Different Concentrations Of Ivermectin Research Articles

The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 μg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

Formulation and evaluation of ivermectin emulgel formulations

Ivermectin, a broad-spectrum antiparasitic agent, has demonstrated significant therapeutic efficacy against various skin conditions. However, it's poor skin permeability and limited stability often hinder its clinical application. Emulgel, a hybrid dosage form combining the advantages of both emulsions and gels, has emerged as a promising topical delivery system to enhance the skin permeation and stability of poorly permeable drugs. In this study, we aimed to formulate and characterize an Ivermectin emulgel to address the challenges associated with its topical delivery. The emulgel was prepared using a two-step method, involving the preparation of an oil-in-water (o/w) emulsion, which was then gelled with suitable gelling agents. Different concentrations of Ivermectin, oil phase, surfactants, and gelling agents were screened to optimize the formulation for maximum drug loading and stability. The optimized Ivermectin emulgel was characterized for its physicochemical properties, including pH, viscosity, spreadability, drug content, and rheological behavior. The study's findings support the potential application of the emulgel in treating various skin conditions caused by parasites and infectious agents. Further, in vivo studies and clinical trials are required to validate its safety and effectiveness in a clinical setting.

Sugar Bait Composition Containing Ivermectin Affect Engorgement and Mortality of the Mosquito Aedes aegypti (Diptera: Culicidae).

Toxins and attractants have been studied and used in sugar baits for mosquitoes. However, little importance has been given to the combination of sugar concentration, bait composition, and their relationship with mosquito engorgement and mortality. Therefore, the present study evaluated the effects of three concentrations of sucrose on baits with and without an attractant (concentrated guava juice), on engorgement and mortality rates of adult Aedes aegypti (Linnaeus, Diptera: Culicidae). Toxic sugar baits (TSB) and attractant toxic sugar baits (ATSB) containing 10, 50, and 70% sucrose and 100 ppm ivermectin (IVM) were prepared to assess engorgement and mortality rates. Subsequently, different concentrations of IVM (0.312-100 ppm) in TSB and ATSB were prepared with sucrose concentrations of 10 and 70% to determine the lethal concentrations (LC50 and LC90) values. Engorgement on the baits was observed under a stereomicroscope, and mortality was followed up to 48 h after feeding. In general, more mosquitoes engorged on TSB regardless of the sugar concentration, while higher concentrations of sugar in ATSBs resulted in higher numbers of mosquitoes engorging. A large increase in the LC90 of IVM was observed for females feeding on ATSBs and TSBs with 70% sucrose relative to those feeding on baits with lower sugar concentrations. No such effect was observed for males.

Potential of ivermectin as an active ingredient of the attractive toxic sugar baits against the Indian malaria vectors Anopheles culicifacies and Anopheles stephensi.

Attractive toxic sugar bait (ATSB) is a novel vector control tool that exploits the sugar feeding behavior of mosquitoes. The current study aims to evaluate the efficacy of ivermectin-based ATSB against insecticide susceptible and resistant strains of major Indian malaria vectors - Anopheles culicifacies and Anopheles stephensi. ATSB with different concentrations of ivermectin were tested against mosquito vectors under standard laboratory conditions. Dose-response analysis of ivermectin-ATSB showed 7.8 and 19.8ppm as 50% and 90% lethal concentration (LC50 and LC90 ) values for insecticide susceptible An. culicifacies. In the case of insecticide susceptible An. stephensi, the LC90 value was 35 ppm which was significantly higher in comparison to the LC90 for An. culicifacies. The LC50 of insecticide-resistant An. culicifacies and An. stephensi were 10.6 and 15.9ppm respectively whereas LC90 values were 36.9 and 61.0. Ivermectin-ATSB resulted in 99 ± 0.8% mortality of An. culicifacies and 93 ± 3.8% mortality of An. stephensi at an ivermectin concentration of 25 ppm. In another set of experiments, the ATSB solution containing standardized dose of ivermectin was sprayed on Allysum plant and mortality of both Anopheline vectors was recorded. Here, we observed > 90% mortality in both An. stephensi and An. culicifacies. Our study demonstrates the potential of ivermectin-based ATSB in killing Indian malaria vectors irrespective of the method of application. Further field trials with ivermectin containing ATSB may pave the way for its usage in the national vector control program. © 2022 Society of Chemical Industry.

