenticides The arerodentici summarizedde products in Table are baits5. in block or paste form [52]. The most common rodenticides are summarized in Table 5. Table 5. Classification of the most common rodenticides. [52].Table The most5. Classification common rodenticidesof the most common are summarized rodenticides. in Table 5. Table 5. Classification of the most common rodenticides. Name PesticideTable Class 5. Classification Chemical of the mostStructure common rodenticides. Mode of Action Name PesticideTable 5. TableClassClassiﬁcation 5. Classification of the Chemical most of the common Structuremost common rodenticides. rodenticides. Mode of Action Name Pesticide Class Chemical Structure Mode of Action Name Pesticide Class Chemical Structure Mode of Action NameName Pesticide Pesticide Class Class Chemical Chemical StructureStructure Mode Mode of Actionof Action Anticoagulant agent depressing hepatic syn- Anticoagulant agent depressing hepatic syn- Chlorophacinone Organochlorine thesisAnticoagulant of prothrombin agent depressing and clotting hepatic factors syn- VII, Chlorophacinone Organochlorine thesisAnticoagulant of prothrombin agent depressing and clotting hepatic factors syn- VII, Chlorophacinone Organochlorine thesisAnticoagulantIX,Anticoagulant and of prothrombin X, inducing agent agent depressinginternaland depressing clotting hemorrhage. hepatic factors syn- VII, Chlorophacinone Organochlorine thesisIX,hepatic andof prothrombin X, synthesis inducingof andinternal prothrombin clotting hemorrhage. factors VII, ChlorophacinoneChlorophacinone OrganochlorineOrganochlorine thesisIX, and of prothrombin X, inducing internaland clotting hemorrhage. factors VII, IX, and X, inducing internal hemorrhage. and clotting factors VII, IX, and X,

IX,inducing and X, inducing internal internal hemorrhage. hemorrhage.

Inhibition of vitamin K epoxide reductase Inhibition of vitamin K epoxide reductase Diphacinone Pyrethroid complexInhibition 1, whichof vitamin is an K essential epoxide enzyme reductase for Diphacinone Pyrethroid complexInhibitionInhibition 1, ofwhich vitamin of vitamin is an K essential epoxide K epoxide enzymereductase for Diphacinone Pyrethroid complexInhibition 1, whichof vitaminactivating. is an K essential epoxide enzyme reductase for DiphacinoneDiphacinone PyrethroidPyrethroid complexreductase 1, which complexactivating. is an 1, essential which is enzyme an for activating. Diphacinone Pyrethroid complexessential 1, which enzyme is an for essential activating. enzyme for activating.

activating.

Anticoagulant agent depressing hepatic syn- AnticoagulantAnticoagulant agent agent depressing depressing hepatic syn- Bromadiolone Pyrethroid thesisAnticoagulant of prothrombin agent depressing and clotting hepatic factors syn- VII, Bromadiolone Pyrethroid thesisAnticoagulanthepatic of prothrombin synthesis agent ofdepressing and prothrombin clotting hepatic factors syn- VII, BromadioloneBromadiolone PyrethroidPyrethroid thesisAnticoagulantIX, and of prothrombin X, inducing agent depressinginternaland clotting hemorrhage. hepatic factors syn- VII, Bromadiolone Pyrethroid thesisIX,and andof clotting prothrombin X, inducing factors andinternal VII, clotting IX, andhemorrhage. factors X, VII, Bromadiolone Pyrethroid thesisIX, and of prothrombin X, inducing internaland clotting hemorrhage. factors VII, IX,inducing and X, inducing internal internal hemorrhage. hemorrhage. IX, and X, inducing internal hemorrhage.

AnticoagulantAnticoagulant agent agent depressing depressing hepatic syn- Anticoagulant agent depressing hepatic syn- Difethialone Pyrethroid thesisAnticoagulanthepatic of prothrombin synthesis agent ofdepressing and prothrombin clotting hepatic factors syn- VII, DifethialoneDifethialone PyrethroidPyrethroid thesisAnticoagulant of prothrombin agent depressing and clotting hepatic factors syn- VII, Difethialone Pyrethroid thesisAnticoagulantIX,and and of clotting prothrombin X, inducing agent factors depressinginternaland VII, clotting IX, hemorrhage. and hepatic factors X, syn- VII, Difethialone Pyrethroid thesisIX, andof prothrombin X, inducing andinternal clotting hemorrhage. factors VII, Difethialone Pyrethroid thesisIX,inducing and of prothrombin X, inducing internal internaland hemorrhage. clotting hemorrhage. factors VII, IX, and X, inducing internal hemorrhage. IX, and X, inducing internal hemorrhage.

InhibitionInhibition of glycine of neurotransmitters, glycine as StrychnineStrychnine PyrethroidPyrethroid Inhibitionneurotransmitters, of glycine asneurotransmitters, glycine and as Strychnine Pyrethroid Inhibitionglycine of glycine and acetylcholine. neurotransmitters, as Strychnine Pyrethroid Inhibitionglycine ofacetylcholine. glycine and acetylcholine.neurotransmitters, as Strychnine Pyrethroid Inhibitionglycine of glycine and acetylcholine. neurotransmitters, as Strychnine Pyrethroid glycine and acetylcholine.

glycine and acetylcholine.

2.2.4. Fungicides 2.2.4. Fungicides2.2.4. Fungicides 2.2.4.Fungicides Fungicides are compounds that kill parasitic fungi or their spores. They permit the Fungicides2.2.4. FungicidesFungicides are compounds are compounds that kill that parasitic kill parasiti fungic orfungi their or spores. their spores. They permit They permit the the control2.2.4.Fungicides Fungicides of fungal are infestations, compounds especially that kill o ccurringparasitic duringfungi or the their whole spores. food They supply. permit Indeed, the controlcontrol of fungalFungicides of fungal infestations, are infestations, compounds especially especially that occurring kill o parasiticcurring duringc fungiduring the whole or the their whole food spores. supply. food They supply. Indeed, permit Indeed, the fungicidescontrolFungicides of fungal are widely areinfestations, compounds applied especiallyin thethat agricultural kill o ccurringparasiti industry.c duringfungi or the Fungicides their whole spores. food interfere Theysupply. permitwith Indeed, vari- the fungicidescontrolfungicides are of widely fungal are widely applied infestations, applied in the agricultural especially in the agricultural o industry.ccurring industry. Fungicidesduring the Fungicides interferewhole food withinterfere supply. various with Indeed, vari- ousfungicidescontrol biochemical of fungal are widely procesinfestations, appliedses within especiallyin the the agricultural fungal occurring cytoplasm industry. during and theFungicides mitochondria.whole food interfere supply. They with Indeed,inhibit vari- biochemicalfungicidesous biochemical processes are widely within proces applied theses fungal within in the cytoplasm the agricultural fungal and cytoplasm mitochondria.industry. and Fungicides mitochondria. They inhibit interfere several They with inhibit vari- severalousfungicides biochemical enzymes are widely andproces proteins appliedses within involvedin the the agricultural fungal for example cytoplasm industry. in the and lipid Fungicides mitochondria. metabolism, interfere fungalThey with inhibit respi- vari- enzymesousseveral and biochemical proteinsenzymes involvedprocesand proteinsses for within exampleinvolved the fungal in for the example lipidcytoplasm metabolism, in the and lipid mitochondria. metabolism, fungal respiration, Theyfungal inhibit respiration,severalous biochemical and enzymes production andproces proteins ofses adenosine within involved the triphosphate fungal for example cytoplasm (ATP) in the [53]. and lipid mitochondria. metabolism, fungalThey inhibit respi- and productionseveralration, andenzymes of production adenosine and proteins triphosphateof adenosine involved (ATP)triphosphate for [example53]. (ATP) in the [53]. lipid metabolism, fungal respiration,severalTable and enzymes 6 production reports and information proteins of adenosine involved on the triphosphate mostfor example common (ATP) in fungicides.the [53]. lipid metabolism, fungal respi- Tableration,6 Tablereports and 6production reports information information of onadenosine the moston thetriphosphate common most common fungicides. (ATP) fungicides. [53]. ration,Table and 6 production reports information of adenosine on the triphosphate most common (ATP) fungicides. [53]. Table 6 reports information on the most common fungicides. Table 6 reports information on the most common fungicides. BioengineeringBioengineering 20212021,, 88,, xx FORFOR PEERPEER REVIEWREVIEW 1111 ofof 2929 BioengineeringBioengineering Bioengineering 2021, 8, 92 2021 20212021, ,8, 88, ,x, xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 11 of 29 111111 of ofof 29 2929 Bioengineering 2021, 8, x FOR PEER REVIEW 11 of 29

