Evaluation of the Essential Oils Used in the Production of Biopesticides: Assessing Their Toxicity toward Both Arthropod Target Species and Beneficial Pollinators

Abstract

Biopesticides, alongside the expansive growth of conventional agriculture, emerged as a hopeful avenue for mitigating the environmental impact of synthetic chemicals. Essential oils derived from various plant species are regarded as environmentally friendly and have been suggested by numerous researchers for combating the pest species. However, in addition to their detrimental effects on harmful species, these essential oils exert varying degrees of impact on non-target species with which they share ecological relationships. This review serves the dual purpose of updating data on the use of the essential oils as biopesticides, considering the substantial increase in research output in the recent years. Simultaneously, it aims to provide a focused synthesis on the essential oils currently approved for commercial use as biopesticides, and shedding light on their impact on beneficial pollinator species, which may inadvertently suffer from the application of substances deemed safe by most users.

1. Introduction

Approximately one third of the planet’s land surface is represented by agroecosystems—the most extensive surface (percentage) is in Asia (54%) and the smallest in Europe (21%) [1]. In recent decades, a growing dilemma has emerged between the imperative to augment food production for the ever-expanding global population and the need to protect the environment, increasingly impacted by the repercussions of the conventional agricultural practices that have become more pronounced. Despite its environmentally sustainable attributes, organic agriculture faces a notable drawback: reduced productivity in comparison to conventional approaches. Achieving an equivalent production necessitates nearly twice the agricultural land area [2].

Although the general perception of the organic agriculture is that it brings only benefits, research has shown that it is necessary to adjust our expectations regarding it [1].

Beyond the constrained food yield resulting from the diminishing availability of the agricultural land due to factors like desertification, soil degradation, and urbanization, a substantial portion of the harvest is lost annually due to a variety of pest species. Among these pests, the arthropods constitute a significant category responsible for considerable damage. However, the relationship between arthropods and crop plants is intricate [3]. In addition to the pest species, there are numerous beneficial ones, such as pollinators, parasites, parasitoids, and predatory insects, whose well-being should not be compromised by the control measures intended for the aforementioned pests [4]. Preserving the pollinator populations is crucial for the regular progression of the biological cycle in traditionally cultivated plants [2].

The essential oils produced by plants have always played an important role in their interaction with harmful and useful arthropods; their structure has been perfected over millions of years of evolution to remove certain unwanted creatures but also to attract the useful ones. Essential oils extracted from higher plants’ organs predominantly contain terpenes—monoterpenes (C10), sesquiterpenes (C15), terpenoids, and phenylpropanoids [5]; in addition to these, we can also find alcohols, acids, aldehydes, acyclic esters, or lactones [6].

The development of biopesticides, which are substances found in some plants, animals or microorganisms [7], and some minerals [8], in general, as well as those based on essential oils, in particular, has given farmers greater hope that they will be able to solve a large part of the problems caused by the pests without significantly affecting the environment. Plants contain numerous secondary metabolites that can be used as biopesticides [9]; the main advantages of using biopesticides in combating harmful species in agriculture reside in their low persistence in the environment [10] and the fact that they are biodegradable, allowing pest control without polluting the environment.

Both conventional pesticides and biopesticides are double-edged weapons introduced by humans into agroecosystems to protect the crops. However, their effects cannot be directed 100% at only one target. This highlights the importance of educating users to use them with a clear understanding of their effects, always considering the benefit to damage ratio.

Despite the considerable amount of research conducted on the use of essential oils from various plant species as biopesticides [4,10,11] only a relatively small number of essential oils have obtained commercial approval. The focus of this review is on those essential oils that constitute at least one of the components of a commercially available biopesticide.

The most used essential oils as biopesticides that have insecticidal/miticidal activity are citronella oil, lemongrass oil, clove oil, thyme oil, mint oil, cinnamon oil, rosemary oil, sweet orange oil, eucalypt oil, and oregano oil [12]. While all of the biopesticide manufacturers assert the reduced toxicity of their products to the non-target species, it is imperative to investigate their actual effects on the non-target organisms on a case-by-case basis.

As we mentioned, the number of studies on biopesticides (in particular, those based on essential oils) have increased significantly over the last 5 years; although there is an important number of reviews on this topic, none of them address, with up-to-date information, the essential oils that are actually marketed to be used as biopesticides. Isman (2020) [13] lists and describes six insecticides/miticides based on essential oils extracted from plants.

The primary goals of this review include the following: (i) the identification of trends in the specialized literature regarding the use of essential oils as biopesticides and their effects on target and non-target species through bibliometric analysis; (ii) a comprehensive review of the essential oils marketed as biopesticides, registered at the level of the U.S. Environmental Protection Agency (EPA) and/or which are included in the EU Pesticides Database; (iii) a summary of the research carried out up to the present moment regarding the action of essential oils marketed as biopesticides on non-target species.

2. Materials and Methods

In order to carry out this study, an extensive investigation of the databases containing specialized papers was carried out.

The bibliometric analysis was carried out using the programs VOS viewer (Leiden University, The Netherlands) (version 1.6.20), available at https://www.vosviewer.com accessed on 7 November 2023, and Bibliometrix (University of Naples Federico II, Italy) (version 4.3.1), available at https://www.bibliometrix.org/home/index.php (accessed on 17 October 2023) [14].

In order to collect the necessary data, the Web of Science database was accessed on 17 October 2023 with the aim of finding out what is the trend of the investigations carried out by researchers regarding the effect of the essential oils (used as biopesticides) on target species but also on non-target species.

The following search settings were used. Search in: Web of Science Core Collection; category: documents; publication date: all years (1975–2023); search terms in all fields: for Group 1—“pest” AND “biopesticides” AND “essential oil”, for Group 2—“non target” AND “biopesticides” AND “essential oil”. We opted to conduct the searches within two distinct groups: one focusing on the papers exploring the effects of the essential oils used as biopesticides on the pest species and another specifically addressing their impacts on the non-target species. In Group 1, 316 papers were initially identified—of which 3 were excluded (1 retracted article and 2 articles relating to the composition of the essential oils). In Group 2, 70 papers were initially identified—of these, 1 was excluded because it referred to silver nanoparticles.

Subsequently, papers specifically related to the 12 essential oils chosen and marketed as biopesticides were selected from the collected literature. To ensure the comprehensive coverage of information, the targeted searches were conducted on Web of Science, Scopus, and Google Scholar. The keywords used included “lemongrass oil”, “citronella oil”, “clove oil”, “thyme oil”, “mint oil”, “cinnamon oil”, “rosemary oil”, “oregano oil”, “sweet orange oil”, “eucalyptus oil”, and “tea tree oil”, in combination with terms such as “biopesticides”, “insects”, “pests”, “pollinators”, and “non-target”. This approach aimed to gather thorough and specific insights for the presented information.

3. Results

3.1. Bibliometric Analysis Regarding the Effect of the Essential Oils as Biopesticides

Numerous studies have focused on examining the impact of the essential oils, either currently utilized or with the potential for use as biopesticides. In Group 1, 313 papers were chosen, while Group 2 comprised 69 selected papers.

Concerning the publication timeframe of the chosen studies in both groups, 62.61% of the papers in Group 1 and 65.21% in Group 2 were released in the past four years (2020–2023); also, for Group 2, the first paper that appears in Web of Science is from 2014. This trend underscores a significant surge in interest toward these subjects in recent times (Figure 1).

Most papers in Group 1 focus on the effects of the essential oils on the target species, exploring lethal doses or inhibitory concentrations estimated through various administration methods (such as direct contact, fumigation, ingestion, and direct application on the insect’s body). Fewer papers delve into the mechanisms of action of the essential oils and how they exert their toxic effects on harmful species.

Unfortunately, very few studies address the issue of the toxicity of essential oils on non-target species from the group of useful insects (Group 2); if there are studies on non-target species, they rather refer to mammals and humans. The rarity of these studies (despite their necessity for the inclusion of essential oils in the commercial circuit as biopesticides) [15] is more due to the general perception that biopesticides generally have low toxicity for the environment and for the living species not targeted by them, rather than the low interest in useful insect species. The vast majority of papers regarding the toxic effects on useful insects, especially pollinators, target honeybees, and they are made at the time of testing the effectiveness of oils on their main parasite—Varroa destructor Anderson & Trueman (Arachnida: Varroidae).