Transgenic Expression of Haemonchus contortus Cytochrome P450 Hco-cyp-13A11 Decreases Susceptibility to Particular but Not All Macrocyclic Lactones in the Model Organism Caenorhabditis elegans.

The number of reported macrocyclic lactones (ML) resistance cases across all livestock hosts is steadily increasing. Different studies in the parasitic nematode Haemonchus contortus assume the participation of cytochrome P450s (Cyps) enzymes in ML resistance. Still, functional data about their individual contribution to resistance or substrate specificity is missing. Via microinjection, transgenic Caenorhabditis elegans expressing HCON_00141052 (transgene-Hco-cyp-13A11) from extrachromosomal arrays were generated. After 24 h of exposure to different concentrations of ivermectin (IVM), ivermectin aglycone (IVMa), selamectin (SEL), doramectin (DRM), eprinomectin (EPR), and moxidectin (MOX), motility assays were performed to determine the impact of the H. contortus Cyp to the susceptibility of the worms against each ML. While transgene-Hco-cyp-13A11 significantly decreased susceptibility to IVM (four-fold), IVMa (2-fold), and SEL (3-fold), a slight effect for DRM and no effect for MOX, and EPR was observed. This substrate specificity of Hco-cyp-13A11 could not be explained by molecular modeling and docking studies. Hco-Cyp-13A11 molecular models were obtained for alleles from isolates with different resistance statuses. Although 14 amino acid polymorphisms were detected, none was resistance specific. In conclusion, Hco-cyp-13A11 decreased IVM, IVMa, and SEL susceptibility to a different extent, but its potential impact on ML resistance is not driven by polymorphisms.

Sustained-release hydrogels of ivermectin as alternative systems to improve the treatment of cutaneous leishmaniasis.

Background: Leishmaniasis is a neglected tropical disease and its cutaneous form manifests as ulcers or nodules, generally in exposed parts of the body. This work aimed to develop ivermectin (IVM) thermosensitive hydrogels as topical formulations to improve cutaneous leishmaniasis treatment. Materials & methods: Hydrogels based on poloxamers 407 and 188 with different concentrations of IVM were prepared and rheologically characterized. The IVM release profiles were obtained and mathematically analyzed using the Lumped model. Results: The formulation containing 1.5% w/w of IVM presented an adequate gelling temperature, an optimal complex viscosity and elastic modulus. Hydrogels allowed to modulate the release of IVM. Conclusion: IVM thermosensitive hydrogels can be considered a valuable alternative to improve the treatment of cutaneous leishmaniasis.

Susceptibility of entomopathogenic nematodes to ivermectin and thiabendazole

The objective of the present study was to determine the susceptibility of entomopathogenic nematodes to ivermectin and thiabendazole. Soil samples collected from the municipalities of Irapuato and León, Guanajuato, Mexico, were obtained, from which the entomopathogenic nematodes (EPNs) of the Steinernematidae and Heterorhabditidae families were isolated. The samples were classified from livestock and nonlivestock soils, and the susceptibility of EPNs to anthelmintics was determined with the larval motility assay (LMA, 24 h) and the larval migration inhibition assay (LMI assay, 48 h). Sterile distilled water (T1) and treatments with 1% ivermectin diluted in 5% DMSO (dimethyl sulfoxide) (T2) and 5% thiabendazole diluted in 5% DMSO (T3) were applied to infective juvenile larvae. Analysis of variance was performed with a factorial design and Tukey’s test at 0.05 probability. In addition, different concentrations of ivermectin (0.1, 0.5, 1, 1.2, 1.5, and 2 μg) and thiabendazole (1, 5, 10, 12, 15, and 20 mg) were evaluated to perform a Probit analysis to determine their LC50. All strains of EPNs were susceptible to ivermectin in both the LMA and LMI assay. The results show that EPNs are susceptible to ivermectin and thiabendazole, and the degree depends on the type of test performed, the chemical product used, and the origin of the strain of EPN.

