Eszter Takács

Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18–2000 times for co-formulants, 8–141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine—POEA and alkyl polyglucoside—APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.

Palladium-catalysed aminocarbonylation of steroidal 17-iodo-androst-16-ene derivatives in N,N′-dialkyl-imidazolium-type ionic liquids

The use of [bmim]+[BF4]−, [bmim]+[PF6]− and [emim]+[PF6]− ionic liquids as solvents in homogeneous catalytic aminocarbonylation of 17-iodo-5α-androst-16-ene at atmospheric carbon monoxide pressure has been investigated. It has been proved that after the extraction of the product with toluene, the ionic liquid–catalyst mixture could be recycled several times. Although there was a loss of catalytic activity in the further cycles, even 94% conversion can be achieved after the fifth run by using [bmim]+[BF4]− ionic liquid. The conversion depended strongly both on the properties of the ionic liquid and those of the catalyst. The activities of the in situ palladium(0) catalysts prepared from Pd(OAc)2 and various phosphine ligands (PPh3, TPPTS, DPPBA) have been compared. The method can be effectively used for the aminocarbonylation of other steroids with 17-iodo-16-ene functionality.

Inter-laboratory comparison of Cry1Ab toxin quantification in MON 810 maize by enzyme-immunoassay

Abstract A laboratory ring trial was performed in four laboratories for determination of Cry1Ab toxin in leaf material of MON 810 maize using a standardised enzyme-linked immunoassay protocol. Statistical analysis was carried out by the ISO 5725-2 guidelines, sample standard deviation and standard error, within-laboratory and inter-laboratory SD and SE were calculated. Measured inter-laboratory average values were 12.5±4.0, 15.3±4.6 and 72.6±17.8 µg/g for three lyophilised samples, and 27.8±4.3 µg/g for a frozen sample, yet, Cry1Ab concentrations ranged 66.5–160.1% of the corresponding IA. Determined concentrations by in-house protocols were statistically not different in one laboratory and different in two laboratories from the corresponding values by the joint protocol. Results emphasise the importance of a standardised protocol among laboratories for comparable quantitative Cry1Ab toxin determination. However, even when using a standardised protocol, significant differences still occur among toxin concentrations detected in different laboratories, although with a smaller range of variation.

Relationships of Helicoverpa armigera, Ostrinia nubilalis and Fusarium verticillioides on MON 810 Maize.

MON 810 maize was developed against Ostrinia nubilalis and is suggested to indirectly decrease Fusarium spp. infestation in maize ears. To evaluate this effect, co-occurrence of insect and fungal pests on MON 810 maize was studied. During 2009, exceptionally high maize ear infestation occurred in Julianna-major (Hungary). From investigation of some thousands of maize ears, the majority of the larval damage originated from Helicoverpa armigera larvae, while O. nubilalis larvae contributed significant damage only at a single plot. Fusarium verticillioides infection appeared only in a small portion (∼20–30%) of the insect damaged cobs. H. armigera and O. nubilalis larvae feeding on F. verticillioides mycelia can distribute its conidia with their fecal pellets. MON 810 maize showed 100% efficacy against O. nubilalis in the stem, but lower efficacy against O. nubilalis and H. armigera in maize ears. The ∼Cry1Ab toxin content of maize silk, the entry site of H. armigera, was lower than that in the leaves/stem/husk leaves of MON 810. Fusarium-infected MON 810 cobs are rarely found and only after larval damage by O. nubilalis. H. armigera larvae could not tolerate well F. verticillioides infected food and attempted to move out from the infected cobs. For further feeding they re-entered the maize ears through the 8–12 husk leaves, but in the case of the MON 810 variety, they usually could not reach the kernels. Apical damage on cobs resulted in only a minor (about one-tenth of the cob) decrease in yield.