TableTable 6.6. ClassificationClassification ofof thethe mostmost commoncommon fungicidesfungicides usedused inin agriculturalagricultural soil.soil. Table 6. Classification of the most common fungicides used in agricultural soil. Table 6. TableClassiﬁcationTable 6. 6. Classification Classification of the most of of the commonthe most most common fungicidescommon fungicides fungicides used in agricultural used used in in agricultural agricultural soil. soil. soil. NameName Pesticide Pesticide ClassClass Chemical Chemical StructureStructure Mode Mode ofof ActionAction Name Pesticide Class Chemical Structure Mode of Action NameNameName Pesticide Pesticide Pesticide Class Class Class Chemical Chemical Chemical Structure StructureStructure Mode Mode Mode of Actionof of Action Action InhibitionInhibition ofof enzymeenzyme activityactivity inin fungifungi byby InhibitionInhibitionInhibition of ofof enzyme enzymeenzyme activity activityactivity in inin fungi fungifungi by byby MancozebMancozeb CarbamateCarbamate formingformingInhibition aa complexcomplex of enzyme withwith activity metal-containingmetal-containing in fungi by en-en- Mancozeb Carbamate formingformingInhibition aa complexcomplex of enzyme withwith metal-containing activitymetal-containing in en-en- MancozebMancozebMancozeb CarbamateCarbamateCarbamate formingformingfungi a a complex complex by forming with with a metal-containing complexmetal-containing en- en- zymes.zymes.

zymes.zymes. with metal-containingzymes. enzymes. zymes.

ChlorothalonilChlorothalonil OrganochlorideOrganochloride ReductorReductorReductor andand and deactivatordeactivator deactivator ofof glutathione. ofglutathione. ChlorothalonilChlorothalonil Organochloride Organochloride Reductor and deactivator of glutathione. ChlorothalonilChlorothalonil OrganochlorideOrganochloride ReductorReductor and andglutathione. deactivator deactivator of of glutathione. glutathione.

CaptanCaptan OrganochlorideOrganochloride ReductionReduction ofof enzymaticenzymatic activity.activity. CaptanCaptan OrganochlorideOrganochloride ReductionReduction of enzymatic of enzymatic activity. activity. CaptanCaptan OrganochlorideOrganochloride ReductionReduction of of enzymatic enzymatic activity. activity.

ReactionReactionReaction andand and inactivationinactivation inactivation ofof of sulfhydrylsulfhydryl ReactionReactionsulfhydryl and and inactivation groupsinactivation of amino of of sulfhydryl sulfhydryl groupsgroupsReaction ofof aminoamino and inactivationacidsacids andand enzymes,enzymes, of sulfhydryl causingcausing ManebManeb CarbamateCarbamate groupsgroupsacids of of amino amino and enzymes, acids acids and and causing enzymes, enzymes, causing causing ManebManebManeb CarbamateCarbamateCarbamate damagedamagegroups of toto amino lipidlipid metabolism,metabolism, acids and enzymes, respiration,respiration, causing andand Maneb Carbamate damagedamagedamage to to lipid lipid to metabolism, lipidmetabolism, metabolism, respiration, respiration, and and damage to lipid metabolism, respiration, and productionproduction ofof adenosineadenosine triphosphate.triphosphate.

productionrespiration, of and adenosine production triphosphate. of

production of adenosine triphosphate. productionadenosine of triphosphate.adenosine triphosphate.

CreationCreation ofof chemicalchemical barrierbarrier betweenbetween thethe ZiramZiram CarbamateCarbamate CreationCreation of of chemical chemical barrier barrier between between the the Ziram Carbamate CreationCreation ofplantplant ofchemical chemical andand a abarrier fungus.fungus. barrier between the ZiramZiramZiram CarbamateCarbamateCarbamate plant and a fungus. between theplantplant plant and and and a a fungus. fungus. a fungus.

Inhibitor of sterol TebuconazoleTebuconazoleTebuconazole OrganochlorideOrganochlorideOrganochloride InhibitorInhibitor ofof sterolsterol 1414αα-demethylase-demethylase Tebuconazole Organochloride InhibitorInhibitor14α -demethylaseofof sterolsterol 1414α-demethylase-demethylase TebuconazoleTebuconazole OrganochlorideOrganochloride InhibitorInhibitor of of sterol sterol 14 14αα-demethylase-demethylase

