THE ECOTOXICOLOGY OF PESTICIDES GROUP OF TRIAZOLE AND THEIR USE TO CONTROL APPLE SCAB (VENTURIA INAEQUALIS)

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1 Review paper UDC :547.79]: THE ECOTOXICOLOGY OF PESTICIDES GROUP OF TRIAZOLE AND THEIR USE TO CONTROL APPLE SCAB (VENTURIA INAEQUALIS) Edlira Shahinasi 1*, Ferdi Brahushi 2, Ariola Devolli 1, Mariola Kodra 1 1 Department of Chemistry, Faculty of Biotechnology and Food, Tirana Agricultural University, Koder-Kamez, 1000 Tirana, Albania 2 Department of AgroEnvironment and Ecology, Agricultural University of Tirana, Koder-Kamez, 1000 Tirana, Albania * eshahinasi@ubt.edu.al Abstract Plant and plant products are affected by a large number of plant pathogens among which fungal pathogens. These pathogens can cause serious damage in agriculture, which can lead in critical losses of yield, quality and profit. This article review covers information over the triazoles which belong to the group of systematic fungicides. Actually, the triazoles play an important role on controlling and treatment of fungal diseases of: vegetables, legumes, grasses and crops in general and of apple scab in particular. This group of pesticides acts by inhibiting sterol synthesis. Many toxicological studies have shown an extensive oral absorption for some triazoles such as myclobutanil, penconazole, difenconazole, tebuconazole, etc. The proposed classification of these fungicides is Xn, R22 harmful if swallowed. The oral LD50 for penconazole varies from 200 mg/ kg bw in rates to 2000 mg/kg bw in rabbits; LD50 for myclobutanil in mammals is 1600mg/kg bw; LD50 for tebuconazole 1700 mg/kg bw; and for difenconazole oral LD50 > 2000 mg/kg bw and 1450 mg/kg bw in birds and mammals, respectively. Their maximum residue limits (MRLs) values are different not only from one product to another, but even in the same product. Thus, MRLs in apples are 0.2, 0.3, 0.6 and 0.8 mg/kg for penoconazole, myclobutanil, tebuconazole and difenconazole, respectively. Reviewed data show that triazoles are effective on controlling fungal plant diseases and presents in general, low ecotoxicity, thus they can be recommended on controlling fungal plant diseases. Although, their use have to be in accordance with level of plant infection and environmental conditions in order to avoid plant and environment pollution. Key words: Triazole, Apple scab, Ecotoxicity, Fungal diseases. 1. Introduction Different agricultural plant and their agricultural products can be affected by a large number of plant pathogens among which are fungal pathogens. Fungi due to their widespread in agricultural plants can cause serious damage in agriculture, resulting in critical losses of yield, quality and profit [1, 2]. Fungicides are important tools for control and managing diseases in: fruits, vegetables, legumes, grasses and other crops. Unlike insecticides and some herbicides which kill established insects or weeds, fungicides are most commonly applied to protect healthy plants from infection by fungal plant pathogens [2]. Among many classes of fungicides azole or conazole fungicides represent a large group of substances which are widely used in agriculture for protection of crop plants, and in pharmaceutical industry in the treatment of various fungal diseases. They are synthetic compounds that can be classified into imidazole or triazole, depending on the number of nitrogen atoms in the five-membered ring [3]. To be effective, fungicides must be applied before infections become established and in a sufficient spray volume to achieve thorough coverage of the plant or treated area. Protection from fungicides is temporary because they are subject to degradation or mineralization over time. Furthermore, in some cases the plant pathogens can be adapted to fungicides. Thus, they have to be reapplied to protect new growth when new disease threatens. Poor disease control with fungicides can result from 36