Effects of neonicotinoid insecticide formulations and their components on Daphnia magna – the role of active ingredients and co-formulants

ABSTRACT Formulating agents used in pesticide products have been regarded as inactive/inert components; however, several studies confirmed additive, synergistic or antagonistic effects between the active ingredients (AIs) and additives, or the individual toxicity of the formulating agents. The worldwide used neonicotinoid insecticides and their components can reach surface waters, due to their physico-chemical properties (e.g. water solubility) or improper application in chemical plant protection technology, and can adversely affect non-target aquatic organisms. Formulated pesticides were analysed for alkane sulphonate surfactants by using liquid chromatography coupled with tandem mass spectrometry. In acute immobilisation tests on Daphnia magna, differences were found among the toxicity of the investigated neonicotinoid AIs and formulations. The toxicity of a formulated insecticide (Apache 50 WG®– AI: clothianidin) was found to be 46.5 times more toxic than explained by its AI, probably due to toxic effect of the formulating agents on D. magna. In contrast, two preparations (Calypso 480SC® – AI: thiacloprid, Actara 240 SC®– AI: thiamethoxam) were 2–3 times less toxic than their AIs. Results indicate possible synergistic/antagonistic interaction with the AIs.

Mycotoxin Biosensor Based on Optical Planar Waveguide

The research aim of this work is to develop a simple and highly sensitive optical biosensor for detection of mycotoxins. This sensor is built on a planar waveguide operating on the polarization interferometry principle, i.e., detecting a phase shift between p- and s-components of polarized light developed during the binding of analyte molecules. The operation of the proposed sensor is similar to that of a Mach–Zehnder interferometer, while its design is much simpler and it does not require splitting the waveguide into two arms. The refractive index sensitivity of the polarization interferometer sensor was in the range of 5200 radians per refractive index unit (RIU). Several tests were conducted to detect ochratoxin A (OTA) at different concentrations in direct immunoassay with specific antibodies immobilized in the sensing window. The lowest concentration of OTA of 0.01 ng/mL caused a phase shift of nearly one period. The results obtained prove high sensitivity of the sensors, which are capable of detecting even lower concentrations of mycotoxins at the ppt (part-per-trillion) level.

Dissipation of the herbicide active ingredient glyphosate in natural water samples in the presence of biofilms

ABSTRACT Dissipation of the herbicide active ingredient glyphosate was investigated in natural waters. To assess combined effects, glyphosate was applied in its pure form (glyphosate isopropylammonium salt) and in preparation Roundup Classic® formulated with polyethoxylated tallowamines (POEA). Standing and running surface water samples originated from Lake Balaton and River Danube between early May and mid-June of 2015. The kinetics of dissipation of glyphosate, measured by high-performance liquid chromatography combined with UV-VIS absorbance detection or tandem mass spectrometry, was investigated under laboratory conditions in aquaria with or without the presence of biofilms. The quantity and the biofilm structure of algal biomass were determined by in vivo fluorimetry and scanning electron microscopy. The presence of POEA affected the dissipation of glyphosate, and dissipation profiles differed in the investigated natural waters. Significantly higher initial concentrations of glyphosate were measured in River Danube for treatment with formulated glyphosate (101.4 ± 6.2 µg L−1), than with glyphosate alone (79.9 ± 6.6 µg L−1), and dissipation to a residual level (57.6 ± 1.4 µg L−1) consequently took longer (approximately by 1 day). Degradation of glyphosate from the initial level (91.24 ± 5.9 µg L−1) in Lake Balaton was not detected. Phytotoxic effects of glyphosate, particularly if enhanced by a formulant on algal biomass, were observed. Thus, 5–18% and 11–33% of algal biomass reduction was determined in River Danube upon treatments with glyphosate and Roundup Classic®, respectively. Corresponding biomass decreases in Lake Balaton were 1.3–13% and 9–14%, respectively, accompanied by an overall decay in the algal biofilms. In River Danube, treatments resulted in the occurrence of 1.4–5.8% of green algae in the algal biomass in 28 days, while green algae were not detected in the untreated control. The results indicate that glyphosate is capable of modifying the structure of the algal community and to induce increased secretion of extracellular polymeric substances matrix in the biofilms assessed.