3.3. DiffusionDiffusion ofof PesticidesPesticides intointo thethe EnvironmentEnvironment andand TheirTheir ToxicologyToxicology 3. Diffusion3.3. Diffusion Diffusion of Pesticides of of Pesticides Pesticides into the into into Environment the the Environment Environment and Their and and Toxicology Their Their Toxicology Toxicology 3. DiffusionPesticidesPesticides of aim aimPesticides toto prevent,prevent, into remove, remove,the Environment andand contcontrolrol and harmfulharmful Their pests,Toxicologypests, butbut theythey maymay bebe harm-harm- PesticidesPesticidesPesticides aim to prevent, aim aim to to prevent, prevent, remove, remove, andremove, control and and harmful cont controlrolrol pests,harmful harmfulharmful but pests, pests, theypests, maybut butbut they betheythey harmful may maymay be bebe harm- harm-harm- fulful totoPesticides thethe environmentenvironment aim to prevent, andand humanhuman remove, health.health. and Their Theircontrol excessiveexcessive harmful use usepests, cancan but givegive they highhigh may concentra-concentra- be harm- to the environmentfulfulful to toto the thethe environment environmentenvironment and human and andand health. human humanhuman Their health. health.health. excessive Their TheirTheir use excessive excessiveexcessive can give use useuse high can cancan concentrations give givegive high highhigh concentra- concentra-concentra- tionstionsful to of ofthe pollutingpolluting environment substancessubstances and human inin thethe environm environmhealth. Theirent.ent. InexcessiveIn thethe years,years, use thethe can WorldWorld give highHealthHealth concentra- Organi-Organi- of pollutingtionstionstions substancesof ofof polluting pollutingpolluting in substances substancessubstances the environment. in inin the thethe environm environmenvironm In the years,ent.ent. In the In the the World years, years, Health the the World World Organization Health Health Organi- Organi- zationzationtions of rankedranked polluting thethe pesticidessubstancespesticides andand in the reportedreported environm theirtheirent. toxicitytoxicity In the andand years, theirtheir the effectseffects World onon Health humanhuman Organi- healthhealth rankedzation thezation pesticides ranked ranked andthe the pesticides reportedpesticides theirand and reported toxicityreported and their their their toxicity toxicitytoxicity effects and andand on their their humantheir effects effectseffects health on onon human [ human54human]. health healthhealth [54].[54].zation ranked the pesticides and reported their toxicity and their effects on human health Through[54].[54].[54]. time, several pesticides have been banned in some countries due to their high [54]. ThroughThrough time,time, severalseveral pesticidespesticides havehave beenbeen bannedbanned inin somesome countriescountries duedue toto theirtheir toxicity. However,ThroughThrough at the time,time, moment, severalseveral their pesticidespesticides production havehave and beenbeen use bannedbanned go on, especially inin somesome countriescountries in developing duedue toto theirtheir highhigh toxicity.Throughtoxicity. However, However,time, several atat thethe pesticides moment,moment, have theirtheir been productionproduction banned andand in some useuse gogo countries on,on, especiallyespecially due to inin their de-de- countries.highhigh toxicity. toxicity. However, However, at at the the moment, moment, their their production production and and use use go go on, on, especially especially in in de- de- velopingvelopinghigh toxicity. countries.countries. However, at the moment, their production and use go on, especially in de- velopingveloping countries. countries. 3.1. Presenceveloping and Distribution countries. into the Environment The3.1.3.1. pesticides PresencePresence persist andand DistributionDistribution in the environment intointo thethe EnvironmentEnvironment and may bioaccumulate and contaminate 3.1.3.1. Presence Presence and and Distribution Distribution into into the the Environment Environment the food chain,TheThe affecting pesticidespesticides human persistpersist health inin thethe and environmentenvironment the environment andand may asmay a whole. bioaccumulatebioaccumulate Pesticide andand tends contaminatecontaminate to TheThe pesticides pesticides persist persist in in the the environment environment and and may may bioaccumulate bioaccumulate and and contaminate contaminate conservethethe its food molecularfoodThe chain,chain,pesticides integrity affectingaffecting persist and humanhuman in chemical, the health healthenvironment physical, andand thethe and and environmentenvironment functionalmay bioaccumulate as characteristicsas aa whole.whole. and PesticidePesticide forcontaminate a tendstends thethethe food foodfood chain, chain,chain, affecting affectingaffecting human humanhuman health healthhealth and andand the thethe environment environmentenvironment as asas a aa whole. whole.whole. Pesticide PesticidePesticide tends tendstends certain timetotothe conserveconserve food after chain, being itsits molecular molecularreleasedaffecting into human integrityintegrity the soilhealth andand [55 chemic].chemicand theal,al, environment physical,physical, andand as functionalfunctional a whole. Pesticide characteristicscharacteristics tends tototo conserve conserveconserve its itsits molecular molecularmolecular integrity integrityintegrity and andand chemic chemicchemical,al, physical, physical, and and functional functional characteristics characteristics Theforforto parameter conserve aa certaincertain thattimeitstime molecular can afterafter be beingbeing considered integrity releasedreleased and to intointo evaluate chemic thethe soilsoilal, its [55].physical,[55]. persistence and into functional the soil characteristics is the forforfor a aa certain certaincertain time timetime after afterafter being beingbeing released releasedreleased into intointo the thethe soil soilsoil [55]. [55].[55]. half-timefor (t aThe Thecertain), that parameterparameter is,time the after time thatthat being required cancan bereleasedbe consideredconsidered for a compoundinto the toto soilevaleval to [55].uateuate halve itsits its persistencepersistence initial concentration. intointo thethe soilsoil isis thethe 1/2TheThe parameterparameter thatthat cancan bebe consideredconsidered toto evalevaluateuate itsits persistencepersistence intointo thethe soilsoil isis thethe Pesticideshalf-timehalf-timeThe with (tparameter(t1/21/2 short),), thatthat half-times is,is, that thethe can timetime accumulate be requiredrequired considered forfor and a ato persistcompoundcompound evaluate less its intototo halvepersistencehalve the soil. itsits initialinitial By into contrast, concentration.concentration. the soil is the half-timehalf-time (t (t1/21/21/2),),), that thatthat is, is,is, the thethe time timetime required requiredrequired for forfor a aa compound compoundcompound to toto halve halvehalve its itsits initial initialinitial concentration. concentration.concentration. pesticideshalf-time with long (t1/2), half-times that is, the are time more required persistent for a compound and this may to halve increase its initial the risk concentration. to PesticidesPesticides withwith shortshort half-timeshalf-times accumulateaccumulate andand persistpersist lessless intointo thethe soil.soil. ByBy contrast,contrast, contaminatePesticides thePesticides environment. with with short short half-times half-times accumulate accumulate and and persist persist less less into into the the soil. soil. By By contrast, contrast, pesticidespesticides withwith longlong half-timeshalf-times areare moremore persistentpersistent andand thisthis maymay increaseincrease thethe riskrisk toto con-con- Altogether,pesticidespesticides they with with can long long be half-times classiﬁed half-times as are are [56 more more]: persistent persistent and and this this may may increase increase the the risk risk to to con- con- taminatetaminatetaminate thethethe environment.environment.environment. taminatetaminate the the environment. environment. • nonpersistentAltogether,Altogether, pesticides, theythey can whencan bebe t classified1/2classifiedis lower asas than[56]:[56]: 30 days; Altogether,Altogether, they they can can be be classified classified as as [56]: [56]: Bioengineering 2021, 8, 92 12 of 29

• moderately persistent, when t1/2 is in the range 30–100 days; • persistent ones, whent1/2 is higher than 100 days. Once present in the soil, pesticides may: (1) be adsorbed by soil particles; (2) be degraded by photochemical, chemical, and microbiological processes; and (3) move from soil to water.

3.1.1. Adsorption by Soil Particles Pesticide molecules can be adsorbed physically (Van der Waals forces) or chemically (electrostatic interactions) on the soil particles. The process can be described with the adsorption isotherms [57–59]. The adsorption constant is evaluated since it provides information about solute mobility. If pesticides have a low afﬁnity for adsorption, they tend to spread more easily into the environment. Several soil parameters inﬂuence the adsorption process, namely soil organic matter content, clay content, clay mineralogy, and pH.

3.1.2. Degradation Processes The pesticides can be degraded and transformed into one or more metabolites through photochemical, chemical, and microbiological processes. Photodegradation is an abiotic process induced by the absorption of light energy that leads to the decomposition of the polluting molecules. This process takes place with more difficulty in the soil, being a heterogeneous system, and it is influenced by soil properties. For example, photodegradation is more efficient with particles having a large size and a high specific area since they promote light diffusion [60]. Chemical and biological degradation occurs by reactions such as hydrolysis, oxidation, reduction, dehydrogenation, dehalogenation, decarboxylation, and condensation. In the biodegradation process, pesticides are degraded by microbial organisms through metabolic or enzymatic action [61]. The evaluation of the kinetics of these reactions gives information on the persistence of pesticides.

3.1.3. Leaching from Soil to Water Leaching is the movement of pesticides within the soil. The soluble contaminants are carried by water downward through permeable soils. This phenomenon is responsible for the contamination of groundwater. The extent of leaching is highly dependent on soil properties, pesticide physicochemi- cal properties, formulation types, distribution of rainfall events or irrigation strategy, and hydrogeological processes [62].

3.2. Toxicity and Short- and Long-Term Damages Several studies report the toxic effects on human health associated with the use of pesticides. Typically, the main routes of human exposure to pesticides are inhalation, ingestion, and dermal contact. Each compound has its toxicity, but the risk increases with increasing dosage and exposure time. The WHO provides guidelines for the classiﬁcation of pesticides, dividing them into ﬁve categories, and considering the lethal dose 50 (LD50) as a benchmark (Table7)[54]. The LD50 value represents the dose required to kill half the tested population after a standardized test duration. The substance route can be given dermal and oral. Bioengineering 2021, 8, 92 13 of 29

Table 7. Criteria for classiﬁcation of pesticides.