The structure of entries by document type in the two groups was as follows: Group 1—articles 227 (72.523%), review articles 62 (19.808%), book chapters 8 (2.555%), proceeding papers 8 (2.555%), early access 7 (2.236%), and editorial material 1 (0.32%); Group 2—articles 52 (75.362%), review articles 14 (20.289%), proceeding papers 2 (2.899%), and early access 1 (1.449%) (source: Web of Science™, Analyze Results).

The most relevant sources (top 5) for Group 1 were Plants-Basel, International Journal of Tropical Insect Science, Industrial Crops and Products, Journal of Pest Science, Pesticide Biochemistry and Physiology (22.04% of total publications; 69 journals), and for Group 2, Environmental Science and Pollution Research, Ecotoxicology and Environmental Safety, Industrial Crops And Products, Plants-Basel, and Biocatalysis and Agricultural Biotechnology (36.23% of total publications; 25 journals). These results show a low concentration of papers in only a few journals, their spread being quite extensive (Figure 2).

For Group 1, the topmost locally cited sources were from Industrial Crops and Products (800), Pest Management Science (587), and Journal of Agricultural and Food Chemistry (473), and for Group 2, Industrial Crops and Products (254), Parasitology Research (244), and Pest Management Science (111) (source: Bibliometrix).

Keyword analysis allows us to observe the trend of research carried out in the investigated field. The full counting method (VOS Viewer 1.6.20 for Microsoft Windows systems) was used to generate a network visualization map of the main words from the text and abstract (Figure 3). The circle’s size indicates the frequency of the term’s occurrence in articles. The closeness of the two interconnected terms signifies their interrelation, determined by the frequency of their co-occurrences. Keywords exhibiting closer proximity were grouped into the same cluster.

The results obtained in Group 1 were the following: out of 9193 terms with at least 25 occurrences/terms (n = 25), 67 met the conditions; among these, 36 items grouped in three clusters were selected. Cluster 1 (red) contains 14 items, the most relevant of which are “essential oil”, “chemical composition”, “insecticidal activity”, and “exposure”; it is focused on the use of insecticidal use of essential oils, emphasizing at the same time their chemical composition. Cluster 2 (green) contains 14 items, of which “biopesticide”, “environment”, “pesticides”, and “treatment” appear more frequently; this predominantly refers to pest management and the impact of these treatments on the environment and human health. Cluster 3 (blue) contains 8 items that refer, in particular, to components of essential oils, “carvacrol”, “cineole”, “linalool”, and “thymol” and their effect on insects (other keywords are represented by “lepidoptera” and “larvae”).

The results obtained in Group 2 were the following: out of 2631 terms with at least 10 occurrences/terms (n = 10), 41 met the conditions; among them, 35 items grouped in three clusters were selected.

Cluster 1 (red) contains 13 items, of which the most frequent are “non-target species”, “control”, and “toxicity”; it groups papers that study the effect of essential oils on non-target species in relation to their use, especially as a mosquito repellent (other keywords being “aegypti”, “dengue”, and “mosquito”). Cluster 2 (green) contains 11 items, the most important being “essential oil”, “compound”, and “treatment”, and Cluster 3 (blue) contains 11 items: “biopesticides”, “effect”, and “non-target organisms”. Noteworthy is the presence of the 2 keywords “non-target species” and “non-target organisms” in clusters 1 and 3, with this fact underlining its power and interrelationships.

Figure 4 depicts a word cloud illustrating the 30 most frequently used “Keywords Plus” (provided by Web of Science). In this representation, the size of each keyword corresponds to its frequency.

In order to have a clearer picture of the connection between the data presented above, we employed thematic evolution analysis and a three-field plot using Bibliometrix software [16].

According to the thematic evolution analysis (Sankey diagram) (Figure 5) for Group 1, we observed that the major themes addressed by researchers varied during the analyzed periods. “Toxicity” was the preferred theme in the period 2005–2018. During this time, research focused on the antimicrobial and antifungal action of the essential oils, as well as on the repellent activity on some species of mosquitoes that transmit various diseases. Very few papers questioned the effective use of essential oils in combating pests in agroecosystems.

From 2018, the number of papers per year started to increase significantly, and new terms, such as “insecticidal activity” and “lepidoptera,” began to gain relevance in research. In the last three years, more attention has been paid to the investigations regarding the “chemical composition” of essential oils. This shift is natural considering the significant compositional variability, which determines varied effects even for the same essential oil derived from plants grown under different conditions.

For Group 2, the number of keywords is reduced, proportional to the smaller number of selected papers. The term “non-target” appears constantly in the analyzed periods (with the exception of the years 2019–2020). The keywords “lethal” and “effects” appear in the period 2019–2020, and in the last year, the word “formulation” stands out in correlation with the increase in the number of papers investigating the effect of nanostructured essential oils. The plot is particularly useful for understanding how the emphasis on specific terms changes across different temporal slices of the literature.

The three-field plot analysis from Bibliometrix allows us to have a general, focused picture of the main investigated aspects; in this case, we correlated, for each of the two groups separately, abstracts (in the middle), the sources—journals (left), and the countries of origin of the authors (right) (n = 15) (Figure 6). The vertical dimension of the rectangular nodes corresponds to the frequency of a particular country, institution, or journal in the collaboration network, while the horizontal dimension of the connecting lines is proportional to the number of connections.

In Group 1, the strongest links in the data flow are between the main terms “essential”, “control”, “compounds”, and “toxicity”, and the main countries with scientific production related to the effects of essential oils as biopesticides are Brazil, India, and the USA.

For Group 2, the main terms “non-target”, “effect”, and “essential” are related to the main countries providing research results on this topic—India, Italy, and Spain.

It is noteworthy that Saudi Arabia, which did not feature in the top 15 in the analysis conducted in Group 1 but now appears in Group 2, positioned comparably to China, Brazil, and the USA—countries with a well-established tradition in the biopesticide research.

Bibliometric analyses reveal a significant disparity, at least up to the present moment, between the research focused on the pesticidal properties of the essential oils and their practical utilization in commercially available preparations reaching farmers for use in agriculture.

3.2. The Main Essential Oils Currently Used as Biopesticides

1.

Citronella oil is extracted from Cymbopogon nardus L. and Cymbopogon winterianus Jowitt ex Bor [17,18]. PC Code 021901 (First Registration 1965: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is not approved for use in the EU (EU Pesticides Database); Expiry of Approval: 31 August 2022. https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/start/screen/active-substances [20] (accessed on 13 November 2023).

Although it is not among the plant families known for the species producing essential oils, in the Poaceae family, there is a group of aromatic plants that belong to the genera Cymbopogon [21] and Chrysopogon [22].

The main components of the essential oil extracted from Cymbopogon nardus and C. winterianus are citronellal, citronellol, and geraniol, in variable proportions, together exceeding 60% [21,23], depending on the variety analyzed [21], by methods of extraction and geographical distribution (Kaur, 2021) [23]; other component elements are camphene, elemol, nerol, linalool, limonene, pinene, and borneol [18,24].

The effects of citronella oil are multiple, it being used both in the treatment of various diseases in human medicine (having antioxidant, anti-inflammatory, antifungal, and antibacterial action) [23,24] and in organic agriculture, due to its insecticidal and herbicidal properties.

Geraniol and cironellol are part of a biopesticide with the trade name BIOMITE™ (Natural Plant Protection, Nogueres, France), which is used to control mites in agricultural crops [25].

The insecticidal effect was shown by Papulwar et al. [26]; a concentration of 500 μg/L determined a 100% mortality of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae after 120 h. Also, the effect of citronella oil (especially the version encapsulated in nanoparticles) on the cotton leaf worm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) was analyzed by Ibrahim et al. [27]; the insecticidal properties were thus amplified compared to the non-encapsulated oil variant.