Aquatic effect duration study of Cry4 toxin with immunoassay and Aedes aegypti larval biotest

Bacillus thuringiensis subsp. israelensis (Bti) preparations are widely used for culicid larvae. There is no suitable commercially available analytical method for Cry4 toxin as active ingredient in Bti preparations. To overcome this limitation, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitative determination of Cry4 toxin allowing a limit of detection (LOD) of ∼2 ng ml−1 in water. Preconcentration of aqueous samples by lyophilisation resulted in low but reproducible recoveries (25.7±6.8%), and the practical LODs for Bti preparations VECTOBAC WDG granulate and VECTOBAC 12 AS suspension were found to be ∼170 ng ml−1 and ∼900 ng ml−1, respectively. ELISA determinations indicated a rapid decay in detectable concentrations of VECTOBAC WDG applied at 400 ng ml−1 concentration in surface water: detected concentrations decreased by 18% and 44% in 4 days in water collected from two locations, and dropped below LOD afterwards. Larval mortality of Aedes aegypti indicated a continuous decrease even thereafter. Thus, quantitative Cry4 toxin detection facilitates proper timing and frequency of treatments to achieve optimal efficacy.

Aquatic toxicity and loss of linear alkylbenzenesulfonates alone and in a neonicotinoid insecticide formulation in surface water

Substance losses of linear alkylbenzene sulfonates (LASs) in a neat surfactant mixture, in an insecticide formulation Mospilan 20 SG, and in solutions with different neonicotinoid active ingredients (AIs) was studied in distilled water and in surface water samples originated from River Danube. Analytical measurements were performed both by HPLC-UV and commercial ELISA methods. Loss rates of LASs were found different in these aqueous matrices, with decomposition rates higher for the neat surfactant mixture than for Mospilan 20 SG (nearly 2- and 9-fold in distilled water and in surface water from River Danube, respectively). Half-lives determined in surface water from River Danube were shown to be affected by the presence of neonicotinoid AIs thiacloprid > imidacloprid > acetamiprid (ACE), while clothianidin and thiamethoxam did not affect LAS decomposition. Aquatic toxicity of Mospilan 20 SG, along with that of its AI ACE and co-formulant LAS, as well as the mixture of ACE and LAS was also investigated in the 48-h acute immobilisation assay on the water flea (Daphnia magna) aquatic indicator organism. LAS appeared to be significantly (8-fold) more toxic in the D. magna test than ACE, and the toxicity of the formulated insecticide was found to be 1.3 and 19.6 times higher than explained by its AI and LAS content, respectively, indicating synergistic toxicity. The strongest synergy between ACE and LASs was observed, when the neat forms of the two substances were applied in combination at concentrations equivalent to those in Mospilan 20 SG.

Label-Free Optical Detection of Mycotoxins Using Specific Aptamers Immobilized on Gold Nanostructures

This work focuses on the development of the novel label-free optical apta-sensors for detection of mycotoxins. A highly sensitive analytical method of total internal reflection ellipsometry (TIRE) combined with Localized Surface Plasmon Resonance (LSPR) phenomenon in nano-structured gold films was exploited here for the first time for detection of aflatoxin B1 and M1 in direct assay with specific aptamers immobilized on the surface of gold. The achieved detection of low molecular weight molecules, such as aflatoxin B1 and M1, in a wide range of concentrations from 100 ng/mL down to 0.01 ng/mL is remarkable for the LSPR method. The study of binding kinetics of aflatoxin molecules to their respective aptamers using dynamic TIRE measurements yielded the values of affinity constants in the range of 10−8–10−7 mol, which is characteristic for highly specific aptamer/target interactions similar to that for monoclonal antibodies. The effect of aptamers’ DNA chain length on their binding characteristics was analyzed.