LD50 for the Rat − Bioengineering 2021, 8, x FOR PEER REVIEWClass Characteristics (mg·kg 1 Body Weight) 13 of 29

Oral Dermal Ia Extremely hazardous <5 <50 IIIb Moderately Highly hazardous hazardous 50–2000 5–50 50–200 200–2000 IIIII Slightly Moderately hazardous hazardous 50–2000>2000 200–2000 >2000 III Slightly hazardous >2000 >2000 U Unlikely to present acute hazard >5000 >5000 U Unlikely to present acute hazard >5000 >5000 3.2.1. Organochlorines 3.2.1. Organochlorines Considering the mode of action, the organochlorines damage the nervous systems, sinceConsidering they alter the the ion mode channels. of action, The the main organochlorines effects are hyperexcitability damage the nervous of brain, systems, convul- sincesions, they tremor, alter the hyperreflexia, ion channels. and The ataxia. main effects are hyperexcitability of brain, convulsions, tremor,Organochlorines hyperreflexia, and have ataxia. high toxicity and fall into the first classes of WHO classifica- Organochlorines have high toxicity and fall into the first classes of WHO classification tion (Figure 5). (Figure5).

−1 Figure 5. LD50 (mg·kg−1) of organochlorines for the rat. Figure 5. LD50 (mg·kg ) of organochlorines for the rat.

ItIt was was noted noted that that the the major major source source for for human human exposure exposure to to organochlorines organochlorines is is food, food, particularlyparticularly fish fish products products [ 63[63].]. The The OCs OCs are are accumulated accumulated in in fish fish muscle, muscle, and and then, then, they they becomebecome bioaccessible bioaccessible to to humans humans during during gastrointestinal gastrointestinal digestion. digestion. SignificantSignificant concentrations concentrations of of OCs OCs were were found found on duston dust particles. particles. This This type type of exposition of exposi- causestion causes cytotoxic cytotoxic effects effects on human on human skin, leadingskin, leading to the to development the development of carcinoma of carcinoma [64]. [64].

3.2.2.3.2.2. Organophosphates Organophosphates TheThe organophosphates organophosphates (OPs) (OPs) are are acetylcholinesterase acetylcholinesterase inhibitors, inhibitors, which which lead lead to having to hav- highing levelshigh levels of acetylcholine. of acetylcholine. The consequence The conseque isnce damage is damage to several to several organs organs such assuch periph- as pe- eralripheral and central and central nervous nervous systems, systems, muscles, muscles, liver, pancreas, liver, pancreas, and brain. and Most brain. of themMost belongof them tobelong the ﬁrst to classesthe first (Ia, classes Ib, and (Ia, II) Ib, of and the II) WHO of the classiﬁcation WHO classification (Figure6). (Figure 6). Bioengineering 2021, 8, 92 14 of 29 BioengineeringBioengineering 2021 2021, 8, x8 ,FOR x FOR PEER PEER REVIEW REVIEW 14 14of of29 29

−1 FigureFigure 6. 6.LD LD50 (mg·kg50 (mg·kg)− of1)1 oforganophosphates organophosphates for for rat. rat. Figure 6. LD50 (mg·kg ) of organophosphates for rat.

HighHighHigh doses doses doses of of of organophosphate organophosphate organophosphate cause cause cause acute acute acute intoxication, intoxication, intoxication, which which which leads leadsleads to toto pancreatitis. pancreatitis.pancreatitis. ThisThisThis is is isdue due due to to to the the the AChE-inhibition-induced AChE-inhibition-induced AChE-inhibition-induced cholinergic cholinergic cholinergic overstimulation, overstimulation, overstimulation, which which which leads leads leads to to to increasedincreasedincreased intraductal intraductal intraductal pressure pressure pressure and and and excess excess excess pancreatic pancreatic pancreatic enzyme enzyme enzyme secretion secretion secretion [65]. [65 [65].]. InInIn the the the long long long term, term, term, one one one of of of the the effects effects ofof of organophosphateor organophosphateganophosphate poisoning poisoning poisoning is is a isa seizure seizurea seizure disorder. disor- disor- der.Chuangder. Chuang Chuang et al. et [etal.66 al.] [66] have [66] have shownhave shown shown that that the that riskthe the ri of skri seizuresk of of seizure seizure development development development is greater is isgreater greater in patients in in pa- pa- tientswithtients organophosphatewith with organophosphate organophosphate poisoning poison poison thaninging inthan than healthy in in healthy healthy individuals. individuals. individuals.

3.2.3.3.2.3.3.2.3. Carbamates CarbamatesCarbamates TheTheThe toxicity toxicity toxicity of of of several several several carbamates carbamates carbamates is is issuch such such as as as to to to make make make them them them moderately moderately moderately and and and slightly slightly slightly hazardous, in some cases unhazardous, as can be seen from the LD values shown in hazardous,hazardous, in in some some cases cases unhazardous, unhazardous, as as can can be be seen seen from from the the LD LD5050 50values values shown shown in in FigureFigureFigure 7.7 . 7.

−1 FigureFigure 7. 7.LD LD50 (mg50 (mg·kg·kg)− of11) ofcarbamates carbamates for for rat. rat. Figure 7. LD50 (mg·kg ) of carbamates for rat.

TheTheThe carbamates carbamates carbamates act act act as as as acetylcholinesterase acetylcholinesterase inhibitorsinhibitors inhibitors andand andendocrine endocrine endocrine disruptors. disruptors. disruptors. Expo- Ex- Ex- posuresureposure to to carbamates to carbamates carbamates causes caus causes respiratoryes respiratory respiratory diseases. diseases. diseases. ToxicologicalToxicologicalToxicological experiments experiments experiments have have have shown shown that that prenatal prenatal andand and postnatalpostnatal postnatal exposureexposure exposure to to carba-to car- car- bamatesmatesbamates can can can affect affect affect fetal fetal fetal and and childand child child development, development, development, causing causing causing damage damage damage to the to hippocampus to the the hippocampus hippocampus [67,68]. [67,68].[67,68]. Bioengineering 2021, 8, x FOR PEER REVIEW 15 of 29 Bioengineering 2021, 8, 92 15 of 29

It was noted that many fruits and beverages are contaminated by ethyl carbamate, a It was noted that many fruits and beverages are contaminated by ethyl carbamate, a carcinogenic chemical causing the development of cancers in various animal species and carcinogenic chemical causing the development of cancers in various animal species and rarely in humans. In particular, this pesticide can induce the production of reactive oxy- rarely in humans. In particular, this pesticide can induce the production of reactive oxygen gen species, depurination of DNAs, and mitochondrial dysfunction [69,70]. species, depurination of DNAs, and mitochondrial dysfunction [69,70].

3.2.4.3.2.4. Pyrethroids Pyrethroids and and Pyrethrins Pyrethrins PyrethrinsPyrethrins and and pyrethroids pyrethroids are are less less toxic toxic to mammalianto mammalian cells cells and and less persistentless persistent in the in environmentthe environment than otherthan other pesticides. pesticides. They They can damage can damage human human health, health, mainly mainly affecting affecting the nervousthe nervous system. system. The effectsThe effects can be can systemic, be systemic, immunological, immunological, neurological, neurological, reproductive, reproduc- developmental,tive, developmental, genotoxic, genotoxic, and carcinogenic, and carcinog andenic, death and [death40]. The [40]. neurobehavioral The neurobehavioral func- tioningfunctioning of fetuses of fetuses and children and children is damaged is damaged by the by exposition the exposition to these to pesticides,these pesticides, since they since havethey an have immature an immature nervous nervous system, system, and their and their brain brain still hasstillto has grow to grow and and develop develop [71]. [71]. A correlationA correlation between between pyrethroid pyrethroid exposure exposure and and the the increased increased risk risk of of diabetes diabetes in in the the adult adult populationpopulation was was found found [72 [72].]. InIn Figure Figure8 ,8, the the LD LD50 valuevalue of of several several pyre pyrenoidsnoids is shown. is shown. These These compounds compounds are more are moretoxic toxic than than pyrethrins pyrethrins since since they they are aresynthesi synthesizedzed with with the the purpose purpose of ofincreasing increasing their their in- insecticidalsecticidal power. power.

