[Retrospective surveys on intoxications with essential oils]. / Enquêtes sur la survenue d'intoxications après l'utilisation d'huiles essentielles, comparaison avec les données du CAP d'Angers (2017­2018).

OBJECTIVES: Non-conventional medicines are not devoid of toxicity and it is relevant to establish an inventory of the general public's knowledge of essential oils. The objective is to identify the profile of the victims of a poisoning, the ways of administration and the symptoms as well as the incriminated essential oils. METHODS: Two surveys, for the general public and health professional, were distributed (January-March 2019). In addition, data from the Angers poison control center for the period 2017-2018 were analyzed and compared with the data from our study. RESULTS: Our surveys gathered 623 and 59 answers. The data of the poison control center of Angers counted 741 intoxications. The precautions for use and contra-indications of essential oils are not well known since 5% of the respondents identified them correctly. Our data show that using a mixture increases the risk of intoxication (P<0.02). The most cited essential oils in case of intoxication are Eucalyptus, Tea tree and Lavender. The symptoms mainly concern a cutaneous application (75%) and remain of short duration and without gravity. Concerning the intoxications referenced to the poison control center in Angers, the same essential oils are involved, the oral route is mostly used (70%) and the symptoms listed for 74% of intoxications concern oropharyngeal, ocular, abdominal and skin pain. CONCLUSION: The delivery of essential oils is not harmless and the data obtained both through our surveys and the processing of data from the poison control center of Angers show that they must be used with caution.

Characterization of tea tree oil nanoemulsion and its acute and subchronic toxicity.

Tea tree oil (TTO) is a popular topical use to treat skin infections. However, its poor aqueous solubility and stability have substantially limited its widespread application, including oral administration that might be therapeutic for enteric infections. In this study, mechanical ultrasonic methods were used to prepare TTO nanoemulsion (nanoTTO) with a mean droplet diameter of 161.80 nm ± 3.97, polydispersity index of 0.21 ± 0.01, and zeta potential of -12.33 ± 0.72 mV. The potential toxicity of nanoTTO was assessed by studying the oral median lethal dose (LD50) and repeated 28-day oral toxicity to provide a reference for in vivo application. Results showed that nanoTTO had no phase separation under a centrifugation test and displayed good stability during storage at -20, 4 and 25 °C over 60 days. Repeated-dose 28-day oral toxicity evaluation revealed no significant effects on growth and behavior. Assessments of hematology, clinical biochemistry, and histopathology indicated no obvious adverse effects in mice at 50, 100 and 200 mg/mL. These data suggest that nanoTTO can be considered a potential antimicrobial agent by oral administration due to its inhibitory effect on bacteria and relatively lower toxicity.

Preliminary Assessment of the Mucosal Toxicity of Tea Tree (Melaleuca alternifolia) and Rosemary (Rosmarinus officinalis) Essential Oils on Novel Porcine Uterus Models.

Antimicrobial resistance, an ever-growing global crisis, is strongly linked to the swine production industry. In previous studies, Melaleuca alternifolia and Rosmarinus officinalis essential oils have been evaluated for toxicity on porcine spermatozoa and for antimicrobial capabilities in artificial insemination doses, with the future perspective of their use as antibiotic alternatives. The aim of the present research was to develop and validate in vitro and ex vivo models of porcine uterine mucosa for the evaluation of mucosal toxicity of essential oils. The in vitro model assessed the toxicity of a wider range of concentrations of both essential oils (from 0.2 to 500 mg/mL) on sections of uterine tissue, while the ex vivo model was achieved by filling the uterine horns. The damage induced by the oils was assessed by Evans Blue (EB) permeability assay and histologically. The expression of ZO-1, a protein involved in the composition of tight junctions, was assessed through immunohistochemical and immunofluorescence analysis. The results showed that low concentrations (0.2-0.4 mg/mL) of both essential oils, already identified as non-spermicidal but still antimicrobial, did not alter the structure and permeability of the swine uterine mucosa. Overall, these findings strengthen the hypothesis of a safe use of essential oils in inseminating doses of boar to replace antibiotics.

Effect of essential oils of Mentha spicata L. and Melaleuca alternifolia Cheel on the midgut of Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae).