Mortality among Apis mellifera L. (Hymenoptera: Apidae) adults was 60% higher than that of the control group when they were fed sugar syrup mixed with this biopesticide; instead, the larvae did not suffer [28]. The body mass of the adults decreased significantly under the conditions of this experiment, despite bees’ avoidance of food sources contaminated with essential oil. The social bee, Tetragonisca angustula (Latreille) (Hymenoptera: Apidae), which fulfills an important role in the pollination of plant species in agroecosystems, is sensitive to citronella oil; it showed a strong repellent effect at a concentration of 10 mL/L [29].

The repellent impact of citronella oil on honeybees has been recognized for an extended period of time; initially, it was employed for its repellent properties against mosquitoes [30,31]. Due to these properties that extend to broader groups of insects, it has been utilized to deter bees from food sources represented by sweet substances resulting from human activities, for instance, in candy factories, animal farms (where by-products from sugar cane are used) [32], gardens, and parks, where their presence is not desired in proximity to people. It is also employed to keep them at a distance from crops that have been sprayed with conventional pesticides which are lethal for bees [33].

An interesting approach to the repellent effect of citronella oil involves the utilization of the plant in situ to deter pests from the crop. Siswanto and Trisawa [34] conducted experiments that yielded positive results, observing a significant reduction in the populations of Thrips sp., which attacks pepper plants, through a cropping pattern incorporating lemongrass and citronella. However, the repellent effect on pests also extended to pollinators; out of the 10 pollinator species inventoried, 8 were not found in these experimental fields (three Diptera and five Hymenoptera). Only two species of Hymenoptera pollinators were identified in the fields with citronella and lemongrass, representing 20% compared to the control fields containing only the cultivated species (pepper) [34].

The toxic effect of citronella oil on pests has been extensively investigated; thus, Caballero-Gallardo et al. [35] reported the modification of the life cycle (including pupation and pupal viability) and injuries in the midgut epithelium of Ceraeochrysa claveri (Navás) (Neuroptera: Chrysopidae) adults. Polyphagous cotton leafworm, Spodoptera littoralis, a very widespread pest whose larvae can reduce production (especially of cotton) by more than 50% through defoliation, has developed a special resistance to synthetic pesticides [36]; citronella oil has a toxic effect on this species (LD₅₀—median lethal dose—being 2664.01 mg/L) by inhibiting the enzymes involved in detoxification.

In the case of citronella oils, its application in the form of nanoemulsions significantly increased the mortality of Spodoptera littoralis larvae [37]: at concentrations reduced by half, the nanoemulsified oil was 12–18% more efficient than bulk oil.

2.

Lemongrass oil is extracted from Cymbopogon flexuosus (Nees ex Steud.) Will.Watson and Cymbopogon citratus (DC.) Stapf PC Code (Pesticide Chemical Code) 040502 (First Registration 1972: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is not approved for use in the EU (EU Pesticides Database) [20].

The main components of lemongrass oil are citral (geranial and neral), β-myrcene, and citronellal [38,39].

The site of volatile oil secretion is represented by oleiferous cells (idioblasts) located in the mesophyll [40]. The oil is found in greater quantity in young leaves and has a richer composition in citral (considered an indicator of superior quality) [41].

The impact of lemongrass oil on various pests has been extensively investigated, while research on the non-target species remains limited. It has been proven to be an effective insecticide against numerous insects that cause damage to the agricultural crops. Lemongrass oil exhibits a strong insecticidal effect on the black cutworm Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), increasing mortality and the developmental duration, as well as the expression level of catalase and lipid peroxidase; at the same time, the treatment shows an inhibition of carboxylesterase and glutathione S-transferase [38]. Another harmful lepidopteran, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), recognized as a key devastating pest of tomatoes, demonstrates increased sensitivity, particularly in the larval stage, to lemongrass oil administered through both direct contact and fumigation routes [39]. The LC₅₀ (lethal concentration, 50%) was recorded at only 0.328 µL/mL, while the LC₉₀ was 1.643 µL/mL.

Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae), one of the key storage pests causing harm to cowpea, is affected by lemongrass oil. This impact results in a decrease in the number of eggs laid and in emergence, a reduction in lipid content in the bodies of adult females, alterations in sexual behavior, and a decrease in the total activity of acetylcholinesterase [42]. On Asian longhorned tick, Haemaphysalis longicornis Neumann (Arachnida: Ixodidae), the acaricidal effect was more pronounced on larvae and nymphs and less on adults. This led to morphological and anatomical changes, including the constriction and shrinking of the midgut, obturation of aeropyles, cuticular disruptions, alterations in sensilla (disjointed from sockets, with irregular spots at the base), and cracks on the cuticular surface, among others [43]. The authors attribute the toxic effect of the oil to anoxic asphyxiation rather than neurotoxicity, which typically occurs predominantly when arthropods interact with essential oils.

Tested on honeybees and its main pest, Varroa destructor, lemongrass oil showed a miticidal effect on the parasite but proved to be safe on the pollinating insect [44]. This is due to the fact that the parasite, Varroa destructor, is sensitive to a much lower dose of Cymbopogon nardus essential oil (LC₅₀ = 7.45 μL/mL vs. 13.11 μL/mL, at 24 h) [45]. We consider the doses tested at 24 h more relevant than those at 48 h because the persistence in the environment of volatile oils administered as such (non-encapsulated) is quite low. No increases were observed in the relative expression of the acetylcholinesterase gene in honeybees after treatment with this essential oil [44].

Lemongrass oil showed an inhibitory effect on Trichogramma pretiosum (Riley) (Hymenoptera: Trichogrammatidae), a very useful insect and the most widely commercialized parasitoid employed in global biological pest control [46]. The mortality rate in T. pretiosum adults was 32.2% at a minimum concentration of 0.01%; also, the parasitism rate against the eggs of Anagasta kuehniella Zeller (Lepidoptera: Pyralidae) decreased to 8% when a concentration of 0.1% was used (although lepidopteran adults had 100% emergence) [46].

3.

Clove oil is extracted from Syzygium aromaticum (L.) Merr. & L.M.Perry (Myrtaceae) (syn Eugenia caryophyllata Thunb.); the main constituent is eugenol; PC Code 102701 (First Registration 1983: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. Clove oil and eugenol are approved for use in the EU (EU Pesticides Database) [20].

It is included in the composition of the biopesticide Pest Out^® (JH BIOTECH, INC., Ventura, CA, USA) (which contains cottonseed oil 40%, clove oil 20%, and garlic oil 10%) [47].

Clove oil contains over 30 component elements, the most important of which are eugenol (at least 50%, but it can also reach 86.7%) [48], eugenyl acetate, β-caryophyllene, and α-humulene [49].

The miticidal action of clove oil was also demonstrated on the species Varroa jacobsoni Oudemans (Arachnida: Varroidae); at a concentration of 1 mg/cage, it induced 100% mortality at 48 h on the parasite but showed a slightly toxic effect on the honeybee (Apis mellifera), where the mortality was only 3% [50]. But in this case, the mortality on the parasitic species was largely attributed to the synergistic effect of the components of the oil; eugenol, tested separately, caused a pest mortality of only 42% at 48 h (at a concentration of 0.62 mg/cage) [50].

The weak toxic effect of the clove oil on honeybees was also confirmed by the studies of other authors; Majeed Kadhim et al. [51] observed similar mortality rates in the control variant when the oil was applied at a concentration of 10 mg/mL. Only quantities of 20 and 40 mg/mL induced higher mortality in bees, reaching 4% and 6.58%, respectively, compared to 2.83% in the control variant (at 15 h). After 64 h, the insecticidal effect of the clove oil completely disappeared, with bee mortality values returning to levels similar to those of the control version, regardless of the concentration used.

Clove oil, however, demonstrated a significant insecticidal effect on various species of harmful insects; 2.0 mL/cm² oil determined 100% mortality at 120 h after the treatment on Blatella germanica L. (Blattodea: Ectobiidae), while the mortality rate at the lowest concentration (0.25 mL/cm²) of clove bud oil reached 45.00% after 120 h [52]. Eugenol and eugenol acetate were tested under the same experimental conditions and separately; at low concentrations (0.25 mL/cm²), eugenol acetate proved to have a much higher toxic potential than that of eugenol, but at high concentrations (4 mL/cm²), the situation reversed, with eugenol having a greater toxic effect.

