Learning impairment in honey bees caused by agricultural spray adjuvants

Background

Spray adjuvants are often applied to crops in conjunction with agricultural pesticides in order to boost the efficacy of the active ingredient(s). The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to spray adjuvants as well. It was previously unknown whether these agrochemicals have any deleterious effects on honey bee behavior.

Methodology/Principal Findings

An improved, automated version of the proboscis extension reflex (PER) assay with a high degree of trial-to-trial reproducibility was used to measure the olfactory learning ability of honey bees treated orally with sublethal doses of the most widely used spray adjuvants on almonds in the Central Valley of California. Three different adjuvant classes (nonionic surfactants, crop oil concentrates, and organosilicone surfactants) were investigated in this study. Learning was impaired after ingestion of 20 µg organosilicone surfactant, indicating harmful effects on honey bees caused by agrochemicals previously believed to be innocuous. Organosilicones were more active than the nonionic adjuvants, while the crop oil concentrates were inactive. Ingestion was required for the tested adjuvant to have an effect on learning, as exposure via antennal contact only induced no level of impairment.

Conclusions/Significance

A decrease in percent conditioned response after ingestion of organosilicone surfactants has been demonstrated here for the first time. Olfactory learning is important for foraging honey bees because it allows them to exploit the most productive floral resources in an area at any given time. Impairment of this learning ability may have serious implications for foraging efficiency at the colony level, as well as potentially many social interactions. Organosilicone spray adjuvants may therefore contribute to the ongoing global decline in honey bee health.     

"Inert" formulation ingredients with activity: toxicity of trisiloxane surfactant solutions to twospotted spider mites (Acari: Tetranychidae)

Organosilicone molecules are important surfactant ingredients used in formulating pesticides. These methylated silicones are considered inert ingredients, but their superior surfactant properties allow them to wet, and either suffocate or disrupt important physiological processes in mites and insects. Aqueous solutions of the tri-siloxane surfactants Silwet L-77, Silwet 408, and Silwet 806 were bioassayed against adult female two-spotted spider mites, Tetranychus urticae Koch, with leaf dip methods to compare their toxicity with organosilicone molecules containing bulkier hydrophobic components. All three tri-siloxanes in aqueous solutions were equivalently toxic (LC50 = 5.5-8.9 ppm), whereas Silwet L-7607 solutions were less toxic (LC50 = 4,800 ppm) and Silwet L-7200 was nontoxic to mites. In another experiment, the toxicity of Silwet L-77 was affected by the wettability of leaf surfaces. The LC50 shifted from 22 to 84 ppm when mites were tested on bean and strawberry leaf disks, respectively. Droplet spreading on paraffin and surface tension were both related to the toxicity of surfactant solutions. Surface tensions of solutions below 23 mN/m caused > 90% mite mortality in leaf dip bioassays. A field test of Conserve SC and its formulation blank, with and without Dyne-Amic adjuvant (a vegetable oil-organosilicone surfactant mixture) revealed that Dyne-Amic had the greatest miticidal contribution, reducing mite populations by 70%, followed by formulation inactive ingredients. Spinosad, the listed active ingredient in Conserve, only contributed miticidal activity when synergized by Dyne-Amic. Researchers should include appropriate surfactant or formulation blank controls when testing insecticides or miticides, especially when using high spray volumes.     

Evaluation of three techniques used to determine surfactant phytotoxicity

Three techniques were used to measure and compare the phytotoxicities of Triton X-45 and Agral 90 (two organic surfactants) with two organosilicone surfactants (Silwet L-77 and L-7607), and to compare these four non-ionic compounds with a cationic tallow amine surfactant (Hyspray). Total ion efflux and ethylene response methods were used in vivo and in vitro, while a betacyanin efflux method was an in vitro system only. The first two methods, using intact leaves, were considered to be more closely related to normal spraying conditions than the betacyanin efflux test which used explant material. However, the use of intact leaves was thought to bias the results in favour of the leaf penetration properties of the surfactants rather than their phytotoxicities. The in vitro pigment efflux method provided a simple way of ranking the surfactants in order of their potential phytotoxicities, especially with regard to effects on membrane permeability. This ranking, in increasing order of toxicity, was: Silwet L-7607, Silwet L-77, Agral 90, Triton X-45 and Hyspray.     