Agricultural pest control is a popular research topic, and essential oils are widely studied because they represent a promising alternative to synthetic insecticides. However, despite the increase in studies on pests, little work has been done on pesticide contamination of the predators feeding on insecticide-affected prey. Therefore, the objective of this study was to evaluate the effects of Lethal Dose 50 (LD50) from the essential oils of Mentha spicata L. and Melaleuca alternifolia Cheel on the histology, including histochemistry (from protein and carbohydrate analysis) and immunohistochemistry (from the evaluation of cellular apoptosis), of the midgut of fifth instar nymphs of Podisus nigrispinus (stinkbug) (Dallas). The periods of analysis were 12, 24, and 48 h after ingestion of Alabama argillacea Hübner caterpillars treated with the respective oils. The oil from M. spicata did not cause histological alterations or apoptosis in the insect. However, there was a reduction in the level of carbohydrates within the 48-h period. After 24 h, the oil of M. alternifolia caused an elongation of digestive cells and, after 48 h, cell lysis with the release of material into the lumen, suggesting tissue necrosis. The immunohistochemical study revealed no apoptotic process. There was a reduction in the neutral carbohydrate levels in the 24- and 48-h periods and in the number of regenerative cells, when compared to the control, after the period of 48 h. These results demonstrate that M. spicata oil has potential for use in cotton fields because it does not affect the vital characteristics of P. nigrispinus. However, the essential oil of M. alternifolia is not suitable for use as a pesticide because it is extremely toxic to predators.

Repellency, Toxicity, Gene Expression Profiling and In Silico Studies to Explore Insecticidal Potential of Melaleuca alternifolia Essential Oil against Myzus persicae.

In the current study, deterrent assay, contact bioassay, lethal concentration (LC) analysis and gene expression analysis were performed to reveal the repellent or insecticidal potential of M. alternifolia oil against M. persicae. M. alternifolia oil demonstrated an excellent deterrence index (0.8) at 2 g/L after 48 h. The oil demonstrated a pronounced contact mortality rate (72%) at a dose of 4 g/L after 24 h. Probit analysis was performed to estimate LC-values of M. alternifolia oil (40%) against M. persicae (LC30 = 0.115 g/L and LC50 = 0.37 g/L respectively) after 24 h. Furthermore, to probe changes in gene expression due to M. alternifolia oil contact in M. persicae, the expression of HSP 60, FPPS I, OSD, TOL and ANT genes were examined at doses of LC30 and LC50. Four out of the five selected genes-OSD, ANT, HSP 60 and FPPS I-showed upregulation at LC50, whereas, TOL gene showed maximum upregulation expression at LC30. Finally, the major components of M. alternifolia oil (terpinen-4-ol) were docked and MD simulated into the related proteins of the selected genes to explore ligand⁻protein modes of interactions and changes in gene expression. The results show that M. alternifolia oil has remarkable insecticidal and deterrent effects and also has the ability to affect the reproduction and development in M. persicae by binding to proteins.

Delivery of adapalene using a novel topical gel based on tea tree oil nano-emulsion: Permeation, antibacterial and safety assessments.

The aim of present study was to design and optimize 0.1% adapalene loaded nano-emulsion to improve the drug efficacy and increase its user compliance. Effect of type and concentration of surfactants was studied on size of 0.1% adapalene loaded nano-emulsion. Optimized formulation was then evaluated for particle size, polydispersity index, morphology, viscosity, and pH. Subsequently, 1% carbopol® 934 was incorporated to the optimized formulation for preparation of its gel form. The efficacy and safety of 0.1% adapalene loaded nano-emulsion gel was assessed compared to marketed gel containing 0.1% adapalene. In-vitro studies showed that adapalene permeation through the skin was negligible in both adapalene loaded nano-emulsion gel and adapalene marketed gel. Furthermore, drug distribution studies in skin indicated higher retention of adapalene in the dermis in adapalene loaded nano-emulsion gel compared with adapalene marketed gel. Antibacterial activity against Propionibacterium acnes showed that adapalene loaded nano-emulsion is effective in reducing minimum inhibitory concentration of the formulation in comparison with tea tree oil nano-emulsion, and pure tea tree oil. In vivo skin irritation studies showed absence of irritancy for adapalene loaded nano-emulsion gel. Also, blood and liver absorption of the drug, histological analysis of liver and liver enzyme activity of rats after 90 days' treatment were investigated. No drug was detected in blood/liver which in addition to an absence of any adverse effect on liver and enzymes showed the potential of adapalene loaded nano-emulsion gel as a novel carrier for topical delivery of adapalene.