Also, the oil had a larvicidal effect on Pericallia ricini (Fabricius) (Lepidoptera: Erebidae), manifesting itself through larval mortality, delayed development duration, formation of non-viable individuals, and appearance of abnormal adults, whose general emergence was inhibited [53]. It also had a larvicidal effect on the species Culex pipiens L. (Diptera: Culicidae), especially in the version encapsulated with chitosan nanoparticles, where the LC₅₀ was 20 ppm, compared to bulk oil where the LC₅₀ was 39 ppm [54]. Acetylcholinesterase activity decreased significantly both in the case of larvae treated with encapsulated oil and with bulk oil, while enzymes with a role in defense (acid phosphatase, alkaline phosphatase, and glutathione-S-transferase) increased.

Clove oil showed high toxicity on the date palm mite, Phyllotetranychus egypticus Sayed (Arachnida: Tenuipalpidae), reducing the number of parasites on the leaves by up to 90% in the autumn season in Egypt (Giza) [55]. But the concentration used experimentally was very high—150 mL of oil/1 L of water.

Insecticidal effects have also been reported for Callosobruchus maculatus, a widespread pest of stored peas and beans [56], Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), Rhyzopertha dominica (Fabricius) (Coleoptera, Bostrichidae), an invasive cereal grains pest [57], etc.

4.

Thyme oil usually originates from Thymus vulgaris L., but it can also be extracted from other species of the genus Thymus, as T. pectinatus Fisch. & C.A.Mey., and T. capitatus (L.) Hoffmanns. & Link [58]. PC Code 597800 (First Registration 2004: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. Thyme oil is not approved for use in the EU, but its constituent, thymol, is approved (EU Pesticides Database) [20].

It is one of the components of the biopesticide Thymox^® used against gray mold Botrytis cinerea Pers. (Fungi: Sclerotiniaceae), and fire blight Erwinia amylovora (Burrill) Winslow et al. (Bacteria: Erwiniaceae) (Laboratoire M2, Quebec, QC, Canada); it is also found in Thymovar^® (Andermat BioVet, Grossdietwil, Switzerland), and together with eucalyptus oil, levomenthol, and camphor in ApiLife Var^® (Chemicals Laif SPA, Vigonza, Italy), it is used to combat Varroa destructor on Apis mellifera.

Thyme oil comprises more than 70 elements, with thymol being the predominant compound, constituting approximately 56–60%, according to most studies. In addition to thymol, it contains varying concentrations of p-Cymene, γ-terpinene, α-pinene, caryophyllene, and other components, the proportions of which depend on the oil’s origin and environmental conditions [59].

The insecticidal effect of thyme oil has been demonstrated on numerous harmful species. For instance, it has shown efficacy against the horn weevil Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) in both raw and encapsulated forms [60]. Additionally, it has proven effective against the carmine spider mite Tetranychus cinnabarinus (Boisduval) (Arachnida: Tetranychidae) [61] and Pochazia shantungensis Chou & Lu (Hemiptera: Ricaniidae), an invasive species recently categorized as a pest (LD₅₀ = 57.48 mg/L, 75.80 mg/L on nymphs and adults, respectively) [62] (Park et al., 2017).

In minute amounts (0.16 ppb), thyme oil stimulates the immune system in honeybees, concurrently reducing the levels of viral infection during experimental contamination with deformed wing virus and flock house virus [63].

The application of thyme oil through fogging significantly increased the mortality of caged bees compared to the control group in an laboratory study [64]. However, Mohammed and Fhad [65] observed insignificant mortality in bee hives when treated with thyme oil administered by fumigation or in the form of bulk oil. The LD₅₀ for thyme oil was only 0.79 µL/cage when using the complete exposure method, while that of thymol was 1.73 µL/cage [66].

In the case of the oil extracted from Thymus kotschyanus, the LC₅₀ for bees (Apis mellifera) was five times higher than the one required for Varroa destructor, specifically 5.08 µL/L air compared to 1.07 µL/L air. This suggests a low degree of toxicity for bees, making it a recommended option for combating Varroa infestation. In contrast, the essential oil from Thymus pulegioides L. exhibited very high toxicity (100%) on A. mellifera at a concentration of 2 µL/L air [58].

The essential oil extracted from Thymus schimperi Ronniger exhibited selective toxicity against V. destructor, approximately 91 times higher than that necessary for Apis mellifera and 27 times higher than for Apis cerana Fabricius [67]. In contrast, the main component of the essential oil, thymol, when tested in its pure form, showed selective toxicity of only 6.5 against A. mellifera and 4.4 against A. cerana. This highlights the synergistic effect of the components of the volatile oil in exerting the miticidal effect.

5.

Mint oil—the main species from which this oil is extracted are spearmint (Mentha spicata L.), peppermint (M. × piperita L.), and corn mint (M. canadensis L.) (Lamiaceae), the last being cultivated exclusively for obtaining the essential oil [6]. PC Code 128800 (First Registration 2000: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. Only spearmint oil is approved for use in the EU (EU Pesticides Database) [20]; peppermint oil is not approved.

The composition of mint oil varies depending on the species. For example, in Mentha × piperita, the oil composition reported by Da Silva et al. [68] includes menthol (55%), menthone (25%), and menthyl acetate (10%). Mentha spicata oil is predominantly composed of carvone (59.6%), limonene (25.59%), and m-cymene (2.77%) [69]. Meanwhile, Mentha canadensis oil’s main components are menthol (19.01%), limonene (18.94%), and l-menthone (10.74%) [70].

Mint oil has demonstrated effectiveness as an insecticide against the olive fruit fly Bactrocera oleae (Rossi) (Diptera: Tephritidae), a pest responsible for significant production losses in olive groves [71]. Additionally, it has shown efficacy against the green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) in early nymphal instars. Application at a 10% concentration resulted in mortality ranging between 85.12% and 88% after 1–3 days [72].

Mentha piperita oil demonstrated remarkable efficacy by causing 100% mortality in Tuta absoluta, a highly destructive pest of tomatoes. The LC₅₀ was observed at a concentration of 1.78 μL/mL [73].

Mint oil, specifically from Mentha suaveolens subsp. timija (Briq.) Harley, has proven to be effective against the Varroa destructor pest. The acaricidal effect of this oil is evident, with an LD₅₀ value of 3.360 mL/L air and an LD90 value of 7.300 mL/L air. Furthermore, its efficacy is significantly enhanced by more than 50% when used in combination with the essential oils from Chenopodium ambrosioides L. (Amaranthaceae) and Laurus nobilis L. (Lauraceae) [74].

Mentha canadensis essential oil exhibits an insecticidal effect on Piophila casei (L.) (Diptera: Piophilidae), inhibiting acetylcholinesterase activity [70].

However, it is essential to point out that not all volatile oils exhibit a more potent insecticidal effect on pests compared to their individual components. Prasannakumar et al. [73] discovered that mint oil demonstrated a lower insecticidal effect when compared to its components levomenthol and α-pinene. Interestingly, these individual components showed considerably greater toxicity when tested independently.

Mint oil has been tested on honeybees in various forms, using different application procedures. The encapsulated variant of the oil (from Mentha piperita) demonstrated a high toxicity on worker bees, and the LC₅₀ value in oral treatment was 2629.85 ppm, having an almost double value in the contact treatment (4246.84 ppm). On the other hand, the crude oil showed an LC₅₀ of 5471.13 ppm in oral treatment and 11,895.65 ppm in contact treatment [75]. The topical application of peppermint oil directly on the body of the insect exhibited a more pronounced toxic effect compared to application through contact with a treated surface, as reported by Da Silva et al. [68]. The differences were notable, particularly concerning LC₉₀ values (17.24% vs. 25.5%). In a test scenario where the peppermint oil was applied directly in test cages at a concentration of 12 mL/cage, bee mortality reached 32% after 3 days, a significant increase compared to the 4% mortality observed in the control group. This heightened mortality rate raises caution regarding the oil’s use, despite its effective performance against the target species, wax moth larvae Galleria mellonlla (L.) (Lepidoptera: Pyralidae), where mortality reached 100% under the same conditions [76].