An ethoxylated alkyl phosphate (anionic surfactant) for the promotion of activities of proteases and its potential use in the enzymatic processing of wool

Pretreatments of wool fabrics with cationic, anionic or non-ionic surfactants were investigated to reduce surface tension and improve the wettability of the fibres in order to promote protease activity on the fibres in subsequent processes. Results showed that an ethoxylated alkyl phosphate (specific anionic surfactant) as well as the widely used non-ionic surfactant was compatible with proteases in the enzymatic treatment of wool. There is therefore a potential for using specific anionic surfactants to achieve efficient enzymatic scouring processes.     

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.     

Silicone antifoam performance enhancement by nonionic surfactants in potato medium

The ability of a silicone antifoam to retard foaming in a liquor prepared from potatoes is enhanced by the addition of ethoxylated nonionic surfactants. The enhancement is non-linear for surfactant concentration, with all 12 surfactants tested possessing a concentration at which foam heights strongly diminish, referred to as the surfactant critical antifoaming concentration (SCAFC). SCAFCs vary between surfactants, with lower values indicating better mass efficiency of antifoaming enhancement. SCAFCs decrease with degree of ethoxylation and decrease with the hydrophilic–lipophilic balance for ethoxylated nonionic surfactants. Surfactant addition produces a mixed water-surface layer containing surfactant and surface-active components in the potato medium. Surface tension reduction does not correlate well with antifoam performance enhancement. A model is proposed where surfactant adsorption promotes desorption of surface-active potato medium components from the water surface. At the SCAFC, desorption is not complete, yet the rate of bubble rupture is sufficiently enhanced to provide excellent foam control. Electronic Publication     

Dependence of buffer acidity and surfactant chain-length on electro-osmotic mobility in thermoplastic microchannels

In this paper, we report the dependence of buffer pH and coating surfactant chain-length on electro-osmotic (EO) mobility in co-polyester microchannels. Thermoplastics co-polyester hydrolyzes to anionic functionality to create electrical double layer on the micro-channel walls. These negatively charged sites are partially or completely screened when long-chain surfactants are added into the buffer. This ancillary technique to modify surface charge polarity to avoid analyte adsorption is known as dynamic coating. We develop a theory to predict the EO mobility tendency on buffer acidity considering the combination of pH-dependent surfactant aggregation and surface dissociation. Our findings of pH-dependent EO mobility in coated channels, using three types of quaternary ammonium surfactants, lauryltrimethyammonium bromide (LTAB), trimethyl (tetradecyl) ammonium bromide (TTAB), and cetyltrimethyammonium bromide (CTAB), agree with our theoretical prediction. We also explain the chain-length dependence of mobility with a collaborative adsorption mechanism of surfactant aggregates.     

Effect of ethyleneoxide groups of anionic surfactants on lipase activity

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant‐enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE₀S) and of sodium lauryl ether sulfate with two EO units (SLE₂S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE₂S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276–1282, 2016     

Evaluation of bacterial surfactant toxicity towards petroleum degrading microorganisms

The acute toxicity of bacterial surfactants LBBMA111A, LBBMA155, LBBMA168, LBBMA191 and LBBMA201 and the synthetic surfactant sodium dodecyl sulfate (SDS) on the bioluminescent bacterium Vibrio fischeri was evaluated by measuring the reduction of light emission (EC(20)) by this microorganism when exposed to different surfactant concentrations. Moreover, the toxic effects of different concentrations of biological and synthetic surfactants on the growth of pure cultures of isolates Acinetobacter baumannii LBBMA04, Acinetobacter junni LBBMA36, Pseudomonas sp. LBBMA101B and Acinetobacter baumanni LBBMAES11 were evaluated in mineral medium supplemented with glucose. The EC(20) values obtained confirmed that the biosurfactants have a significantly lower toxicity to V. fischeri than the SDS. After 30 min of exposure, bacterial luminescence was almost completely inhibited by SDS at a concentration of 4710 mg L(-1). Growth reduction of pure bacterial cultures caused by the addition of biosurfactants to the growth medium was lower than that caused by SDS.     