Administración accidental de aceite puro esencial de arbol de té (Melaleuca alternifolia) en lactantes / Accidental pure essential tea tree oil (Melaleuca alternifolia) administration in two infants

El aceite de árbol de té es una sustancia que se obtiene mediante la destilación de las hojas y ramas frescas del árbol Melaleuca alternifolia. En los últimos años se ha popularizado su uso sobre todo como agente antiinfeccioso tópico en una gran variedad de enfermedades. Se trata de una sustancia natural que tiene un potencial efecto tóxico demostrado sobre todo si se ingiere. Presentamos dos casos de pacientes atendidos en nuestro Servicio de Urgencias tras administración e ingestión accidental de aceite esencial de árbol de té, en ambos casos los padres confundieron el envase con el de la vitamina D. La atención de estos pacientes nos llevó a realizar una búsqueda bibliográfica de casos publicados de intoxicación por ingesta de aceite de árbol de té y a poner de manifiesto la ausencia de advertencias de seguridad en los envases de aceite de árbol de té que actualmente se comercializan en nuestro país (AU)

Tea tree oil is an essential oil obtained by steam distillation of the leaves and terminal branches of Melaleuca alternifolia. In recent years, it has become popular as an antimicrobial agent against a large number of diseases. It is a natural substance that has a potential toxic effect especially if ingested. We report two cases of infants who came to our Emergency Department because they were accidentally given tea tree oil. In both cases parents mistook the tea tree oil bottle with the D vitamin bottle. The care of these patients led us to perform a literature search of published cases of tea tree oil poisoning and highlight the absence of safety warnings on tea tree oil packages currently marketed in our country (AU)

Molecular and structural changes induced by essential oils treatments in Vicia faba roots detected by genotoxicity testing.

Over the last few years, there has been an increased interest in exploiting allelopathy in organic agriculture. The aim of this investigation was to examine the effects of essential oil mixtures in order to establish their allelopathic use in agriculture. Two mixtures of essential oils consisting respectively of tea tree oil (TTO) and clove plus rosemary (C + R) oils were tested. Phytotoxicity and genotoxicity tests on the root meristems of Vicia faba minor were performed. A phytotoxic influence was particularly relevant for C + R mixture, while genotoxicity tests revealed significant results with both C + R oil mixture and TTO. Phenotypic analysis on Vicia faba minor primary roots following C + R oil mixture treatment resulted in callose production, an early symptom attributed to lipid peroxidation. The approach described in this study, based on genotoxicity bioassays, might identify specific DNA damage induced by essential oil treatments. These tests may represent a powerful method to evaluate potential adverse effects of different mixtures of essential oils that might be useful in alternative agriculture. Future studies are focusing on the positive synergism of more complex mixtures of essential oils in order to reduce concentrations of potentially toxic components while at the same time maintaining efficacy in antimicrobial and antifungal management.

In vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells.

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

In vitro genotoxicity of Melaleuca alternifolia essential oil in human lymphocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: The volatile essential oil derived from the plant Melaleuca alternifolia, also called tea tree oil (TTO), is largely employed for its antimicrobial properties against several human pathogens. It is used in many topical formulations to treat cutaneous infections. AIM OF THE STUDY: Since very few studies have been done on the safety and toxicity of the crude Melaleuca alternifolia essential oil, current investigation evaluates the possible genotoxic effects of TTO in human lymphocyte cultures. MATERIAL AND METHODS: The composition of current TTO sample was determined by GC/MS and NMR. The level of cytotoxicity in TTO treated cultures was determined by decrease of mitotic index when compared to that in negative control. The genotoxic potential of TTO was assessed by the in vitro mammalian cell micronucleus and the chromosome aberrations (CA) tests. RESULTS: Twenty-seven compounds were identified, accounting for 98.9% of the constituents. Terpinen-4-ol (42.8%), γ-terpinene (20.4%), p-cymene (9.6%), α-terpinene (7.9%), 1,8-cineole (3%), α-terpineol (2.8%) and α-pinene (2.4%) were the major compounds of the oil sample. None of the tested TTO concentrations (95µg/ml, 182µg/ml and 365µg/ml) caused a significant increase in the observed frequencies of micronuclei when compared to those in the untreated cultures (negative control). Additionally, no significant differences regarding the frequencies of CA were observed among the tested TTO concentrations and the negative control. CONCLUSIONS: Results demonstrate that TTO, in the tested concentrations, is not genotoxic in in vitro mammalian cells.

Mosquitocidal essential oils: are they safe against non-target aquatic organisms?