Genotoxicity and carcinogenicity of ivermectin and amoxicillin in vivo systems

Antiparasitic substances are chemicals used to control or kill endoparasites and ectoparasites. Based on the premise that Ivermectin (IVM) and Amoxicillin (AMX) are commonly considered in parasitic control in mammals, the present study aimed to evaluate the carcinogenic and genotoxic potential of different concentrations of IVM and AMX through the detection of epithelial tumor test in Drosophila melanogaster. Third-instar larvae descending from the cross between wts/TM3, Sb1 females and mwh/mwh males were treated with different concentrations of IVM (2.9, 5.8, 11.6 and 23.2 x 10−17 mM) or AMX (1.37, 2.74, 5.48 and 10.9 x 10−16mM). The results revealed that IVM increased the frequency of epithelial tumor in D. melanogaster considering all evaluated concentrations, while AMX showed no carcinogenic effect. Furthermore, the Micronucleus (MN) test in Tradescantia pallida was used to evaluate the genotoxic effect of IVM and AMX. T. pallida individuals were exposed for 8 hours at different concentrations of IVM (5.71, 11.42, 22.84 and 45.68 x 10−5mM) or AMX (5.13, 10.26, 20.52 and 41.05 x 10−3mM). Findings showed an increase in the frequency of micronuclei in T. pallida treated with 11.42, 22.84 and 45.68 x 10−5mM of IVM. We conclude that chronic exposure to IVM is directly associated with events resulting from genetic instability (genotoxicity and carcinogenicity). On the other hand, AMX was neither carcinogenic nor genotoxic for D. melanogaster and T. pallida.

Suppressing ROS-TFE3-dependent autophagy enhances ivermectin-induced apoptosis in human melanoma cells.

Melanoma is an aggressive skin malignancy with a high mortality rate; however, successful treatment remains a clinical challenge. Ivermectin, a broad-spectrum antiparasitic drug, has recently been characterized as a potential anticancer agent due to its observed antitumor effects. However, the molecular mechanisms of ivermectin remain poorly understood. In the current study, we tested the involvement of autophagy in the ivermectin mechanism of action in human melanoma cells. We exposed SK-MEL-28 cells to different concentrations of ivermectin (2.5, 5, and 10 μM) for 24 hours. Here, ivermectin-induced apoptosis, as evidenced by the upregulation of cleaved poly (ADP-ribose) polymerase, BAX expression, and caspase-3 activity and downregulation of BCL-2 expression. In line with the apoptosis response, ivermectin triggered autophagy. Pharmacological or genetic inhibition of autophagy further sensitized SK-MEL-28 cells to ivermectin-induced apoptosis. Mechanistically, ivermectin-induced TFE3(Ser321) dephosphorylation, activated TFE3 nuclear translocation and increased TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes, and subsequently, initiated autophagy in SK-MEL-28 cells. Moreover, N-acetyl-cysteine, an reactive oxygen species (ROS) scavenger, abrogated the effects of ivermectin on TFE3-dependent autophagy. Taken together, we demonstrated that ivermectin increases TFE3-dependent autophagy through ROS signaling pathways in human melanoma cells and that inhibiting autophagy enhances ivermectin-induced apoptosis in human melanoma cells.

Ivermectin alters reproductive success, body condition and sexual trait expression in dung beetles

Ivermectin is a very common parasiticide used in livestock. It is excreted in the dung and has negative effects on survival and reproduction of dung-degrading organisms, including dung beetles. Here we exposed the dung beetle Euoniticellus intermedius to different concentrations of ivermectin in the food and evaluated reproductive success and the expression of traits associated with survival and reproduction under laboratory conditions. It is the first time the effects of ivermectin were evaluated on offspring physiological condition and the expression of a secondary sexual trait. We also registered the number of emerged beetles, sex ratio and body size of emerged adult beetles. Besides reducing the number of emerged beetles and body size, as found in the same and other insects, ivermectin at high doses reduced muscle mass while at intermediate doses it increased lipid mass. Ivermectin changed offspring sex ratio and at high doses increased the size of male horn, which is an important trait defining the male mating success. Our results highlight the importance of regulating parasiticide usage in livestock in order to maintain ecosystem services provided by dung beetles and confirm that contaminants impose new environmental conditions that not only impact on wild animal survival, but also on evolutionary processes such as sexual selection.

Ivermectin efficacy against Biomphalaria, intermediate host snail vectors of Schistosomiasis.