Factors Affecting the Design of Slow Release Formulations of Herbicides Based on Clay-Surfactant Systems. A Methodological Approach

A search for clay-surfactant based formulations with high percentage of the active ingredient, which can yield slow release of active molecules is described. The active ingredients were the herbicides metribuzin (MZ), mesotrione (MS) and flurtamone (FL), whose solubilities were examined in the presence of four commercial surfactants; (i) neutral: two berols (B048, B266) and an alkylpolyglucoside (AG6202); (ii) cationic: an ethoxylated amine (ET/15). Significant percent of active ingredient (a.i.) in the clay/surfactant/herbicide formulations could be achieved only when most of the surfactant was added as micelles. MZ and FL were well solubilized by berols, whereas MS by ET/15. Sorption of surfactants on the clay mineral sepiolite occurred mostly by sorption of micelles, and the loadings exceeded the CEC. Higher loadings were determined for B266 and ET/15. The sorption of surfactants was modeled by using the Langmuir-Scatchard equation which permitted the determination of binding coefficients that could be used for further predictions of the sorbed amounts of surfactants under a wide range of clay/surfactant ratios. A possibility was tested of designing clay-surfactant based formulations of certain herbicides by assuming the same ratio between herbicides and surfactants in the formulations as for herbicides incorporated in micelles in solution. Calculations indicated that satisfactory FL formulations could not be synthesized. The experimental fractions of herbicides in the formulations were in agreement with the predicted ones for MS and MZ. The validity of this approach was confirmed in in vitro release tests that showed a slowing down of the release of a.i. from the designed formulations relative to the technical products. Soil dissipation studies with MS formulations also showed improved bioactivity of the clay-surfactant formulation relative to the commercial one. This methodological approach can be extended to other clay-surfactant systems for encapsulation and slow release of target molecules of interest.     

Effect of surface-active substances on bioluminescence intensity of bacteria

The study of sensitivity of luminous bacteria isolated from the Black and Azov seas to surfactants from various classes was carried out. It was shown that cationic surfactants had a strong inhibition effect on bacterial luminescence in contrast to anionic and in particular nonionic surfactants. To increase the luminous bacteria sensitivity to the action of OP-10 (nonionic surfactant) and ABS (anionic surfactant), which are widely used in industry, several approaches have been developed. They include modulation of bacterial sensitivity by the additives of cationic substances, use of luminous bacteria at a logarithmic stage of growth, realization of biotesting at low pH = 5.5. The use of these approaches allows to lower effective concentrations of OP-10 and ABS, which caused a decrease of bioluminescence by 50%, 3-200 times and opens perspectives for the use of the bioluminescent method to study these surfactants toxicity on the principle of biosensorics.     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

Rotational dynamics of coumarin 153 in non-ionic mixed micelles of n-octyl-β-D-thioglucoside and Triton X-100

Rotational diffusion of the neutral probe coumarin 153 has been examined in a mixed surfactant system containing n-octyl-β-D-thioglucoside (OTG) and Triton X-100 (TX100), two non-ionic surfactants belonging to the alkyl glucoside and polyoxyethylene alkyl ether families, respectively. Both steady-state and time-resolved fluorescence measurements indicate that the polarity of the dye decreases slightly as the amount of ethoxylated surfactant in the mixed micelle increases. This behaviour can be attributed to migration of the probe towards the inner region of the micellar palisade layer as a result of the increasing hydration induced by the presence of TX100. The anisotropy decay curves, r(t), were well fitted to a biexponential function, characterized by two reorientational times of the probe in the micellar pseudophase. The experimental data were analyzed on the basis of the two-step and wobbling-in-a-cone model, and the results obtained correlated with the changes that occur in the palisade layer of the mixed micelles due to the different structure and nature of the head groups of both surfactants. It was found that, although the average reorientation time of the probe molecule is almost unaffected with the participation of the ethoxylated surfactant, the observed increase in the generalized order parameter suggests a certain rise in the compactness of the mixed micelles.     