The essential oil of Mentha spicata, at a concentration of 2%, resulted in only a 2% mortality rate for bees under laboratory conditions, compared to a 58% mortality rate for Varroa destructor [77]. Another study found that when Mentha longifolia (L.) Huds. oil was applied at a rate of 5.5 μL/L of air, it caused a mortality rate of 65.53% among Varroa mites and 10.13% among honeybees after a 10 h exposure period [78].

The supplementary feeding of honeybees with sugar syrup to which mint essential oil was added at a concentration of 0.1 mL/L resulted in an increased honey production per hive by stimulating the feeding process (32.16 kg honey per honeybee colony compared to 25.09 kg in the control variant) [79].

6.

Cinnamon oil—the main biologically active substances are cinnamaldehyde and eugenol. For cinnamaldehyde PC Code is 040506 (First Registration in 1994: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. Cinnamon oil is not approved for use in the EU, but its constituent, cinnamaldehyde, is pending for approval (EU Pesticides Database) [20].

Cinnamon oil is extracted from the bark of Cinnamomum species; true tree cinnamon, also called Ceylon or Sri Lankan cinnamon, is the species Cinnamomum verum J.Presl (Lauraceae), synonymous with C. zeylanicum Blume, but a large part of the cinnamon on the market comes from Chinese cinnamon or cassia (Cinnamomum cassia (L.) J.Presl) [80].

The main components of the oil extracted from the stem bark of C. verum are cinnamaldehyde (60 to 80%), cinnamyl acetate, camphor, eugenol, linalool, and pinene [80], while the oil extracted from the leaves contains eugenol (76.60%), linalool, piperitone, and cinnamyl acetate [81]. The bark of C. cassia contains a greater concentration of cinnamaldehyde than C. verum [81].

It is included in the commercial biopesticides Akabrown^® (Green Corp Biorganiks, Saltillo, Mexico) (contains 1.25% cinnamon oil, 1.0% peppermint oil, 0.5% clove oil, and 0.25% oregano oil) and Eko Postforte Silver 23.5 OD (Agrointesa Internacional S.R.L., Santiago, Dominican Republic) (Clove Extract 23.5%).

The insecticidal effects of cinnamon oil have been studied on pest species from different taxonomic groups. It exhibited a moderate effect on the building termite, Odontotermes assamensis Holmgren (Isoptera: Termitidae) [82], with mortality reaching approximately 50% at a dose of 2.5 mg/g after 8 days. In contrast, Subekti and Saniaturrohmah [83] found that the oil extracted from Cinnamomum osmophloeum Kaneh, at a concentration of 10%, induced 100% mortality in the fungus-growing termite Macrotermes gilvus (Blattodea: Termitidae). It is worth noting that the oil concentration tested in the second study was almost 40 times higher, highlighting the importance of interpreting insecticidal effects of essential oils in accordance with the specific experimental conditions employed.

Against Callosobruchus maculatus, the LC₅₀ of the cinnamon oil was 131 μL/kg [84]. Experimental studies in the laboratory have shown that even a dose of LC₂₀ is sufficient to produce a significant decline in the number of pests and to reduce bean mass losses.

Physiological (respiratory rate) and behavioral (locomotory) responses induced by cinnamon essential oils were investigated by Gonzales Correa et al. [85] on maize weevil, Sitophilus zeamais; the population of insects resistant to phosphine and pyrethroids also showed resistance to treatment with the essential oil, while the population susceptible to these two insecticides also showed sensitivity to cinnamon oil. The LD₅₀ for this species was set at 0.02 mg/cm² [86].

Both the oil from the bark and from the green leaves of cinnamon (at a concentration of 0.5%) induced a mortality rate of 82.3% and 82.9%, respectively, on the citrus flatid planthopper Metcalfa pruinosa (Say) (Hemiptera: Flatidae) [87].

Cinnamon oil shows a fairly high degree of toxicity to honeybees (A. mellifera) [66]. In tests against Ascosphaera apis (Maasen ex Claussen) Olive et Spiltoir (a fungal pathogen that invades the honeybees), this oil manifested a selectivity ratio (SR) close to 1 (where SR = LD₅₀ A. mellifera/MIC₅₀ A. apis). This means that a minimum inhibitory dose for the pathogen is almost equal to the lethal dose of substances that resulted in a 50% mortality rate in honeybees.

The high toxicity of cinnamon oil to honeybees was also confirmed by Hýbl et al. [88]; in a parallel study on the parasite Varroa destructor and its host, Apis mellifera, the oil presented a reduced selectivity ratio of 2.461 at 4 h and 2.96 at 48 h, this increasing only at 72 h to 4.542.

7.

Rosemary oil—from Salvia rosmarinus Spenn. (Rosmarinus officinalis L.) (Lamiaceae); PC Code 597700 (First Registration 1998: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is not approved for use in the EU (EU Pesticides Database) [20].

The essential oil’s primary constituent elements exhibit significant variations based on the plant’s collection location, the climatic conditions, the organs of the plant used, and the investigated variety; for rosemary leaves collected from Taizhou, China, the following essential oil composition was reported: 1,8-cineole (26.54%), α-pinene (20.14%), camphor (12.88%), camphene (11.38%), and β-pinene (6.95%) [89]. When the oil was extracted from the aerial part (stem, leaves, and flowers), the composition of the oil for the species harvested from Konya, Turkey, was p-cymene (44.02%), linalool (20.5%), γ-terpinene (16.62%), thymol (1.81%), β-pinene (3.61%), α-pinene (2.83%), and eucalyptol (1,8-Cineole) (2.64%) [90].

It is included in the commercial biopesticides EcoTrol (KeyPlex, Winter Park, FL, USA) (10% rosemary oil), TetraCURB Max (Kemin Industries, Des Moines, IA, USA) (50% rosemary oil), and Sporan (KeyPlex, Winter Park, FL, USA) (17.6% rosemary oil) [13,91].

The pesticidal action of rosemary oil is well known. The twospotted spider mite, Tetranychus urticae Koch is a polyphagous species which attacks more than 15 species of cultivated plants [92]. Rosemary oil has proven to be effective against this harmful species, with an LC₅₀ of 13.19 mL/L (or 33.09 µg/cm²) [91]. The investigations were carried out concurrently for a host plant, tomatoes, against which the oil did not show toxicity at the administered dose. It is noteworthy that the biopesticides mentioned above, which contain rosemary oil in combination with other essential oils, were more effective than the oil administered alone. This demonstrates the synergistic action of the component elements. EcoTrol [13] contains, in addition to 10% rosemary oil, 5% geraniol, and 2% peppermint oil; for this biopesticide, the LC₅₀ was 5.51 mL/L [91].

Against Callosobruchus maculatus, the rosemary oil had an LC₅₀ of 0.9709 µL/50 mL [93]; significant increases in catalase, glutathione S-transferase, and acetylcholinesterase were also recorded.

The acaricidal effect of rosemary oil is quite low. In a study conducted by Ariana et al. [77] on both honeybees (A. mellifera) and their parasite, V. destructor, the oil exhibited significantly increased mortality in bees (4% vs. 0.7% in the control variant) at a concentration of 2%. However, the mortality compared to mites was statistically insignificant (27% vs. 14%), which does not recommend the rosemary oil in the fight against varroosis. The moderate miticidal effect of the rosemary oil was also confirmed by Hýbl et al. [88] (below 50% at a concentration of 0.075%). In this case, the effect on honeybees was not analyzed, as the oil was considered insufficiently effective for combating mites.

But rosemary oil exerts a highly toxic action on bees as well. Tests conducted with the oil obtained from the leaves of Rosmarinus officinalis collected from Buenos Aires province (Argentina) showed that when it is extracted from air-dried plant material, (27 °C) the LC₅₀ for honeybees (A. mellifera) is similar to that for V. destructor mites [94]. Only in the case of the oil extracted from oven-dried leaves at (50 °C), which also contains 18.8% camphor (this is absent in the oil extracted at 27 °C), were the differences between the LC₅₀ for honeybees and for V. destructor significant. Considering the significant variability in the components comprising rosemary volatile oil, it is essential to recognize that not all variants are safe for pollinators.

8.