−1 Figure 8. LD (mg·kg −1 ) of pyrethroids for rat. Figure 8. LD5050 (mg·kg ) of pyrethroids for rat. 4. Biological Techniques for Pesticide Removal 4. Biological Techniques for Pesticide Removal Bioremediation reduces pesticide contamination of agricultural soils by biodegradation processesBioremediation via the metabolic reduces pesticide activities contamination of microorganisms. of agricultural It is an efficient, soils by cost-effective, biodegrada- andtion environment-friendly processes via the metabolic treatment. activities of microorganisms. It is an efficient, cost-effective,During and environment-friendly the bioremediation processes, treatment. the microorganisms use the pesticides as cosub- stratesDuring in their the metabolic bioremediation reactions processes, together with the othermicroorganisms nutrients, thus use eliminating the pesticides them as fromcosubstrates the environment. in their metabolic The efficiency reactions of these toge processesther with depends other nutrients, on the characteristics thus eliminating of pesticides,them from such the asenvironment. their distribution, The efficiency their bioavailability, of these processes and their depends persistence on the in characteris- soil. It is necessarytics of pesticides, to promote such the as availability their distribution, of pesticides their bioavailability, to microorganisms: and their this ispersistence negatively in affectedsoil. It is by necessary the adhesion to promote of pesticides the availability to soil particles of pesticides and their to microorganisms: low water solubility this is [ 73neg-]. Inatively addition, affected the soil by the characteristics adhesion of andpesticides the environmental to soil particles conditions, and their low such water as pH, solubility water content,[73]. In microbialaddition, diversity,the soil characteristics and temperature, and influencethe environmental the bioremediation conditions, efficacy. such as pH, water content, microbial diversity, and temperature, influence the bioremediation efficacy. Bioengineering 2021, 8, 92 16 of 29

4.1. Mechanisms of Microbial Degradation During biodegradation processes, pesticides are transformed into degradation products or completely mineralized by microorganisms, which use the pollutant compounds as nutrients for their metabolic reactions. A key role in the biotransformation mechanisms is carried out by enzymes, such as hydrolases, peroxidases, and oxygenases, that inﬂuence and catalyze the biochemical reactions. The degradation process of pesticides can be divided into three phases, which can be summarized in: • Phase 1: Pesticides are transformed into more water-soluble and less toxic products through oxidation, reduction, or hydrolysis reactions. • Phase 2: The Phase-1 products are converted into sugars and amino acids, which have higher water solubility and lower toxicity. • Phase 3: Conversion of the Phase-2 metabolites into less toxic secondary conjugates. The microorganisms involved in the degradation process are bacteria or fungi, which may generate intra- or extra-cellular enzymes. The degradation time is a relevant parameter to be assessed when a bioremediation activity is planned. It is typically interpreted by the ﬁrst-order model [74], which depends on the pollutant concentration at the beginning and end of the process. This approach has limits because several parameters condition the process, such as microbial activity, temperature, water content, availability, and leaching of pesticide in the soil [61].

4.1.1. Bacterial Degradation In the years, several bacterial strains were identified as capable of degrading the pesticides present in the soils. Each bacterium has a specificity that makes it particularly suitable for a degradative process. The operative conditions, such as temperature, pH, water content, and types of pollutants, affect the adaptation, development, and role of a bacterial strain. Moreover, during the degradation process, metabolites can form and cause additional environmental problems, since they may be more difficult to remove than the original compound, and this must be considered a drawback. As an example, chlorpyrifos, an organophosphate used as an insecticide, is hydrolyzed by microorganisms, and the primary and major degradation product is 3,5,6-trichloro-2-pyridinol (TCP). TCP has greater water solubility than chlorpyrifos and causes widespread contamination in soils and aquatic environments. Few microorganisms can degrade the pesticide and its metabolite and among them the bacterium Ochabactrum sp. JAS2 is capable of hydrolyzing both compounds [75]. In many cases, the degradation is easier when a bacterial consortium is used compared to using an isolated pure culture [76,77]. In nature, the bacteria coexist and depend on each other for their viability. In the metabolic pathways of pesticide degradation, each bacterium can generate metabolites that may be used as a substrate by others.

4.1.2. Fungal Degradation The agricultural soils are populated by many fungi, which can be exploited to biode- grade pesticides. This class of microorganisms includes yeast, molds, and filamentous fungi. Fungal degradation is promoted by its capacity to produce many enzymes involved in degradative processes [78]. These microorganisms also can influence the soil properties, modifying soil permeability, and ion exchange capability. Fungi can be better degraders than bacteria due to their characteristics, such as specific bioactivity, growth morphology, and high resistance even at high concentrations of pollutants. A common approach is to use both fungi and bacteria to enhance degradation since fungi can transform pesticides into an easier and accessible form for bacteria [79]. Bioengineering 2021, 8, 92 17 of 29

4.1.3. Enzymatic Degradation Enzymatic biodegradation is due to the enzymes produced during the metabolic processes of microorganisms or plants. Enzymes are biological macromolecules that can catalyze biochemical reactions involved in pesticide degradation. These molecules act in the rate of reaction by lowering the activation energy of the reaction itself [80]. The main metabolic reactions, where they are involved, are oxidation, hydrolysis, reduction, and conjugation. • Oxidation, which is the ﬁrst step of the degradation of pesticides, consists of the transfer of an electron from reductants to oxidants. Oxygenase and laccase enzymes may be involved in this reaction. Oxygenases catalyze the oxidation reaction by incorporating one or two molecules of oxygen; laccases cleave the ring present in aromatic compounds and reduce oxygen to water and produce free radicals. During the reaction, heat or energy is generated, and it is utilized by microorganisms for their metabolic activities. • Hydrolysis permits the cleavage of bonds of the substrate by adding hydrogen or hydroxyl groups from water molecules. The pesticide molecules are thus divided into smaller chain compounds than the original ones. Typical enzymes involved in the hydrolysis pathways are lipases, esterases, and cellulases. For example, Luo et al. [81] have identiﬁed and cloned an esterase gene from Rhodopseudomonas palustris PSB-S capable of decomposing several synthetic pyrethroids, such as fenpropathrin, and tolerates temperature and pH changes. The enzyme is involved in the key step of hydrolysis, namely the cleavage of the ester bond in the fenpropathrin compound. • Reduction permits the transformation through reductive enzymes (nitroreductase). • The conjugation reaction is carried out using existing enzymes, and it is typical of fungal biodegradation. It involves the addition of exogenous or endogenous natural compounds to facilitate the mineralization of pesticides. This process includes reactions such as xyloxylation, alkylation, acylation, and nitrosylation. Table8 reports some microorganisms able to degrade widely used pesticides.

Table 8. Microorganisms capable to degrade several pesticides.