Compatibility of OMRI-certified surfactants with three entomopathogenic fungi

The Organic Materials Review Institute (OMRI) is a non-profit organisation providing an independent review of products intended for use in organic production systems to certify compliance with US national organic standards. Since all adjuvants to be used in organic agriculture production are required to meet these standards, OMRI's certified list of products is a convenient starting point when developing organic pest control formulations. In the current study, six OMRI-certified surfactants are tested for their compatibility with three common entomopathogens: Beauveria bassiana, Metarhizium brunneum and Isaria fumosorosea. The fungi were evaluated in two common propagule forms, solid-state produced conidia and liquid-media produced blastospores. The results show that most of the surfactants are compatible with the fungi at a high surfactant concentration (2% w/v). In general, the conidia showed a higher susceptibility (greater reduction in spore germination) to the surfactants than the blastospores under these conditions. In addition, the surface tension and foaming properties of the surfactants were determined.     

Chromatographic behaviour in reversed-phase high-performance liquid chromatography with micellar and submicellar mobile phases: effects of the organic modifier

Continuing our earlier study of the retention behaviour in reversed-phase systems with aqueous mobile phases containing surfactants in concentrations lower (submicellar systems) and higher (micellar systems) than the critical micellar concentration (CMC), we investigated the chromatographic behaviour of various non-ionic solutes in mixed aqueous-organic micellar and submicellar mobile phases and their dependence on the methanol concentration. CMC values were measured for two cationic surfactant and one anionic surfactant in mixed aqueous-methanolic solvents, and were found to increase slightly with increasing methanol concentration. Depending on the character of the surfactant, a limiting concentration of methanol was found, above which micelles do not occur anymore. Sorption isotherms of the surfactants on an octylsilica gel column were measured as a function of the concentration of methanol in aqueous-methanolic solvents. A modified Langmuir equation was used to describe the distribution of the surfactants between the stationary and the mobile phases in the concentration range below CMC. The retention of several polar solutes was measured on an octylsilica gel column both in micellar and submicellar mobile phases containing methanol. The dependencies of the capacity factors of the solutes studied on the concentration of methanol in the mobile phase can be suitably described by the same form of equation as that conventionally used for aqueous-organic mobile phases that do not contain surfactants, but the slopes of the dependencies for a given solute are different in the two ranges of surfactant concentrations. The ratio of the two slopes is controlled by the interaction with micelles and is approximately equal to, below or above 1, depending on whether the solutes do or do not associate with the micelles, or are repulsed from them. Simultaneous control of the concentrations of the organic solvent and of the surfactant in the mobile phase can be used for fine tuning the selectivity of separation as a complement to commonly used adjusting concentrations of two organic solvents in ternary aqueous-organic mobile phases. These effects are illustrated by practical examples of submicellar HPLC with mobile phases containing methanol.     

The effect of varying rates of glyphosate and an organosilicone surfactant on the control of gorse

The herbicidal effect of glyphosate applied to gorse (Ulex europaeus L.) was improved by the addition of increasing amounts (0.5–20 g/litre) of Silwet L-77, an organosilicone surfactant. Increasing the rate of herbicide also enhanced control. There was a highly significant interaction between surfactant rate and herbicide dosage; as the amount of Silwet L-77 was increased the rate of glyphosate could be reduced without loss of herbicide efficacy. However, without any added organosilicone surfactant, glyphosate did not provide more than 73% control of gorse at any rate up to 6.5 kg a.i./ha. With the addition of Silwet L-77, complete mortality of all plants could be achieved with 2.2 kg glyphosate/ ha.     

The Effects of Surfactants on Lipase-Catalysed Hydrolysis of Esters: Activities and Stereoselectivity

The effect of surfactants on the hydrolysis of prochiral and chiral substrates by crude and purified porcine pancreatic lipase (PPL, EC 3.1.1.3)) has been studied. Rather than accelerating the reactions, surfactants slowed down (“inhibited”) the reactions relative to the rate in the absence of surfactant. Surfactants varied in the extent to which the reaction was inhibited. With the crude enzyme there was a correlation between degree of inhibition and the optical purity of the product of hydrolysis of an achiral diester substrate 1. There was no special effect associated with use of surfactants in the concentration range corresponding to critical micelle formation, nor was there any increase in rate of reaction when stable emulsions were formed by using mixtures of surfactants to generate an appropriate hydrophile-lipophile (HLB) balance. A study of the effect of sodium dodecyl sulphate (SDS) on the hydrolysis of the diester 1 by crude PPL showed that the rate of the reaction steadily decreased with increasing surfactant concentration, but that the optical purity of the product first fell and then rose gain, an effect attributed to the differential denaturing action of the surfactant on at least three hydrolytic enzymes. In general, there would seem to be no advantage to be gained from the use of surfactants in the hydrolysis by PPL of compounds of low water solubility; the use of an immiscible co-solvent is more effective.     