Oregano oil—mainly extracted from Origanum vulgare L. (Lamiaceae); PC Code 004300 (First Registration 2011: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is not approved for use in the EU (EU Pesticides Database) [20]. Oregano extract is used in the production of the biopesticide Banaforce 21 Od. (Aspeagro Global SL, Mutxamel, Spain).

Oregano oil is extracted from different Oregano species. Analyses conducted on the oil from the aerial organs of this species collected from Santarem (Ribatejo, Portugal) revealed the following composition: carvacrol (14.5%), thymol (12.6%), β-fenchyl alcohol (12.8%), δ-terpineol (7.5%), γ-terpinene (11.6%), and α-terpinene (3.7%) [95]. Carvacrol emerged as the primary component in the oil extracted from three subspecies and varieties of O. vulgare: ssp. hirtum, var. creticum, and var. samothrake (ranging from 70.0% to 77.4%) [96]. Significant variations in the percentage of components of oregano oil (also from aerial organs of the O. vulgare species, collected from Aseer Province, southern Saudi Arabia) were observed; it contained carvacrol (61.2%), γ-terpinenes (9.6%), thymol (8.3%), and p-cymene (5.7%) [97]. In O. vulgare subsp. virens, hydrocarbon (Z)-α-bisabolene (39.17–42.64%), a sesquiterpene, was identified as the major constituent of the essential oil [98].

Confusion with Mexican oregano (Lippia graveolens Kunth), a species with a similar common name but belonging to the Verbenaceae family [99], must be avoided.

The essential oil of oregano demonstrated a strong insecticidal activity on the maize weevil, Sitophilus zeamais (more than 80% mortality), but at a rather high concentration of 20% [100]. In the case of another species of weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), the LC₅₀ was determined at a value of 3.053 µg/insect (the amount was applied directly to the thorax of the adult insect) [101].

Complete mortality (100%) was recorded when using an oil dose of 25 µL/L air for the flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) (LC₅₀ = 7.52 µL/L air), and 6 µL/l air for the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) (LC₅₀ = 4.6 µL/L air) [102]. The bean weevil, Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae), showed a special resistance to this oil, with a dose of 195 µL/l air and 144 h being necessary to obtain 100% mortality (LC₅₀ = 55.94 µL/L air) [102]. Another species of coleoptera, the yellow mealworm beetle, Tenebrio molitor, proved to be sensitive to treatment with oregano oil (LD₅₀ = 6.124 µg/insect) [101].

In a very small amount, 0.02 µL/cm², the oil showed an intense repellent action on the red flour beetle, Tribolium castaneum (Herbst)(Coleoptera, Tenebrionidae) of over 80% at 15 min, but a weak effect as low as 5% at 180 min [103], with the rapid loss of the effect indicating its heightened volatility.

The field experiments conducted by Sabahi et al. [104] showed that oregano oil administered in hives by means of an electric vaporizer (with a rate of 2.13 g/day in the first part of the experiment and up to 0.55 g/day in the second part) had a significant miticidal effect on the species V. destructor, but the effect on mortality in bee pollution was insignificant. In contrast, a mixture of 7% solution of oregano oil and clove oil, administered by evaporation from a pad soaked with 1 g of each oil, had a lower miticidal effect but determined higher mortality among bees. Considering the fact that the clove oil, administered separately, demonstrated a reduced toxicity to bees [50,51], we can conclude that in this case, the effect is generated by the synergistic action of those two oils.

9.

Sweet orange oil is extracted from Citrus × aurantium f. aurantium (L.) Osbeck (syn. Citrus sinensis); PC Code 040517 (First Registration 1974: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is approved for use in the EU (EU Pesticides Database) [20].

It is obtained from the peel of sweet Citrus × aurantium f. aurantium, which represents more than 60% of the total production of the sweet orange oil [105]. The main component of the oil from ripe fruits is limonene, constituting 95.13%. In young fruits, it is found in lower concentrations (31–53%), along with sabinene (21–34%). The latter almost disappears in mature fruits (0.12%) [105].

It is included in the commercial biopesticide Prev-AmR (Oro Agri, Fresno, CA, USA) at a concentration of 5–6% of orange oil [13]; Citrus aurantifolia extract comprises the primary component, constituting 28.7% in the composition of the biopesticide Eko Postforte 32.7 Sl (Agrointesa Internacional S.R.L., Santiago, Dominican Republic). This formulation also contains Ananas comosus (L.) Merr. extract at a concentration of 4.0%.

The insecticidal effect was manifested on the species of red imported fire ants Solenopsis invicta Buren (Hymenoptera: Formicidae). When treated with 5 mg/tube and 10 mg/tube for 12 h, the mortality rate was 95.33% and 100%, respectively [106] (Hu et al., 2017).

Acetonic extracts of fruit peels from Citrus sinensis and Citrus aurantium induced 100% mortality of Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Rhyzopertha dominica at 8.5 mg/cm² after 72 h of exposure [107].

In the form of nanoemulsion, Citrus sinensis oil exhibited an aphicidal effect on cotton aphids, Aphis gossypii [108]. Cotton aphids are a serious pest of citrus, causing damage through feeding and serving as a vector for several viruses [109]. When administered at concentrations of 4% and 6%, the oil extracted from citrus fruits from the same orchard induced a mortality of over 90% in the aphid population. Additionally, it exerted a phytotoxic effect on infested citrus trees, reducing the photosynthetic rate by 52% and 51% [108].

In concentrations ranging from 1 to 40 µL/cage, C. sinensis oil did not induce mortality in the bee population but caused moderate mortality in Varroa destructor, ranging between 16.7% and 40% [110]. The LC₅₀ for mites after 24 h was 377.00 µL/cage. The findings affirming the low toxicity to bees are further supported by the studies conducted by Souza et al. [111], wherein concentrations of 1 mg/mL and 2 mg/mL did not lead to significant mortality among them.

In experimental conditions, limonene, the main component of the essential oil, when administered individually, disrupted nutritional biochemical parameters and induced histopathological modifications in the midgut cells of the honeybees. It led to a decrease in soluble proteins, lipids, and total sugar in the bodies of the honeybees [111].

10.

Eucalyptus oil—PC Code 040503 (First Registration 1994: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is not approved for use in the EU (EU Pesticides Database) [20].

It is extracted from the leaves of Eucalyptus trees, which comprise over 800 species [112]. The primary source for extracting the essential oil is Eucalyptus globulus Labill (syn. E. globulosus) (Myrtaceae), but it can also be obtained from other species such as E. citriodora (Hook.) Hill & Johnson, E. polybractea Baker [113], E. camaldulensis Dehnh., and E. cinerea Muell. ex Benth. [114].

It is included in the biopesticide Eco-oil^® (Organic Crop Protectants, Clayton, VIC, Australia) containing a 2% blend of tea tree (Melaleuca) and eucalyptus oils [13].

The main component of the oil is eucalyptol (1.8-cineole), with its proportion varying significantly depending on the analyzed species, environmental conditions, and geographical area. For instance, in E. microtheca Muell, it represents 12.1% [115], in E. pimpiniana Maiden, it constitutes 71.6% [116], and in E. globulus from the south of Brazil, it is 77.52% [117]. In the case of E. globulus cultivated in Spain, the essential oil primarily consists of 1,8-cineole (63.1%), p-cymene (7.7%), α-pinene (7.3%), and α-limonene (6.9%) as its main constituents [118].

In some Eucalyptus species or chemotypes, substances other than eucalyptol have been reported as dominant elements in the essential oil. For example, (-)-spathulenol constituted 32.66% in E. globulus from Algeria [119] and 37.46% in E. camaldulensis [120]. Additionally, α-pinene was found to be present at 30.4% in Eucalyptus grandis Hill, while β-pinene was at 39.4% in E. tereticornis Sm. [121].