2 several causes including insufficient application rate, inherently low effectiveness of the fungicide on the target pathogen, improper timing or application method, and excessive rainfall [2]. The intensive or extensive use of these compounds can generate a lot of residues that may potentially lead to substantial plant and environmental contamination. Thus, triazole residues or triazole metabolites may occur in plant or environment and should be considered as a risk for human health due to their possible transport to food chain [4]. 2. Ecotoxicology of pesticides group of triazole and their use to control apple scab 2.1 Classification of fungicides Fungicides can be classified in a number of different ways. Their classification is based on: 1. The origin of fungicides mobility in the plant (biologically based fungicides known as biofungicides; chemically based fungicides, which are synthesized from organic and inorganic chemicals). 2. Classification according to similarities of chemical group or structure, relevant activity and mode of action - MOA [5]. Different letters from A to I, are used to distinguish fungicide groups according to their biochemical mode of action in the biosynthetic pathways of plant pathogens. The grouping was made according to processes in the metabolism starting from nucleic acids synthesis (A) to secondary metabolism, melanin synthesis (I) at the end of the list. According to Fungicide Resistance Action Committee code list, triazoles belong to Group 3 and sub-group G1 (see the Table 1) [6]. Based on the results of the acute oral LD50 in rabbits and rats, triazoles are considered harmful if swallowed and should be classified as: Acute tox. 4 H302 according to Regulation (EC) 1272/2008 and Xn; R22 according to Directive 67/548/EEC. 2.2 Triazole mode of action and transport in the plant In devising a fungicide strategy, it is important to recognize that all principal foliar fungicides have different modes of action, which influence the performance of these fungicides during the application to the plant. The triazoles, as it is mentioned above, belong to Group 3 family of fungicides, and sub-group G1 [6, and 7], are very specific in their mode of action. They inhibit the biosynthesis of sterol, a critical component for the integrity of fungal cell membranes. Because their site of action is very specific, there are resistance concerns [1] Mode of Action in fungi Triazoles belong to the most used agricultural fungicides due to their efficacy as well as their curative properties [2]. The fungicidal activity of azole fungicides is due to an inhibition of the fungal cytochrome P-450-(CYP)-enzyme ergosterol synthesis. Studies have shown that myclobutanil [α-butyl-α- (4-chlorophenyl)1H-1,2,4-triazole-1-propanenitrile] which is a DMI triazole disrupts fungal membranes by inhibiting sterol biosynthesis. Specifically, it acts on the 1Terg111T gene responsible for encoding sterol C14-demethylase (cytochrome P450 isozyme CYP51), inhibiting demethylation in membrane sterol biosynthesis and, thereby, preventing synthesis of ergosterol, a major membrane component in fungi [8]. Since most triazoles are not completely specific, they also inhibit mammalian CYP-enzymes [9, 10]. Especially aromatase, an enzyme that converts androgens to estrogens, e.g. testosterone to estradiol, is a known target for unspecific inhibition by triazoles, but other CYP-enzymes, many of which contribute to steroid biosynthesis, are also affected. Several experiments have shown that triazoles are able to act in a dose additive manner regarding CYP-enzyme inhibition [11, 12, and 13] Transport in plant Triazole fungicides diffuse into the leaf surface and are transported in the xylem vessels. This movement of fungicide ensures the products have a degree of systemic activity within the plant that can be used to control fungal infection in the latent phase of development. Movement in the xylem vessels means that foliar fungicides only move in an upwards direction to the leaf tip. This limits the systemic activity of these fungicides to protection of only tissue that has emerged at the time of application. The triazole fungicides are therefore unable to move back down the leaf in order to provide protection of leaf tissue that had not emerged at the time of application [14]. The uptake and movement of triazole fungicides into the cereal leaf varies amongst active ingredients found in the Group 3 family of fungicides [15]. 2.3 Role of triazoles on management of apple diseases Apple scab, caused by the fungus Venturia inaequalis, is one of the most economically devastating diseases of apple. The scab pathogen can infect both leaves and fruit, and can cause severe defoliation of apple trees if poorly managed. The disease negatively affects fruit size and quality, due to blemishes and poor ripening. Overtime, repeated defoliation caused by the disease reduces tree vigor, growth and yield [16]. Many studies have shown that application of triazole fungicides on plant have significantly increased the fruit production. Halley and Percival [17] have noticed that after spraying apple trees with synthetic fungicide penconazole the values of fruit yield and chlorophyll content were 51.3% and 88.8% higher than control respectively. On the other hand, the experiment demonstrated, that reductions of leaf and fruit scab severity where 88.9% and 100% lower than control 37