Target Pesticide Microorganism References Chlorpyrifos Ochrobactrum sp. JAS2 [75] Cypermethrin Bacillus subtilis [82] DDT Fomitopsis pinicola and Ralstonia pickettii [79] Insects Deltamethrin Streptomyces rimosus [74] Fentopropathrin Rhodopseudomonas palustris PSB-S [81] Brevibacterium frigoritolerans, Bacillus Phorate [77] aerophilus and Pseudomonas fulva Acetochlor Tolypocladium geodes and Cordyceps [11] Glyphosate Fusarium [83] Herbs Glyphosate and its metabolites Pseudomonas fluorescens [84] Penoxsulam Aspargillus flavus and Aspargillus niger [85] Pseudomonas, Rhodobacter, Ochrobactrum, Epoxiconazole and fludioxonil Comamonas, Hydrogenophaga, Azospirillum, [86] Fungi Methylobacillus, and Acinetobacter Tebuconazole Serratia marcescens [87]

4.1.4. Mineralization The mineralization process permits the degradation of pesticides into inorganic matter, namely, carbon dioxide, salts, minerals, and water. The microorganisms use the pesticide compounds as a source of nutrients. Also in this case, the degradation is inﬂuenced by several factors, such as microbial species, soil characteristics, and type of pollutants. The mineralization rate depends on the concentration of microbial community; namely, a decrease in microbial population does Bioengineering 2021, 8, 92 18 of 29

not promote the degradation [88]. For example, chlorothalonil (CTN), an organochlorine fungicide, is degraded in CO2, but if the soil microbial community is reduced, several metabolites can form, which are more toxic, persistent, and mobile than CTN itself. This is due to the absence of actively-degrading groups or the decrease in soil biodiversity that leads to low microbial activity. In glyphosate mineralization, the soil properties inﬂuence the mineralization process. Nguyen et al. [89] have tested agricultural soils, differing for some soil parameters, such as soil texture, soil organic matter content, pH, and exchangeable ions. By the univariate and multiple regression analysis, they have found the parameters that inﬂuence the glyphosate mineralization, namely: the cation exchange capacity, determined as the sum of exchangeable base cations and exchanges acidity (expressed as Al3+ and H+); the exchangeable base cations (expressed as Ca2+); and the available form of potassium, determined by ammonium lactate extraction. The low mineralization of glyphosate in soils with high exchangeable acidity could be due to either the formation of strong chemical bonds with the carboxylic or phosphonic acid groups of the glyphosate itself, reducing its bioavailability, or the toxic effects of exchangeable aluminum to soil microorganisms.

4.1.5. Co-Metabolism Co-metabolism is the biotransformation, through a series of reactions, of an organic compound that is not used to support microbial growth. The pesticides are transformed by microorganisms and enzymes into useful compounds for other biological, chemical, and physical transformations, and ﬁnally degraded thanks to this synergistic effect [90]. In the co-metabolic process, the involved enzymes can be: • hydrolytic enzymes (esterases, amidases, and nitrilases); • transferases (glutathione S-transferase and glucosyl transferases); • oxidases (cytochrome P-450s and peroxidase); • reductases (nitroreductases and reductive dehalogenases). Ma et al. [91] have studied the co-metabolic transformation of imidacloprid (IMI), an insecticide, testing different types of substrates used as an energy source: carbohydrates and organic acids. P. indica CGMCC 6648 is the tested bacterium, capable of degrading IMI through the hydroxylation pathway, and forms two metabolites: one oleﬁn and 5- hydroxy IMI.

4.2. Application of Microbial Remediation The bioremediation techniques may be carried out in situ, ex situ, or on-site. In the in situ approach, the treatment is carried out in the contaminated zone, and typically the process is aerobic. For this, it is necessary to provide oxygen to the soil. The main in situ techniques are: • Natural attenuation, which exploits the microﬂora present in the polluted soil. • Biostimulation, where the amounts and kind of nutrients to stimulate and promote the growth of indigenous microorganisms are optimized. • Bioaugmentation, which is the addition of microbial strains or enzymes into the polluted soils. • Bioventing, where oxygen is fed through unsaturated soil zones to stimulate the growth of indigenous microorganisms capable of degrading the contaminants. • Biosparging, based on the injection of air under pressure into the saturated soil zone to increase the oxygen concentration and stimulate the microorganisms to degrade the pollutant. These methods are very effective and cheap. Their main advantage is that the polluted soil is not moved. Vice versa, in ex situ techniques, the contaminated soil is removed from polluted sites and transported to the site where the clean-up will occur. The main techniques are: Bioengineering 2021, 8, 92 19 of 29

• Bioreactors, which treat the contaminated soil with wastewater to obtain a slurry and promote the microbial reactions capable of removing the pollutants. • Composting, where the contaminated soil is mixed with amendments to promote the aerobic degradation of the pesticides. Landfarming and biopiles are included in this technique. In on-site methods, the soil is removed and processed in the area close to the polluted site. For example, the landfarming treatment can also be effectuated on-site, reducing the operation cost comparing to the ex situ approach. In all bioremediation processes, nutrients, oxygen, pH, water content, and temperature must be controlled to maximize removal efﬁciency.

4.2.1. Natural Attenuation Natural attenuation is a natural process where pollutants are degraded by indigenous microorganisms present in the soil. The natural processes include biological degradation, volatilization, dispersion, dilution, radioactive decay, and sorption of the contaminant onto the organic matter and clay minerals in the soil. For example, Guerin [92] demonstrated that endosulfan diol and endosulfan sulfate, two metabolites of insecticide endosulfan, are both mineralized through the microbial activity present in the contaminated soils.

4.2.2. Biostimulation The biostimulation process consists of the addition of nutrients (nitrogen, phosphorus, carbon, and oxygen) to promote the growth of the indigenous microorganisms. These nutrients are essential for the life of microorganisms and allow them to have energy, microbial population, and enzymes to degrade the pollutants. Typically, nitrogen and phosphorus are added since they stimulate biodegradation and increase the diversity of microbial species. Betancur-Corredor et al. [93] have studied the degradation of DDT, DDD, and DDE, stimulating the microbial population and adding a surfactant. The number of nutrients supplied must be kept under control throughout the process, since a reduced or excessive quantity of stimulants could reduce microbial activity and their diversity. Ba´cmagaet al. [94] have studied the degradation of tebuconazole in soil using the biostimulation process. The tebuconazole negatively inﬂuences the enzymatic activity and microbial proliferation; for this, its concentration in the soil must be reduced. A high concentration of pesticides leads to a decrease in the microbial population. The experimental tests by these authors have evaluated the effects of two different biostimulation substances (compost and bird droppings) on the remediation process. The results have shown that both substances had a positive effect on the development of soil microorganisms and enzymatic activity. The tebuconazole degradation was more intense in the soil fertilized with bird droppings than with compost.

4.2.3. Bioaugmentation The bioaugmentation process involves the inoculation of microbial consortia or single strains into the soil, by augmenting the microbial diversity. In this way, microorganisms with specific metabolic capabilities promote the biodegradation processes. The concentration of pesticides in the soils is a parameter that conditions the process since high doses of pesticides inhibit the vital functions of soil microorganisms. Doolotkeldieva et al. studied the bacterial degradation of pesticide-contaminated soils in dumping zones. In a preliminary study [10], Doolotkeldieva et al. found that several bacterial strains were present in the studied soils. Then, they tested the degradation of aldrin, that is a diffused chlorinated hydrocarbon pesticide. The results have demonstrated that bacteria strains with specific genes (cytochrome P450), namely Pseudomonas fluorescens and Bacillus polymyxa, were capable of degrading aldrin in a relatively short time. The selection of specific bacterium, the optimization of soil conditions such as temperature, pH, and the nutrients available in the soil, were used for the development of the next Bioengineering 2021, 8, 92 20 of 29

experimental tests. In particular, mesocosms were set up with soil contaminated with several pesticides and inoculated with the microbial consortium [76]. In contaminated soil, the pesticide concentrations can vary at different depths since the pesticides leach into the subsurface of soil and adsorb on the soil particles, making them less bioavailable. Odukkathil and Vasudevan [95] have evaluated the bioaugmentation treatment in an experimental test set up in a glass column with a volume of 4500 cm3. The results have shown that the pesticide concentrations in the bottom soil were high, due to the downward drift of pesticides during the water seepage, whereas the low concentrations in the central soil could be due to higher microbial activity favoring the degradation.