In latest years, the importance of the Melaleuca alternifolia essential oil (EO) has been greatly empathised due to its anti-microbial and anti-inflammatory effects, as well as to its toxic properties towards many arthropods of great medical and veterinary importance. In this research, the EO extracted from aerial parts of M. alternifolia was evaluated for its toxicity against larvae of the most invasive mosquito worldwide, Aedes albopictus (Diptera: Culicidae), and towards adults of the water flea, Daphnia magna (Cladocera: Crustacea), a non-target aquatic organism that share the same ecological niche of A. albopictus. The chemical composition of M. alternifolia EO was investigated by GC-MS analysis. Tea tree EO was mainly composed by oxygenated monoterpenes, with 1,8-cineole as the major constituent. M. alternifolia EO exerted toxic activity against A. albopictus larvae, with a LC50 = 267.130 ppm. However, this EO had a remarkable acute toxicity also towards adults of the non-target arthropod D. magna, with a LC50 = 80.636 ppm. This research provide useful information for the development of newer and safer mosquito control tools, highlighting that the non-target effects against aquatic organisms that share the same ecological niche of A. albopictus larvae are crucial in the development of ecofriendly mosquito control strategies. Further research is needed to investigate the chronic and/or reproductive toxicity of M. alternifolia EO both towards target and non-target aquatic arthropods.

Development of a novel synergistic thermosensitive gel for vaginal candidiasis: an in vitro, in vivo evaluation.

The singular aim of the proposed work is the development of a synergistic thermosensitive gel for vaginal application in subjects prone to recurrent vaginal candidiasis and other microbial infections. The dual loading of Itraconazole and tea tree oil in a single formulation seems promising as it would elaborate the microbial coverage. Despite being low solubility of Itraconazole in tea tree oil, a homogeneous, transparent and stable solution of both was created by co-solvency using chloroform. Complete removal of chloroform was authenticated by GC-MS and the oil solution was used in the development of nanoemulsion which was further translated into a gel bearing thermosensitive properties. In vitro analyses (MTT assay, viscosity measurement, mucoadhesion, ex vivo permeation, etc.) and in vivo studies (bioadhesion, irritation potential and fungal clearance kinetics in rat model) of final formulation were carried out to establish its potential for further clinical evaluation.

Insecticidal and repellent effects of tea tree (Melaleuca alternifolia) oil against Lucilia cuprina.

Laboratory studies were conducted to assess the effect of tea tree oil (TTO) from Melaleuca alternifolia (terpinen-4-ol chemotype) against different stages of the Australian sheep blowfly Lucilia cuprina. When applied to wool, 3% TTO formulation repelled gravid female L. cuprina and prevented oviposition for six weeks. Formulations containing 1% TTO caused 100% mortality of L. cuprina eggs and 1st instar larvae and 2.5% TTO caused mortality of most second and third instar larvae in agar feeding assays. In experiments where third instar larvae were dipped in TTO formulations for 60s, concentrations of up to 50% TTO gave less than 50% kill. TTO at concentrations of 0.5%, 2% and 5% was strongly repellent to third instar larvae and caused them to evacuate treated areas. Inclusion of TTO in formulations with diazinon, ivermectin and boric acid reduced mortality in comparison with the larvicides used alone, at least partially because of avoidance behaviour stimulated by the TTO. Addition of TTO to wound treatments may aid in wound protection and myiasis resolution by preventing oviposition by L. cuprina adults, insecticidal action against L. cuprina eggs and larvae, stimulating larvae to leave the wound and through antimicrobial and anti-inflammatory properties that aid in wound healing.

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia).

AIMS: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. METHODS AND RESULTS: Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed. CONCLUSIONS: TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.

What you see may not always be what you get--bioavailability and extrapolation from in vitro tests.

In human risk assessment, bioavailability needs to be considered when relying on in vitro toxicity results. For single chemicals, this quantitative challenge is often handled through a bioavailability factor. For mixtures, however, things are more complicated. Thus, individual constituents may not only interact toxicodynamically and toxicokinetically, but the composition of constituents reaching the target site may also differ from what was present at the site of exposure due to the differences in their bioavailabilities. A recent study concluded on the in vivo potential of Australian tea-tree oil (TTO) to act as an endocrine disruptor based on an in vitro protocol measuring the growth of MCF-7 cells following chemical exposure to TTO. TTO is primarily used topically in humans, and is not a single chemical but is a mixture with some constituents penetrating the skin which others do not. The present study evaluated in an identical in vitro model to what extent TTO and its skin penetrating constituents affected the growth of MCF-7 cells. The estrogenic potency of TTO was confirmed, but none of the bioavailable TTO constituents demonstrated estrogenicity. The present study, therefore, cautions in vitro to in vivo extrapolations from the mixtures of constituents with potentially varying bioavailabilities.

Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells.

AIMS: The aim of this study was to compare both the antimicrobial activity of terpinen-4-ol and tea tree oil (TTO) against clinical skin isolates of meticillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) and their toxicity against human fibroblast cells. METHODS AND RESULTS: Antimicrobial activity was compared by using broth microdilution and quantitative in vitro time-kill test methods. Terpinen-4-ol exhibited significantly greater bacteriostatic and bactericidal activity, as measured by minimum inhibitory and bactericidal concentrations, respectively, than TTO against both MRSA and CoNS isolates. Although not statistically significant, time-kill studies also clearly showed that terpinen-4-ol exhibited greater antimicrobial activity than TTO. Comparison of the toxicity of terpinen-4-ol and TTO against human fibroblasts revealed that neither agent, at the concentrations tested, were toxic over the 24-h test period. CONCLUSIONS: Terpinen-4-ol is a more potent antibacterial agent against MRSA and CoNS isolates than TTO with neither agent exhibiting toxicity to fibroblast cells at the concentrations tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Terpinen-4-ol should be considered for inclusion as a single agent in products formulated for topical treatment of MRSA infection. However, further work would initially be required to ensure that resistance would not develop with the use of terpinen-4-ol as a single agent.

Habituation to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia) is associated with reduced susceptibility to antibiotics in human pathogens.

OBJECTIVES: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic susceptibility profiles of significant human pathogens and commensals. METHODS: The study compared the antibiotic susceptibility (Etest) patterns of Escherichia coli, Staphylococcus aureus/methicillin-resistant S. aureus (MRSA) and Salmonella spp. after broth culture for 72 h in the presence or absence of sub-lethal concentrations of TTO (0.25%, 0.25% and 0.1%). RESULTS: All habituated cultures (exposed to sub-lethal concentrations of TTO) displayed reduced susceptibility to a range of clinically relevant antibiotics compared with non-habituated (control) cultures. CONCLUSIONS: Although TTO may be an effective antimicrobial agent when appropriately used at bactericidal concentrations, its application at sub-lethal concentrations may contribute to the development of antibiotic resistance in human pathogens.

A review of the toxicity of Melaleuca alternifolia (tea tree) oil.

The essential oil of Melaleuca alternifolia, also known as tea tree or melaleuca oil, is widely available and has been investigated as an alternative antimicrobial, anti-inflammatory and anti-cancer agent. While these properties are increasingly well characterised, relatively limited data are available on the safety and toxicity of the oil. Anecdotal evidence from almost 80 years of use suggests that the topical use of the oil is relatively safe, and that adverse events are minor, self-limiting and occasional. Published data indicate that TTO is toxic if ingested in higher doses and can also cause skin irritation at higher concentrations. Allergic reactions to TTO occur in predisposed individuals and may be due to the various oxidation products that are formed by exposure of the oil to light and/or air. Adverse reactions may be minimised by avoiding ingestion, applying only diluted oil topically and using oil that has been stored correctly. Data from individual components suggest that TTO has the potential to be developmentally toxic if ingested at higher doses, however, TTO and its components are not genotoxic. The limited ecotoxicity data available indicate that TTO is toxic to some insect species but more studies are required.

Effect of Australian tea tree oil on Gyrodactylus spp. infection of the three-spined stickleback Gasterosteus aculeatus.

Gyrodactylus spp. infections of commercially farmed fishes are responsible for significant economic losses. Existing treatments have proved uneconomic, stressful to the fishes, and ecologically damaging. Essential oils are naturally occurring compounds that exhibit a wide range of anti-microbial and anti-fungal activities. This study explored the possibility of using Australian tea tree (Melaleuca alternifolia) oil (TTO) to treat Gyrodactylus spp. infection on the three-spined stickleback Gasterosteus aculeatus. In the presence of 0.01 % Tween 80 as an emulsifier, TTO treatments at concentrations between 3 and 30 ppmv (parts per million by volume) lowered the prevalence and significantly reduced the parasite burden of sticklebacks naturally infected with Gyrodactylus spp. In addition, Tween 80 alone exhibited parasiticidal activity against Gyrodactylus spp. These findings show the potential of TTO in combination with Tween 80 as an effective treatment of Gyrodactylus spp. infection of fishes.