3 Table 1. The characteristics of Group G fungicides MOA G: Sterol biosynthesis in membranes Target site and code G1: C14- demethylase in sterol biosynthesis (erg11/cyp51) Group name Chemical group Common name Comments DMI-fungicides (DeMethylation Inhibitors) (SBI: Class I) piperazines pyridines pyrimidines imidazoles triazoles triazolinthiones triforine pyrifenox pyrisoxazole fenarimol nuarimol imazalil oxpoconazole pefurazoate prochloraz triflumizole azaconazole bitertanol bromuconazole cyproconazole difenoconazole diniconazole epoxiconazole etaconazole fenbuconazole fluquinconazole flusilazole flutriafol hexaconazole imibenconazole ipconazole metconazole myclobutanil penconazole propiconazole simeconazole tebuconazole tetraconazole triadimefon triadimenol triticonazole prothioconazole There are big differences in the activity spectra of DMI fungicides. Resistance is known in various fungal species. Several resistance mechanisms are known incl. target site mutations in cyp51 (erg 11) gene, e.g. V136A, Y137F, A379G, I381V; cyp51 promotor; ABC transporters and others. Generally wise to accept that cross resistance is present between DMI fungicides active against the same fungus. DMI fungicides are Sterol Biosynthesis Inhibitors (SBIs), but show no cross resistance to other SBI classes. Medium risk. FRAC code 3 G2: Δ 14 -reductase and Δ Δ 7 isomerase in sterol biosynthesis (erg24, erg2) amines ( morpholines ) (SBI: Class II) morpholines piperidines spiroketal-amines aldimorph dodemorph fenpropimorph tridemorp fenpropidin piperalin spiroxamine Decreased sensitivity for powdery mildews. Cross resistance within the group generally found but not to other SBI classes. Low to medium risk. 5 G3: 3-keto reductase, C4-demethylation (erg27) (SBI: Class III) hydroxyanilides aminopyrazolinone fenhexamid fenpyrazamine Low to medium risk. Resistance management required 17 G4: squaleneepoxidase in sterol biosynthesis (erg1) (SBI class IV) thiocarbamates allylamines pyributicarb naftifine terbinafine Resistance not known, fungicidal and herbicidal activity Medical fungicides only 18 (Source: FRAC code list [6]). 38

4 respectively. Almost the same result was taken from another experiment made by Percival [18]. According to him, application of 0.6mL/L fungicide myclobutanil reduced leaf scab severity with 80%. Within the UK and Ireland, myclobutanil is fully approved for scab control of ornamental and fruiting apples, identified as possessing protective and curative action and anti-sporulation activity [18] The fungicide residues Pesticides are widely used during the production of fruits, vegetables, and cereals. In apple production, fungicides and insecticides are used both pre- and postharvest to protect the fruit from a range of pests and fungi, and to preserve quality. Food authorities, [e.g., the Commission of the European Communities (CEC)] have established Maximum Residue Levels (MRL) for all the pesticides used in the production of food. The Table 2 presents MRLs of triazoles in some fruits, vegetables and cereals. 2.4 Environmental fate of triazole fungicides Different studies have demonstrated that triazole fungicides have different behavior in soils. Thus, difenconazole exhibits moderate to high forming metabolite 1,2,4 - triazole (low to moderate ) and CGA medium to high ; myclobutanil exhibits high to very high ; tebuconazole moderate to medium ; and penconazole moderate to high. The kinetic degradation of these fungicides belongs to single first order and half-life values vary from 19.9 days to 1216 days. Mobility in soil depend on the type of metabolite for example metabolite 1,2,4-triazole (CGA 71019) that is a metabolite formed by degradation of difenconazole demonstrates high to very high mobility in soil with a K foc that varies from 43 to120 ml/g. In contrary to this penconazole shows low to slight mobility in soil. In the Table 3 we have presented some data related to environmental characteristics and ecotoxicity data of triazole fungicides and their metabolites. The 1,2,4 triazole metabolite is present during degradation of almost all triazole fungicides. The studies in soil have demonstrated that absorption of this metabolite was not ph dependent. 