The insecticidal effect has been studied in numerous species of different orders. Significant results were obtained for the species: the lesser grain borer, Rhyzopertha dominica, with E. microtheca essential oil—LC₅₀ at 24 h being 25.261 µL/L of air and significantly decreased to 18.995 µL/L at 72 h [115]; for stored food pest Ephestia kuehniella (Lepidoptera, Pyralidea) with E. globulus oil—LC₅₀ = 0.013 and LC₉₅ = 0.081 µL/cm² [122]; for the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), with the oil of E. cinerea—LC₅₀ = 0.028 μL/insect, and LC₉₀ = 0.373 μL/insect [123]. With E. campaspe Moore and E. torquata Luehm. oils, applied topically, the mortality of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) at 24 h was 92.5 and 70 µL/cm, respectively [124]; for Tribolium confusum Jacquelin du Val (Coleoptera, Tenebrionidae), essential oil nanocapsules from E. globulus exhibited significantly higher toxicity, with an LC₅₀ of 19.030 μL/L, compared to the contact toxicity of pure oil (LC₅₀ of 3.770 μL/L) [125].

The oil of E. globulus, with a high content of 1,8-cineole (63.49–79%), harvested from two regions of Argentina (Mar del Plata and Valle de Conlara) showed different miticidal action on the species V. destructor—36% vs. 76% (at a concentration of 20 μL per cage at 72 h). The variant with a higher eucalyptol (1,8-cineole) content demonstrated significantly greater efficacy [126]. The same concentration of oil induced a low mortality rate in bees (A. mellifera) tested in parallel of only 4% at 72 h (this being from 0 to 24, respectively, at 48 h). The effect of the essential oils extracted from six species of Eucalyptus with a variable content of 1,8-cineole on Brazil cabbage caterpillars Ascia monuste (L.) (Lepidoptera: Pieridae) were moderate [127]; in this study, no directly proportional relationship was found between the mortality of larvae and the content of 1,8-cineole in the oil.

E. camaldulensis oil also showed a miticidal effect (LC₅₀ = 1.7 µL/L air) but also an insecticidal effect against Apis mellifera (LC₅₀ = 3.1 µL/L air); relative toxicities (based on LC₅₀ values) indicated that eucalyptus essential oil was only 1.8 times more toxic to mites than to bees [128]. The variant of E. camaldulensis oil tested in this experiment had an unusually high content of 1,8-cineole (74.7%), the species known for having a great variability in the essential oil constituents [129]. The authors continued the tests, conducting experiments with the same essential oil under conditions similar to those in the beehive, using wooden cages. In this case, there was a 71.06% mortality rate in Varroa mites and a 12% mortality rate in A. mellifera after a 10 h exposure period to 5.5 µL/L air [78]. However, a high content of 1,8-cineole increased the toxicity of the volatile oil not only in insects but also in other organisms, such as shrimps (Artemia salina (L.) (Crustacea: Artemiidae) [130], rats [131], etc.

11.

Tea tree oil—PC Code 028853 (First Registration 2014: Source EPA, https://www.epa.gov (accessed on 13 November 2023)) [19]. It is not approved for use in the EU (EU Pesticides Database) [20].

The oil is extracted from the leaves of Melaleuca alternifolia (Maiden & Betche) Cheel (Myrtaceae); the tea tree is a small tree native to Australia [132]. The dominant constituents are terpinen-4-ol (40.3%), γ-terpinene (11.7%), 1,8-cineole (7.0%), and p-cymene (7.0%).

It is one of the components of the biopesticide Eco-oil^® (Organic Crop Protectants, Clayton, VIC, Australia) [13].

The insecticidal effect of tea tree oil has been documented on some species harmful to agricultural crops, for example, the Mediterranean fruit fly, Ceratitis capitata, a fruit-eating pest found in subtropical and tropical regions globally [133]. The effect of the oil was tested in parallel both on the harmful insect and on the parasitoid Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae), which is a natural enemy. In ingestion trials, the essential oil administered at concentrations of 0.75%, 1.00%, and 2.00% caused a mortality of 90–100% in Ceratitis capitata, with only a 20–30% mortality in the parasitoid Psyttalia concolor. However, contact toxicity trials yielded different results, with oil concentrations of 1.50, 2.00, and 3.00 µL oil/cm² causing similar mortality in the two species. The same trend was observed after applying the oil through fumigation at concentrations of 12.00, 15.00, and 18.00 µL oil/L air [132].

On the African cotton leafworm, Spodoptera littoralis, Melaleuca alternifolia oil showed a stronger antifeeding and larval development inhibiting effect than its main components, terpinen-4-ol and γ-terpinene, tested separately [134].

The effect of tea tree oil and its components terpinen-4-ol, γ-terpinene, linalool, and eugenol was tested on the African cotton leafworm, Spodoptera littoralis (Lepidoptera: Noctuidae) [135]. In doses of 0.025 and 0.1 μg/larva, the crude oil induced 100% mortality in the first larval stages, while its separate components (with the exception of terpinen-4-ol) caused only 66.5% to 33.33% mortality. It had an insecticidal action close to that of the oil, demonstrating that it is the main component responsible for the high mortality of the African cotton leafworm.

On the species Sitophilus oryzae and S. zeamais, tea tree oil had a weak insecticidal effect at a concentration of 137 μL/L air [136].

Table 1. Effect of essential oils used as a biopesticide on pollinating insects.

Essential Oil	Species	Effect	Reference	Other Observations
Citronella oil	Apis melifera	It increases mortality in adults but not in larvae; body mass decreases.	[28]	Changes foraging activities.
		It is non-repellent for Africanized honeybees.	[137]
		It is repellent for honeybees in natural conditions.	[33]	Recommended for use together with neonicotinoid pesticides.
	Apis cerana indica	LC50 = 61.1 (µg/bee)	[138]
	Nannotrigona testaceicornis	2% mortality observed after six days of contact exposure with the oil at the recommended concentration.	[29]	No repellent effects.
	Tetragonisca angustula	75% mortality recorded after six days of contact exposure with the oil at the recommended concentration.	[29]	No repellent effects.
	Pheidole pallidula	Significantly repellent in liquid form.	[139]
Lemongrass oil	Apis melifera	Low toxicity for larvae and adults (LD50 = 12,900 and 54,844 µg/mL),	[44]
		Low toxicity for adults (LC50 = 13.11 μL/mL at 24 h).	[45]
Clove oil	Apis melifera	Low mortality—3% at 48 h at a concentration of 1 mg/cage.	[50]
		Weak insecticidal effect (20 and 40 mg/mL induced a mortality of 4 and 6.58% at 15 h).	[51]	After 64 h, the insecticidal effect is no longer registered.
		Selectivity ratio at 72 h = 2.218 (on Varroa destructor).	[88]
Thyme oil	Apis mellifera	Essential oil delivered by fogging significantly increased caged bee mortality.	[64]
		Immunostimulant and reduced levels of viral infection at a concentration of 0.16 ppb.	[63]
		LD50 was 0.79 µL/cage.	[66]
		High toxicity of 100% at A concentration of 2 μL∙L−1.	[58]	Essential oil from Thymus pulegioides.
		LC50 5.08 s µL/L air.	[128]	Oils from Thymus kotschyanus.
		LD50 = 3.3%.	[68]	Tymus vulgaris; topical exposure.
		LD50 7.9 µg/bee.	[67]	Thymus schimperi.
	Apis cerana	2946 µg/Ml.	[67]	Thymus schimperi.
Mint Oil	Apis melifera	Decrease in the amount of proteins and lipids in the insect’s body.	[75]	The negative effect is accentuated by use of the oil in the encapsulated form in nanoparticles. Mentha piperita.
		Low pollinator toxicity.	[68]	Mentha piperita.
		Slight increase in mortality of adult workers.	[76]
		Oil at concentration of 2% generated a mortality rate of only 2% in bees.	[77]	Mentha spicata.
		10.13% mortality in honeybees at 5.5 μL/L.	[78]	Mentha longifolia.
		Selectivity ratio at 72 h = 9.651 (on Varroa destructor).	[88]
	Trigona hyalinata	Low pollinator toxicity; LD50 = 21.61%.	[68]	Topical exposure; commercial oil.
Cinnamon oil	Apis melifera	High degree of toxicity.	[66]
		Selectivity ratio of 2.461 at 4 h and 2.960 at 48 h.	[88]
Rosemary oil	Apis melifera	LC50 for honeybees is similar to that for V. destructor.	[94]
		Oil at a concentration of 2% generated a mortality rate of 4% in bees (vs. 0.7% in control).	[77]
Oregano oil	Apis melifera	Selectivity ratio of 1.985 at 4 h and 5.830 at 72 h.	[88]
		Administration with electric vaporizer (with a rate of 2.13 g/day) does not induce significant mortality.	[104]
		2 µL/L induces a mortality of 100%.	[140]	O. vulgare subsp. hirtum (67.4% carvacrol).
Sweet orange oil	Apis melifera	No significant mortality at a concentration of 1 to 40 µL/cage.	[110]
		No significant mortality at a concentration of 1 mg/mL and 2 mg/mL.	[111]
		No significant mortality at a concentration of 0.5–2 mg/mL.	[141]
		No significant mortality at a concentration 1 to 40 µL/cage,	[110]
Eucalyptus oil	Apis melifera	4% mortality at a concentration of 20 μL per cage at 72 h,	[126]	Complete exposure method.
		LC50 = 3.1 µL/L air,	[128]	Fumigation; E. camaldulensis oil.
		5.5 µL/L air caused 12% mortality in honeybees after 10 h of exposure.	[78]	E. camaldulensis oil.
	Apis cerana indica	LC50 = 55.20 (µg/bee).	[134]
	Nannotrigona testaceicornis	0% mortality after six days of exposure by contact with the recommended concentration.	[29]	No repellent effects.
	Tetragonisca angustula	50% mortality after six days of exposure by contact with the recommended concentration.	[29]	No repellent effects.