The impact of ivermectin on adult snails of the genus Biomphalaria (B. glabrata, B. tenagophila and B. straminea), B. glabrata infected with Schistosoma mansoni, snail egg-masses cercariae and miracidia, as well as on guppy fish (Poecilia reticulata) was examined and evaluated. Biomphalaria snails, egg-masses, parasite stages and guppies were all exposed to different concentrations of ivermectin for 24 h, followed by regular observations of mortality. The calculated lethal doses of ivermectin were around an LD50 of 0.03 μg ml-1, and an LD90 of 0.3 μg ml-1 for the three species of snails. Specimens of B. glabrata actually shedding parasite cercariae all died when exposed to ivermectin at a concentration of a mere 0.01 μg ml-1. Ivermectin B1a, the major (80%) component of commercially available ivermectin, proved to be inactive, and it was the minor (20%) component, ivermectin B1b, which caused snail death. Snail egg-masses were not affected, even at the highest concentration of 100 μg ml-1. With respect to S. mansoni parasite stages, 0.2 μg ml-1 ivermectin killed 50% of cercariae and miracidia within five minutes, rising to 90% after 30 min. Mortality of guppy fish within 24 h of exposure to ivermectin at concentrations of 0.5 μg ml-1 and 0.01 μg ml-1, were 100% and 30%, respectively. The concentration of 0.01 μg ml-1 that killed Schistosoma mansoni-infected snails only caused 30% mortality in guppy fish. Ivermectin can be considered a promising molluscicide, especially as it is more potent against infected snails than uninfected ones, although it has no impact on egg-masses. Ivermectin and its derivatives could be explored in the search for a new agent to help control schistosomiasis transmission.

Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

BackgroundTicks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.FindingsIRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84 % and 92 % with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.ConclusionsThis is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

Cuantificación de ivermectina eliminada en materia fecal de novillos tratados

<p>El objetivo del presente estudio fue cuantificar la ivermectina eliminada en la materia fecal del ganado bovino, la cual fue detectada mediante la técnica de Cromatografía Líquida de Alta Resolución (CLAR), en la granja experimental Tunguavita, en la vereda Salitre de la ciudad de Paipa (Boyacá). Se experimentó en 10 novillas de diferentes razas y pesos con ivermectina comercial al 1% en una dosis de 0,2 mg/kg de peso vivo, por vía subcutánea en una sola aplicación. Se observó que al segundo día de haber aplicado el antiparasitario se obtuvo la concentración más alta (87.6%), y a los 20 días, la más baja (0,17%); es decir, la concentración de ivermectina fue disminuyendo, aproximadamente, en 0.0535 ppm por día. No se encontraron diferencias significativas entre razas y pesos de los animales. Los resultados de este estudio muestran la presencia de ivermectina en las heces del ganado bovino, presencia que genera un impacto ambiental negativo frente a la entomofauna coprófaga, encargada de la degradación de la materia fecal, ya que las diferentes concentraciones de ivermectina continúan con su función insecticida al ser eliminadas en la materia fecal.<br />De otro lado, se determinó que cierta parte del desparasitante está siendo asimilado por diferentes órganos internos del individuo, por lo tanto, no se eliminan en su totalidad.</p>

Ivermectin as an inhibitor of the plasma membrane and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases in rat vas deferens

ObjectiveThe present work investigated the effect of ivermectin on Ca2+ content and on the Ca2+-ATPase activity (represented by the plasma membrane Ca2+-ATPase and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase present in rat vas deferens.MethodsThe assays were carried out using ultracentrifuged homogenate preparations from rat vas deferens in the presence or absence of the 12-kDa FK506-binding protein-Ca2+ release channel complex. Measures of Ca2+ content and Ca2+ ATPase activity were then carried out in function of different concentrations of ivermectin. ResultsThe data show that ivermectin (10 μM) reduces the sarcoplasmic reticulum Ca2+ content in FK506-binding protein (+) and FK506-binding protein (-) fractions of ultracentrifuged homogenate from rat vas deferens (inhibition of 50% and 40%, respectively, p<0.05) and inhibits both the activities of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPases pumps (33% and 16%, respectively, p<0.05).ConclusionThese data suggest that ivermectin effects Ca2+ handling in the rat vas deferens, indicating that this drug could alter the contractility of this smooth muscle. Therefore, ivermectin could be an interesting pharmacological tool to alter the physiological function of vas deferens and to manipulate the fertility status of male rats.Indexing terms: Calcium. Ivermectin. Rats.

Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones

Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

Lethal and sub‐lethal effects of ivermectin on north temperate dung beetles,Aphodius aterandAphodius rufipes(Coleoptera: Scarabaeidae)

Abstract. 1. Ivermectin is an anthelmintic veterinary medicine, and is excreted in the dung of treated livestock in a mainly unmetabolised form. Ivermectin is known to have toxic effects on dung beetles, but most studies to date have been conducted on tropical and sub‐tropical species. Relatively few laboratory studies have focused on the specific effects of ivermectin on survival and development of north temperate dung beetles.2. In this study, we experimentally investigated the effect of ivermectin concentration on various life stages of twoAphodiusdung beetle species. Dung was collected from cattle groups that had been treated with a subcutaneous injection of ivermectin. Laboratory bioassays were conducted by feeding adults of two beetle species (Aphodius aterandAphodius rufipes) with dung that contained different concentrations of ivermectin. Adult survival and oviposition were measured, and the subsequent development and survival of produced larvae was monitored over time.3. Larval development rates were significantly slowed by ivermectin. Ivermectin had significant negative effects on the survival of larvae. Overall, ivermectin concentration caused large and significant reductions in the cohort size from an individual dung pat that would potentially contribute to the next generation of beetles.4. In general, ivermectin concentration did not have significant negative effects on adult survival. The number of eggs per femaleA. rufipeswas significantly reduced by ivermectin concentration in one of two bioassays, but the magnitude of the effect was not large. The actual impacts on dung beetle population dynamics in farmland would depend on several other factors, which are discussed.

Fasciolagigantica: Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether

Objective To evaluate the in vitro effects of different concentrations of ivermectin and/or artemether on Fasciola gigantica worms and to study the parasitological changes and tegumental alterations using scanning electron microscopy (SEM). Methods Fasciola gigantica worms were incubated in vitro for 24 and 48 h with three concentrations of either ivermectin or artemether (10, 20 and 50 μg/ml) or both in half concentration of either (5, 10 and 25 μg/ml). Results Exposure of Fasciola worms to 25 + 25 μg/ml of combined drug regimens or to 50 μg/ml of either ivermectin or artemether for 48 h led to 100%, 41.7% and 75% worm killing which were accompanied by a significant reduction in egg laying capacity and significant increase in dead eggs maximally recorded in combined drug regimens. SEM of the flukes incubated for 48 h with combined drug regimens showed maximal tegumental disruption with swelling of the worm body, roughness, blebbing, sloughing and complete loss of spines. Disruption to the tegument of the flukes induced by artemether was more than that of ivermectin. Conclusions Artemether alone or combined with ivermectin in half doses had potent fasciocidal activities. Besides, half doses of combined drug regimens had higher ovicidal effects than each drug alone. In vivo studies are recommended to explore the efficacy of combined regimens against Fasciola infection.

New screening test to predict the potential impact of ivermectin-contaminated cattle dung on dung beetles

According to European Union recommendations, a test method has been developed to evaluate the effects of veterinary pharmaceuticals on dung feeding insects. This test method was evaluated with the dung beetle Aphodius constans by using fecal residues of ivermectin after a pour-on administration. Dung of different age (and thus containing different concentrations of ivermectin) as well as mixtures of highly-contaminated spiked dung with untreated control dung were studied in five test runs in two laboratories. The concentration of ivermectin (active substance; a.s.) in the dung samples was verified analytically. The main test endpoint was the survival of first instar larvae. The LC50 using dung directly obtained from treated cattle ranged from 470 to 692 microg a.s. kg(-1) dung (dry weight; d.w.) and 67 to 97 microg a.s. kg(-1) dung (fresh weight; f.w.). Using mixtures, the outcome of two tests was almost identical: 770 to 781 microg a.s. kg(-1) dung (d.w.); 109 to 132 microg a.s. kg(-1) dung (f.w.). In comparison to the LC50 values obtained when ivermectin was spiked in control dung at several concentrations (LC50 880-985 microg a.s. kg(-1) dung (d.w.)), the LC50 values were again very similar. Three conclusions can be drawn from these results. The proposed test method seems to be robust and allows for the initiation of an international validation process (including ringtesting). Because of only small differences found in tests in which the test substance was spiked into control dung and those in which dung from treated cattle was applied, the use of a standard test method is proposed. The effects of ivermectin on ecologically relevant dung beetles obtained in a standardised test method reflect the results from field studies and are in the range of environmentally relevant concentrations.