2.5 Mammalian toxicity Many studies have shown that triazoles fungicides are extensively absorbed from the gastro-intestinal tract and widely distributed without bioaccumulation in body tissues. The highest levels of these fungicides are detected in liver, kidney, adrenal glands and thyroid. Most of triazole fungicides are not toxic via dermal and inhalation routes; are not skin irritant or a skin sensitizer. The major part of triazoles is not irritant to the eyes, except the myclobutanil that is classified as Irritating to eyes [19, 20, 21, and 22] Acute, short and long term toxicity To determine the toxicity of triazole fungicides are used several species such as rats, rabbits, mice and in some cases even dogs and birds. Experimental results indicate that oral LD 50 for penconazole varies from 200 mg/kg bw in rates to 2000 mg/kg bw in rabbits; LD 50 for myclobutanil in mammals is 1600mg/kg bw; LD 50 for tebuconazole 1700 mg/kg bw; and for difenconazole oral LD 50 > 2000 mg/kg bw, and 1450 mg/kg bw in birds and mammals. The primary target organ is the liver. The use of myclobutanil induces liver weight increase associated with hepatocellular hypertrophy in rats, mice and dogs [19, 20, 21, and 22]. During the degradation of triazole fungicides it is obtained the 1,2,4 metabolite (CGA 71019). In number of studies this metabolite was seen to cause neurotoxicity in mice and rats. In a 28 day toxicity study in mice, the Non-Observed-Adverse Effect Levels (NOAEL) were slight testicular degeneration accompanied by apoptotic bodies at 356 mg/kg bw per day. No effects were observed in females at doses up to and including 479 mg/kg bw per day. The NOAEL in mice was equal to 90 mg/kg bw per day. In a 90-day study of toxicity in mice, decreased body weights, tremors (observed from day 30), decreased body weight, and loss of cerebellar Purkinje cells were observed in males and females at 988 mg/kg bw per day. Decreased testicular weights and histopathological findings in testes similar to the 28-day study were observed in males at 3000 and 6000 ppm. The NOAEL was 1000 ppm, equal to 161 mg/kg bw per day, on the basis of tremors, decreased brain weights, decreased Table 2. MRLs in some fruits, vegetables and cereals Name of fungicide Pomes and stone fruits Vegetables Cereals Apples Pears Peaches Plums Cucumber Broccoli Carrots Tomatoes Wheat Rice Myclobutanil Penconazole Tebuconazole Difenconazole (Source: European pesticide database [24]). Maize/ corn 39

5 Table 3. The characteristics and ecotoxicity of some triazoles Compound (name & code) Persistence DT 50 (days) Mobility in soil K foc (mg/g) Ecotoxicological activity NOEC/EC 50 (mg a.s./l) Difenconazole Difenconazole Moderate to high * ** Immobile to medium Acute and chronic risk to earthworms assessed as low (Daphnia magna) 1,2,4 triazole (CGA 71019) Low to moderate 6-12* High to very high Acute and chronic risk to earthworms assessed as low - Toxic to aquatic 3.2 (fish) CGA Medium to high * Immobile to low Acute and chronic risk to earthworms assessed as low 0.74 (fish) Myclobutanil Myclobutanil High to very high Medium to low Risk to soil dwelling assessed as low Myclobutanil butyric acid Very high Penconazole Penconazole Moderate to high Low to slight Low risk for earthworms CGA Low to moderate Very high Low risk for the earthworms - Very toxic to fruit crops 45 Grapes and cucurbits CGA Low High to very high Low risk for the earthworms 498 (fish) Tebuconazole Tebuconazole Moderate to medium High to low Risk to soil dwelling assessed as low - Risk to aquatic assessed as low 1, 2,4 -triazole Very high to high Risk to aquatic assessed as low *(20 0 C, 40-48% MWHC soil moisture). ** (non-normalized soil). 40