Application of Natural Attenuation, Biostimulation and Bioaugmentation Several studies have been conducted to evaluate and compare the biodegradation of pesticides through natural attenuation, biostimulation, and bioaugmentation strategies. For example, Bhardawaj et al. [96] have analyzed the biodegradation of atrazine with three different techniques. Each mesocosm was set up with 100 kg of soil and contaminated with a concentration of atrazine equal to 300 mg·kg−1 of soil. They have found that despite the natural attenuation indicating that the soil microbiome possessed an inherent potential for atrazine biodegradation, the natural process was slow. Conversely, with biostimulation and bioaugmentation treatments, the atrazine was completely removed after 35 days. Moreover, the bioaugmentation strategy was more rapid than biostimulation since after 21 days the pollutant was degraded. Authors recommend this method for the treatment to be fast and cheap. The bioremediation of contaminated soils might be more efﬁcient when coupling bioaugmentation and biostimulation treatments [97]. Raimondo et al. [98] have tested 1 kg mesocosms polluted with lindane at a concentration equal to 2 mg·kg−1 of soil. They have demonstrated that the removal of lindane increases and the half-life of pesticide can be reduced using simultaneously bioaugmentation and biostimulation.

4.2.4. Bioventing Bioventing is an in situ bioremediation technique that promotes the degradation of organic pollutants adsorbed to the soil. The microbial activity is enhanced by the introduction of air or oxygen flow, and nutrients into the unsaturated zone of soil through specifically constructed wells into contaminated soils. The ventilation is light, and it is necessary to provide the only oxygen needed to sustain microbial activity and avoid the volatilization of contaminants. Bioventing can be realized in active or passive mode, with regards to the aeration: in Bioengineering 2021, 8, x FOR PEER REVIEWthe first case, the air is driven into the soil with a blower, while, in the passive method,21 theof 29

gas exchange through the vent wells occurs only by the effect of atmospheric pressure. The schemes of the two aeration methods are shown in Figure9.

(a) (b)

FigureFigure 9.9.Scheme Scheme ofof bioventingbioventing process:process: ((aa)) activeactive technologytechnology andand ((bb)) passivepassive technology. technology.

Bioventing remediation may last from 6 months to 5 years, depending on the kind and concentration of contaminant, biodegradation rates, and characteristics of soil, such as permeability and water content.

4.2.5. Biosparging In the biosparging technique, the biodegradation process occurs by stimulating the indigenous microorganisms through the injection of air in groundwater to increase the oxygen concentration. The method is similar to bioventing, except that in the biosparging the air is injected in the saturated zone. This can cause upward movement of volatile organic compounds to the unsaturated zone to promote biodegradation. Two parameters that influence the effectiveness of the process are (1) soil permeability, which determines pollutant bioavailability to microorganisms, and (2) pollutant biodegradability.

4.2.6. Composting Composting is an approach for the bioremediation of pesticides. It consists of mixing the contaminated soil with nonhazardous organic amendments to promote the development of bacterial and/or fungi population, able to degrade the pesticides through a co- metabolic pathway. This approach is particularly indicated when pesticide concentration is low. In composting, the microbial bioaccessibility to the pollutant is crucial. For this reason, it is important to control the water content, soil composition, and properties of the added amendment. In contaminated soils, biochar can be used as an amendment to promote the degradation processes. Biochar is black carbon produced by the thermal conversions of biomass under limited oxygen conditions (gasification) or in the absence of oxygen (pyrolysis). It is characterized by high porosity and a large surface area; these two properties promote the adsorption of pesticides. Moreover, biochar is a carbon source that stimulates microbial activity, promoting biodegradation. It has been noted that biochar application increases the soil water holding capacity and improves aeration conditions in soil [99]. Sun et al. [100] have studied its application for the biodegradation of tebuconazole. This allowed the immobilization of Alcaligenes faecalis WZ-2, the bacterial strain involved in the degradation process. Composting can be carried out with two techniques: landfarming and biopiles.

Landfarming Landfarming is an aerobic bioremediation process carried out for a long time. Con- taminated soils are transported to a landfarming zone, incorporated into the soil surface over large areas, and periodically tilled to aerate the mixture. The kinetics of the degrada- Bioengineering 2021, 8, 92 21 of 29

4.2.5. Biosparging In the biosparging technique, the biodegradation process occurs by stimulating the indigenous microorganisms through the injection of air in groundwater to increase the oxygen concentration. The method is similar to bioventing, except that in the biosparging the air is injected in the saturated zone. This can cause upward movement of volatile organic compounds to the unsaturated zone to promote biodegradation. Two parameters that inﬂuence the effectiveness of the process are (1) soil permeability, which determines pollutant bioavailability to microorganisms, and (2) pollutant biodegradability.

4.2.6. Composting Composting is an approach for the bioremediation of pesticides. It consists of mixing the contaminated soil with nonhazardous organic amendments to promote the development of bacterial and/or fungi population, able to degrade the pesticides through a co-metabolic pathway. This approach is particularly indicated when pesticide concentration is low. In composting, the microbial bioaccessibility to the pollutant is crucial. For this reason, it is important to control the water content, soil composition, and properties of the added amendment. In contaminated soils, biochar can be used as an amendment to promote the degradation processes. Biochar is black carbon produced by the thermal conversions of biomass under limited oxygen conditions (gasiﬁcation) or in the absence of oxygen (pyrolysis). It is characterized by high porosity and a large surface area; these two properties promote the adsorption of pesticides. Moreover, biochar is a carbon source that stimulates microbial activity, promoting biodegradation. It has been noted that biochar application increases the soil water holding capacity and improves aeration conditions in soil [99]. Sun et al. [100] have studied its application for the biodegradation of tebuconazole. This allowed the immobilization of Alcaligenes faecalis WZ-2, the bacterial strain involved in the degradation process. Composting can be carried out with two techniques: landfarming and biopiles.

Landfarming Landfarming is an aerobic bioremediation process carried out for a long time. Contam- inated soils are transported to a landfarming zone, incorporated into the soil surface over large areas, and periodically tilled to aerate the mixture. The kinetics of the degradation process is slow and may also take years. During the process, leaching and/or volatilization of toxic compounds (original compound and metabolites) must be controlled or prevented. To avoid any risk of inﬁltration, a waterproof cover must be in place on the soil before the start of the treatment. This treatment is applied especially to soils contaminated with a mixture of pollutants.

Biopiles Biopiles are piles of contaminated soil, equipped with a piping system that permits the aeration. During the process, air or oxygen is sent, and a solution containing nutrients is applied to the soil surface to stimulate microbial activity. The parameters that inﬂuence the process are water contents, temperature, pH, and concentration of nutrients and oxygen that must be controlled to enhance biodegradation.

4.2.7. Slurry Bioreactors In slurry bioreactors, the contaminated soil is mixed with wastewater up to obtain a slurry with aqueous suspensions between 10% and 30% w·v−1. The bioreactor can be oper- ating under aerobic or anaerobic conditions. Baczynski et al. [101] studied the anaerobic Bioengineering 2021, 8, 92 22 of 29

biodegradation of organochlorine pesticides. They used methanogenic granular sludge as inoculum for anaerobic treatment of soil contaminated by γ-hexachlorocyclohexane, methoxychlor, o,p’- and p,p’-DDT. The pollutants were removed to a good extent at all tested temperatures (12, 22, and 30 ◦C) without a lag phase. Low temperature reduces the removal rates of pesticides. This might be due to the reduction of desorption rates of slowly desorbing fractions of these pollutants. In another study [102], the same authors found that the rapidly desorbing fractions were not a good indicator for the evaluation of the bioremediation process. Instead, the determination of slowly desorbing fractions can be better used for this purpose.

4.3. In-Field Applications At present, few studies report information on real case studies. Table9 summarizes some examples. Unfortunately, the ﬁndings and results of large- scale remediation are usually neither published nor widely publicized, limiting the knowledge of experiences on real cases. A similar situation occurs for the costs of the clean-up.

Table 9. Some examples of case studies.