summarizes the effects of the investigated essential oils on beneficial pollinating insects. While citronella, thyme, and mint oils have been moderately studied for their impact on non-target insects, studies on cinnamon and rosemary oils are scarce, and those on tea tree oil are entirely absent.

4. Discussion

Essential oils and their primary components, terpenes and terpenoids, have been extensively studied by various authors for their insecticidal and acaricidal effects, as well as their potential as antifeedants and repellents [142]. These effects, whether observed individually or in combination, depending on the context, represent various approaches through which essential oils can be employed to combat arthropods which are harmful to agriculture. However, it is crucial not to overlook the fact that numerous species, closely related to those considered harmful, are ultimately, and sometimes absolutely, necessary for agriculture. Therefore, it is anticipated that the mechanisms through which essential oils act on pests may also be effective in the case of beneficial related species.

The trend in research on the use of essential oils as biopesticides is continually evolving. In addition to the substantial number of papers published in the recent years, researchers’ interests are acquiring new significance. Consequently, it becomes increasingly compelling to investigate the side effects of biopesticides which have been considered safe for the environment and non-target organisms for several decades [143].

Until recently, many studies only investigated the effect of the essential oils on harmful species, without extending the study to non-target species as well. Typically, this occurs during the testing of oils on parasitized species, such as A. mellifera infested with Varroa sp., where side effects on the parasitized species are also examined [68,94,104]. However, other beneficial species coexisting in ecosystems with the harmful species targeted by the treatment have been largely overlooked.

Not all the essential oils currently marketed and utilized as biopesticides in agroecosystems exert the same level of harmful effects against pests. Thyme oil, mint oil, lemongrass oil, and clove oil demonstrate significant miticidal action against the Varroa species while exhibiting a reduced toxic effect toward bees [45,63,68,88]. Conversely, eucalyptus, rosemary, and cinnamon oils [88,94,128] display toxic effects on pollinators; for this reason, they must be used with caution in order not to affect useful species in agroecosystems.

Special attention must also be paid to the synergistic effect that essential oils and/or their components sometimes have; Sabahi et al. [104] observed that the combination of oregano oil and clove oil produces an increased toxic effect toward bees, although the two oils used separately showed weak toxicity toward this species.

Only in recent years has the number of studies aimed at establishing the degree to which biopesticides influence taxonomically close species to the target species increased. A volatile oil may be completely harmless to humans or other vertebrate species but may significantly interfere with the metabolism of the species of insects that belong to the same family due to the structural and functional similarities between them [4,144,145].

The method of administration of essential oils can have a major influence on toxic doses, both for harmful species and for non-target species.

Because of their elevated volatility, there has been a growing trend in recent years to suggest encapsulating the essential oils used in commercial insecticides [11,145]. This method has both advantages and disadvantages; it favors the persistence of the essential oils in the environment and the extension of their action time. But most of the toxicological tests carried out by researchers, especially in laboratory conditions but also in field conditions, had as their object the effect of oils administered, and as such (in different concentrations and by various methods), there are few studies comparing the effect of raw oil vs. encapsulated oil [4,60,146]. These studies, most of the time, revealed a considerable increase in the toxicity of the essential oils compared to the non-target, beneficial species, along with an increase in the toxic effect compared to the harmful insect species [60].

As a rule, the studies on the effect of oils encapsulated in nanoparticles were carried out on the target species, where an increase in the insecticidal effect compared to the oil used was demonstrated; however, it is necessary to expand the investigations also to non-target species [27] in order to highlight the possible negative side effects on them.

The effect of volatile oils on insects is related to their ability to penetrate the cuticle and reach their body. Encapsulation of essential oils leads to higher toxicity to insect species by increasing their water solubility [147]; thus, the procedure reduces their physicochemical limitations [144], such as poor water solubility [10], stability over time [147], etc.

The not always consistent results obtained by various authors after testing the degree of toxicity of different volatile oils on insect species are due, on the one hand, to the different work methodologies (mode of administration) but, on the other hand, to the different components of the tested oil depending on the origin. In correlation with the environmental conditions and the variety of plants used to extract the oil, its composition can vary, sometimes significantly.

Another problem that arises when translating the results obtained from scientific experiments into agricultural practice is derived from the fact that the vast majority of studies are conducted in laboratory conditions. In these conditions, the tested insects are confined in various devices without the possibility of avoiding contact with the biopesticide. This is significant because the repellent effect of the essential oils on insects has been reported by numerous authors [33,34,52], and in the natural environment, there is a possibility that they come into contact to a much lesser extent with the administered active substances.

The results obtained by various researchers who analyzed the influence of the essential oils as such or used as biopesticides both on harmful species and on useful species, affected collaterally, are not always constant, with some results being apparently contradictory. But there are a number of factors that influence the results obtained in the experiments:

• The origin of the essential oil: The oils used experimentally are obtained from very diverse sources by the researchers; in some cases, they are procured from trade or from industrial production [88]. In this case, it is not always possible to know the exact species from which the oil was extracted. Given the significant variability in oil components, which depends on factors such as species, variety, area of origin, climatic conditions, and extraction method, it is understandable to obtain different results in the experiments conducted [114,118,126].
• The method of administration of treatments with the essential oils: They can be applied by direct contact [39,75,134], by fumigation [39,65,128], in food [68], or topically [148]. Many authors have noticed significant variations in LD₅₀ depending on how the same oil was applied [68,149].
• Performing treatments in the laboratory or in the open field: Most experimental studies are conducted under laboratory conditions [58,93,145,150], with fewer directly in the field [55,65] or comparatively (in the laboratory and field) [108]. The latter emphasizes the idea that laboratory studies cannot always be extrapolated to field conditions; in order to certify a certain effect of an oil (both against harmful species and against the non-target ones, especially the useful ones), additional investigations are necessary.

5. Conclusions

Undoubtedly, essential oils as a whole have a lower negative effect on the non-target species than synthetic pesticides. However, this fact should not be overestimated until we can consider them completely harmless to the beneficial species. Although the studies on the effects of essential oils on pests are not as extensive, those conducted on beneficial insect species, especially pollinators such as bees, indicate the need for cautious use in organic agriculture. The choice of oils (or combinations of oils, considering the frequently observed synergistic effect) must be made carefully, and doses must be established to ensure minimal damage to the target species. It is recommended to conduct a pre-treatment evaluation of the risk/benefit ratio of the chosen options.

Although it is not the primary cause of the increasingly apparent decline in pollinators observed in recent decades, the essential oils used as biopesticides must be administered with caution. When they come into contact with insects, especially young ones, at certain concentrations and through various administration methods, biopesticides containing essential oils can cause significant damage to pollinator populations. Therefore, understanding the effects of various essential oils on both harmful and beneficial species can assist in choosing the most appropriate doses and the right times of administration to minimize these damages as much as possible.