6 testicular weights and histopathological changes in the testes seen in males at the NOAEL equal to 487 mg/ kg bw per day [23]. Because of toxic properties that are demonstrated during the use of high concentration of triazole pesticides European Commitment has established the levels of the Acceptable Daily Intake (ADI), the Acceptable Operator Exposure Level (AOEL) and the Acute Reference Dose (ARfD) in humans. These levels are presented to the Table 4. Table 4. Levels of ADI, AOEL and ARfD in humans Name of fungicide ADI mg/kg bw/ day (AOEL) mg/kg bw/ day ARfD mg/kg bw Myclobutanil 0,025 0,31 0,03 Penconazole Tebuconazole Difenconazole (Source: European database for pesticides [24]) 3. Conclusions - The application of pesticides on agriculture is the main key on controlling of plant pathogens. - Among different classes of pesticides, triazoles are the fungicides classified as modern systemic fungicides. They protect leaf and fruit from fungal diseases and increase the fruit quality and productivity. - Triazoles have in general low ecotoxicity and their properties make them preferable for extensive application in the agricultural sector. - Furthermore, the application of triazoles to control apple scab (Venturia inaequalis) is wide recommended due to their efficacy and low ecotoxicity. 4. References [1] Fishel M. F. (2014). Pesticide Toxicity Profile: Triazole Pesticides. IFAS Extension, University of Florida. <URL: Accessed 25 June [2] Damicone J. Fungicide Resistance Management. <URL: dsweb/get/document-2317/epp-7663web.pdf.accessed 25 June [3] Faro R. L. (2010). Neurotoxic Effects of Triazole Fungicides on Nigrostriatal Dopaminergic Neurotransmission. In: Carisse O. (Ed.), Fungicides, InTech, pp [4] Filipov M. N., and Lawrence A. D. (2001). Toxicological Highlight. Developmental Toxicity of a Triazole Fungicide: Consideration of Interorgan Communication. Toxicological Sciences, 62, pp [5] Rouabhi R. (2010). Introduction and Toxicology of Fungicides. In: Carisse O. (Ed.), Fungicides, InTech, pp [6] Fungicide Resistance Action Committee. (2016). FRAC code list 2016: Fungicides sorted by mode of action (including FRAC Code numbering). <URL: Accessed 25 July [7] Poole N. F., and Arnaudin M. E. (2014). The role of fungicides for effective disease management in cereal crops. Canadian Journal of Plant Pathology, 36, 1, pp [8] Elksus A. A. (2014). Toxicity, sublethal effects, and potential modes of action of select fungicides on freshwater fish and invertebrates. <URL: ofr pdf. Accessed 25 July [9] Sanderson J. T., Boerma J., Lansbergen G. W., van den Berg M. (2002). Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol., 182, pp [10] Vinggaard A. M., Hass U., Dalgaard M., Andersen H. R., Bonefeld-Jorgensen E., Christiansen S., Laier P., Poulsen M. E. (2006). Prochloraz: an imidazole fungicide with multiple mechanisms of action. Int. J. Androl., 29, pp [11] Kjaerstad M. B., Taxvig C., Andersen H. R., Nellemann C. (2010). Mixture effects of endocrine disrupting compounds in vitro. Int. J. Androl., 33, pp [12] Hass U., Scholze M., Christiansen S., Dalgaard M., Vinggaard A. M., Axelstad M., Metzdorff S. B., Kortenkamp A. (2007). Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ. Health Perspect., 115, pp [13] Prutner W., Nicken P., Haunhorst E., Hamscher G., Steinberg P. (2013). Effects of single pesticides and binary pesticide mixtures on estrone production in H295R cells. Arch. Toxicol., 87, pp [14] Bartlett D. W., Clough J. M., Godwin J. R., Hall A. A., Hamer M., and ParrDobrzanski B. (2002). The strobilurin fungicides. Pest. Manag. Sci., 58, pp [15] Kendall S. J., Hollomon D. W., Stormonth D. A. (1994). Towards the rational use of triazole mixtures for cereal disease control. In: Brighton Plant Protection, Pests, and Diseases Conference Proceedings; Brighton, UK, 2, pp [16] Nicholson I. R., and Beckerman J. (2008). Towards a Sustainable, Integrated Management Of Apple Diseases. In: Cianco A., and Mukerji G. K., (Eds.), Integrated Management of Diseases Caused by Fungi, Phytoplasma and Bacteria, Springer, Netherlands, pp [17] [Hailey E. L., and Percival C. G. (2014). Comparative Assessment of Phosphite Formulations for Apple Scab (Venturia inaequalis) Control. Arboriculture & Urban Forestry, 40, (4), pp [18] Glynn C., Percival C. G. (2010). Effect of Systemic Inducing Resistance and Biostimulant Materials on Apple Scab Using a Detached Leaf Bioassay. Arboriculture & Urban Forestry, 36, (1), pp [19] European Food Safety Authority. (2011). Conclusion on the peer review of the pesticide risk assessment of the active substance difenoconazole. EFSA Journal, 9. 41

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