Bioremediation Pesticides Description References Technique Contaminated soil with HCH isomers (>5000 mg·kg−1) derived from lindane production was studied in the field for Hexachlorocyclohexane 11 months, setting up two plots (each 2 m × 10 m). The α- Landfarming [103] (HCH) isomers (insecticides) and γ-HCH isomers were decreased by 89 and 82% of the initial concentration, respectively. The concentration of the most persistent β-isomer remained essentially unaffected. Experimental tests were conducted on vineyard plots. In the crops, an agricultural formulation of pesticides by foliar spray was applied. After one h of pesticide application, vines were sprayed with a suspension of four Bacillus Myclobutanil, tetraconazole, Bioaugmentation strains. DR-39, CS-126, TL-171, and TS-204 were tested. [104] and flusilazole. Residue analysis of field samples showed 87.4 and >99% degradation of myclobutanil and tetraconazole, respectively, by the strain DR-39, and 90.8% degradation of flusilazole by the strain CS-126 after 15−20 days of treatment. The bioremediation process was studied in 12 experimental plots, including greenhouse and open field soils. Each plot (area of 6 m2) was inoculated with Stenotrophomonas sp. Bioaugmentation DDT DDT-1 supplemented with 2% yeast powder. The results [105] have shown that this microorganism is efficient for DDT degradation and does not adversely affect soil microbial activity. The Borello Property is a 14 acre area treated with soil amendment to help the indigenous bacteria to metabolize the pesticides. For the analysis, the area was divided into Organochlorine pesticides: zones and in each of them, the soil samples were collected toxaphene; DDT; DDE; DDD; Biostimulation from four soil depths (0.5, 1, 1.5, and 2 ft). At the end of the [106] endosulfan II; γ-chlordane; test, OCPs were not detected; toxaphene, DDT, and DDE α-chlordane; dieldrin. were detected in a single sample; dieldrin was detected in five samples at concentrations ranging from 1.2 to 1.8 µg·kg−1. The Mantegani Property is a 0.8 acre area treated with soil Organochlorine pesticides: amendment to help the indigenous bacteria to metabolize toxaphene; DDT; DDE; DDD; the pesticides. High concentrations of DDT and dieldrin Biostimulation [106] endosulfan II; γ-chlordane; were present. After treatment, DDT was degraded by 97% α-chlordane; dieldrin. and dieldrin by 73%, while the concentrations of other OCPs were below their preliminary remediation goals. Bioengineering 2021, 8, 92 23 of 29

5. Legislation The necessity of having sustainable food production and a reduction, or even a ban, of the use of pesticides, has made it so that each country in the world is committed to implement measures and laws in this regard. Dealing with the topic at the world level, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) have released a pair of updated guidelines on pesticide legislation and labeling. A code for pesticide legislation has been published in 2020 [107], aiming to guide governments that seek to review, update, or design national pesticide legislation.

5.1. European Union In the European Union, the sustainable use of pesticides is ruled by Directive 2009/128/ EC [108]. The aim is to reduce the risks and impacts of these chemicals on health and the environment. Moreover, Regulation (EC) 1107/2009 [5] contains the criteria and rules to authorize the active substances of pesticides and plant protection products (PPPs) and their placement on the European market. Pesticides cannot be marketed and used if they have not been previously approved and authorized by this procedure. The institution which deals to evaluate active substances contained in pesticides is the European Food Safety Authority (EFSA). EFSA supports the European Commission, the European Parliament, and the EU member states in taking effective and timely risk management decisions to ensure the protection of the health and the safety of the food and feed chain. Each EU member state evaluates and authorizes the products with national laws. In the member states, Regulation (EC) No. 1107/2009 [5] has been underpinned by national laws through the formulation of more precise stipulations at the national level. In particular, the National Action Plans include quantitative objectives, targets, measures, and timetables, and they are to be reviewed at least every 5 years. The ﬁrst National Action Plans were communicated to the Commission in 2012, and a report by the EU Commission [109] has shown that in 2017 all Member States had plans in place.

5.2. United States of America In the United States, the regulation and monitoring of pesticides are ruled by three government agencies: • The U.S. Environmental Protection Agency (EPA) registers and approves the use of pesticides and establishes the maximum amounts of residues that are permitted inside or on food. • The U.S. Department of Agriculture (USDA) is responsible for the enforcement of pesticide tolerances primarily in meat, poultry, and certain egg products. • The U.S. Food and Drug Administration (FDA) is responsible for the enforcement of pesticide tolerances in other food categories, both domestic and imported ones. The EPA regulates the use of pesticides considering two federal statutes: (1) the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), enacted in 1947 and updated with new amendments [110]. It regulates the registration, distribution, and use of pesticides, including the toxicology, environmental fate, and ecotoxicology and residue chemistry; (2) the Federal Food, Drug, and Cosmetic Act (FD&C). It establishes tolerances or safe levels of pesticide residues in raw agricultural commodities. The updated act is FD&C 2021 [111]. The EPA works cooperatively with the state agencies to review pesticide safety data, register products, educate professional applicators, monitor compliance, and investigate pesticide problems. State governments develop regulations that are stricter than the federal regulation given by EPA.

5.3. India In India, the pesticide regulation was applied for the ﬁrst time in the insecticide act [112], by the Ministry of Agriculture, Department of Agriculture and Cooperation in Bioengineering 2021, 8, 92 24 of 29

1968. Since 1971, the insecticide rules [113] have been in force: they regulate the import, sale, transportation, manufacture, and use of persistent pesticides. Under this act, all insecticides must be recorded by the Central Insecticides Board (CIB) and Registration Committee (RC). The CIB and RC scrutinize and periodically review all pesticides, before authorizing their sale and use. Moreover, they have the authority to ban environmentally threatened pesticides even after their registration.

5.4. Regulation in Other Countries The regulations on pesticides in other countries are more recent. In China, the Ministry of Agriculture is responsible for the enforcement of the Law on Quality and Safety of Agricultural Products and the Regulation on the Control of Agro- chemicals. The National Food Safety Standard—Maximum Residue Limits for Pesticides in Foods [114] was approved in 2012 and updated several times in the following years to add other maximum residue limits of pesticides in foods, until the current version released in February 2021 [115]. In Japan, the pesticide regulation is contained in the Food Safety Basic Law, enacted in 2003 [116]. This law sets the principles for developing a food safety regime and deﬁnes the maximum acceptable concentration limits of residual pesticides. A limit of 0.01 mg·kg−1 of soil applies to compounds for which tolerance limits have not been established.

6. Conclusions After the Second World War, the use of pesticides and plant protection products has grown heavily, both in developed and developing countries. Unfortunately, all these compounds are toxic to different extents and impact human health and the environment. Moreover, many of them are persistent; that is to say, their degradability is very limited and occurs for long times. Soil bioremediation for their removal can be carried out exploiting either specific or indigenous microorganisms (bacteria and fungi), or enzymatic degradation. While at a laboratory scale, many findings on soil bioremediation are available in the literature, few data on real-scale activities can be found. Unfortunately, this is mainly due to the poor cooperation among research laboratories, local authorities imposing a given soil clean-up, and companies involved in the sector of bioremediation in soils polluted with pesticides. It would be beneficial that more and more this cooperation becomes united, to dissem- inate the experiences and results. Moreover, the cost data are lacking, too. As for other pollutants, when required, the pesticide removal must take into account the chemical and toxicological characteristics of the compounds, without disregarding the national legislation. To this purpose, it must be outlined that several countries are still lacking in legislative acts, and this is the main drawback when polluted areas must be remediated.

Author Contributions: Writing—original draft preparation, C.M.R.; writing—review and editing, C.M.R. and F.C.; supervision, F.C. Both authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest. Bioengineering 2021, 8, 92 25 of 29

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