Edible lipid nanoparticles: Digestion,absorption, and potential toxicity

Food-grade nanoemulsions are being increasingly used in the food and beverage industry to encapsulate, protect, and deliver hydrophobic functional components, such as oil-soluble flavors, colors, preservatives, vitamins, and nutraceuticals. These nanoemulsions contain lipid nanoparticles (radius <100 nm) whose physicochemical characteristics (e.g., composition, dimensions, structure, charge, and physical state) can be controlled by selection of appropriate ingredients and fabrication techniques. Nanoemulsions have a number of potential advantages over conventional emulsions for applications within the food industry: higher stability to particle aggregation and gravitational separation; higher optical transparency; and, increased bioavailability of encapsulated components. On the other hand, there are also some risks associated with consumption of lipid nanoparticles that should be considered before they are widely utilized, such as their ability to alter the fate of bioactive components within the gastrointestinal tract and the potential toxicity of some of the components used in their fabrication (e.g., surfactants and organic solvents). This article provides an overview of the current status of the biological fate and potential toxicity of food-grade lipid nanoparticles suitable for utilization within the food and beverage industry.     

Studies on octylphenoxy surfactants: X. Effect of oxyethylene chain length on ethylene production by cowpea and mung bean and comparison with linear alcohol hydrophobes

The effects of ethoxy (EO) chain length on surfactant-induced ethylene production for selected octylphenoxy (OP) and linear alcohol (LA) surfactants were established using primary leaves of cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata Dixielee) seedlings. OP surfactant-induced ethylene production was concentration dependent and decreased log linearly with increasing EO content. C_12–15 LA-induced ethylene production decreased log linearly with increasing EO content at 0.1%; however, at 1.0% the relationship was curvilinear with maximum response at 7 EO. Relationships for the C_9–11 and C₉ LA series were nonlinear with greatest biological activity at intermediate (8–12) EO content. Short EO chain length OP surfactants were only slightly water soluble, and induced low levels of ethylene production and phytotoxicity. Addition of OP+1EO to a long chain, water soluble, non-ethylene inducing surfactant (OP+40EO) solution significantly increased ethylene production by OP+1EO in cowpea. A similar response was found for surfactant-induced phytotoxicity and EO chain length as between ethylene production and EO content. Similar EO chain length and ethylene production relationships were found for germinating mung bean (Vigna radiata (L.) R. Wilcz) seeds as for ethylene production and phytotoxicity in cowpea. Radicle growth was markedly inhibited by OP surfactants with an EO chain length of 10 or less and, in some cases, radicles were irreversibly damaged by ethylene inducing surfactants.     

Adjuvant effects of nonionic block polymer surfactants on liposome-induced humoral immune response

The ability of several surface-active agents to stimulate the humoral immune response in mice against haptenated liposomes was tested. The surfactants were block copolymers of hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) that differed in m.w., percentage of POE, and mode of linkage of POP to POE. The liposomes were haptenated with tripeptide-enlarged dinitrophenyl coupled to phosphatidylethanolamine, which was incorporated into the liposomal membrane. Additional injection of mice with surfactant stimulated serum hemagglutination titers and splenic plaque-forming cell (PFC) numbers to varying extents. Block polymers with POP chains flanking a POE center, as well as polymers with POE chains flanking a POP center, displayed high adjuvant activity. These block polymers stimulated the antibody response in a dose-dependent manner. They stimulated the antibody response with both high and low antigen doses. Furthermore, the addition of one of these adjuvants (25R1) reduced the amount of carrier lipid required in the liposome in order to obtain an optimal antibody response. The surfactants, which displayed high adjuvant activity, did not interfere with liposome stability as measured with a liposome lysis assay. Moreover, in vitro preincubation of liposomes with a block polymer did not affect their immunogenicity. Optimal adjuvant activity was observed when both adjuvant and liposomes were administered by the same route. Simultaneous injection of both components, however, is not a prerequisite. Conclusively, it can be stated that nonionic block polymer surfactants are potent adjuvants for stimulation of the antibody response against haptenated liposomes.