Acute exposure to urban air pollution impairs olfactory learning and memory in honeybees Abstract While the ecological effects of pesticides have been well studied in honeybees, it is unclear to what extent other anthropogenic contaminants such as air pollution may also negatively affect bee cognition and behaviour. To answer this question, we assessed the impacts of acute exposure to four ecologically relevant concentrations of a common urban air pollutant—diesel generated air pollution on honeybee odour learning and memory using a conditioned proboscis extension response assay. The proportion of bees that successfully learnt odours following direct air pollution exposure was significantly lower in bees exposed to low, medium and high air pollutant concentrations, than in bees exposed to current ambient levels. Furthermore, short- and long-term odour memory was significantly impaired in bees exposed to low medium and high air pollutant concentrations than in bees exposed to current ambient levels. These results demonstrate a clear and direct cognitive cost of air pollution. Given learning and memory play significant roles in foraging, we suggest air pollution will have increasing negative impacts on the ecosystem services bees provide and may add to the current threats such as pesticides, mites and disease affecting colony fitness.

Correction to: Spatial variation in aquatic invertebrate and riparian songbird mercury exposure across a river-reservoir system with a legacy of mercury contamination An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cadmium toxicity and its relationship with disturbances in the cytoskeleton, cell cycle and chromosome stability Abstract This study aimed to investigate the mode of action of cadmium (Cd) toxicity at cell level, especially at early stages of plant exposure. Tomato seedlings were cultivated in growth media containing from 0.1 to 70 µM CdCl2 for 24 h. Mitotic index, chromosome abnormality, DNA integrity and organization of tubulin-based structures were assessed in root cells. As higher the Cd concentration in the growth media, higher was the DNA damage intensity and the occurrence of chromosomal abnormalities that included chromosome lost, bridges, stickiness, C-metaphase and polyploidy. The profile of chromosomal aberrations also varied with elevated Cd concentration, being observed increases in the frequency of chromosome stickiness. The mitotic index was reduced at the lowest Cd concentration, but such reduction was statistically similar to that detected at the highest concentration, suggesting that mitotic depression is a rapid outcome and, at same time, a Cd-induced effect that is limited at the first 24 h of direct root exposure to this metal. Under exposure to 20 µM CdCl2, heterogenous distribution of the spindle fibers, formation of two spindle complexes in both of the cell poles, absence of centrosome center, polarization of the spindle fibers during cell division, and non-uniform tubulin deposition in microtubule and phragmoplast were noticed. The results indicate that the tubulin-dependent components of cytoskeleton are Cd targets, and the sensitivity of tubulin-based structures to Cd exposure depends on cell cycle phase. Moreover, DNA damage intensity and chromosomal abnormality profile can be employed as markers of Cd toxicity level.

Modulation of brain serotonin by benzyl butyl phthalate in Fundulus heteroclitus (mummichog) Abstract Endocrine-disrupting chemicals have been known to alter important animal behaviors by modulating serotonin (5-hydroxytryptamine, 5-HT) and dopamine. F. heteroclitus (mummichog) brain serotonin and dopamine levels were quantified by enzyme-linked immunosorbent assay (ELISA) following a 28-day exposure regimen involving daily doses of either 0.1 mg l−1 benzyl butyl phthalate (BBP) dissolved in acetone or acetone alone (0.1 mg l−1). No differences in mean brain mass or total protein homogenate were induced by exposure to the acetone vehicle or BBP in acetone. The acetone vehicle had no effect on dopamine, serotonin, or tyrosine hydroxylase levels, but acetone did decrease tryptophan hydroxylase levels (p = 0.011). Exposure to BBP in acetone decreased dopamine (p = 0.024), increased serotonin (p < 0.001), reduced tryptophan hydroxylase as compared to the acetone vehicle alone (p < 0.001), and had no significant effect on tyrosine hydroxylase levels. This study is the first to report modulation of F. heteroclitus brain serotonin and its enzyme tryptophan hydroxylase following sub-lethal exposure to BBP in an acetone vehicle. In addition, modulation of brain dopamine in F. heteroclitus, sans simultaneous modulation of tyrosine hydroxylase, was also observed. These findings support the use of F. heteroclitus for assessing sub-lethal BBP exposure.

Acute toxicity of copper to the larval stage of three species of ambystomatid salamanders Abstract Copper (Cu) appears to be consistently more toxic to anuran species relative to other vertebrate taxa. There are limited Cu toxicity data for salamanders; of the few studies conducted on salamanders, most examined Cu effects on the embryonic, but not the larval, stage. We performed acute toxicity experiments, to quantify LC50s, on Harrison stage 46 larvae (free swimming hatchlings with egg yolk completely absorbed) of three ambystomatid salamander species. Each LC50 experiment used exposure concentrations of 10, 20, 30, 40, 50, and 60 µg/L with 10 replicates per concentration each containing one larva. We found very high toxicity for all species compared to previously published research on the embryo stage. Specifically, the 4-d LC50s for Ambystoma tigrinum and A. opacum were 35.3 and 18.73 µg/L, respectively. The same Cu concentrations caused similar toxicity to A. talpoideum (LC50 = 47.88 µg/L), but exposures required up to 48 d to elicit the same level of mortality. A time-to-event analysis indicated that time to mortality was significantly affected by Cu concentration. Additionally, for A. talpoideum, we observed that elevated levels of Cu decreased growth rate. Comparisons with previously reported Cu toxicity for embryos suggest that, as with fish, Cu may be more toxic to larval salamander stages than for embryos. Further, our data suggest that Cu is an important environmental contaminant that deserves increased scrutiny on the potential for population-level effects where contamination has occurred in wetlands and streams inhabited by salamanders.

Behavioral and immunotoxic effects of Prograf® (tacrolimus) in the male Siamese fighting fish Abstract Siamese fighting fish (Betta splendens) has been extensively exploited in the behavioral and physiological toxicology studies of drugs. Tacrolimus is an immunosuppressant drug largely used in liver and renal transplantations. Here we found that a 7-day exposure of male B. splendens to concentrations of 0.05 and 0.1 µg/mL Prograf® (tacrolimus) caused alterations in aggression and immunity indexes. Tacrolimus exposed fish presented lower opercular display in a mirror test which is indicative of reduced aggression. In addition, serum levels of lysozyme, IgM, alternative complement, and bactericidal activity of subjects exposed to 0.1 µg/mL tacrolimus were lower than those from the control treatment. These results showed the behavioral impairment and immunotoxic impacts of tacrolimus in a model of aquatic toxicology. The results suggest fishes provide a possible model for better understanding of the drug action in vertebrates, and possible consequences for the environment via its effects on non-target organisms in an ecotoxicology context.

Oral acute toxicity and impact of neonicotinoids on Apis mellifera L. and Scaptotrigona postica Latreille (Hymenoptera: Apidae) Abstract Wild and managed bees are essential for crop pollination and food production. However, the widespread use of insecticides such as neonicotinoids may affect the survival, development, behavior, and maintenance of bee colonies. Therefore, in this study we evaluated the impacts of three neonicotinoid insecticides on the survival and walking abilities of the Africanized honeybee A. mellifera and stingless bee S. postica. A. mellifera was more susceptible than S. postica to all neonicotinoids tested. The median lethal concentrations LC50 values estimated for acetamiprid, imidacloprid, and thiacloprid were 189.62, 22.78, and 142.31 ng µL–1 of diet for A. mellifera, and 475.94, 89.11, and 218.21 ng µL–1 of diet for S. postica, respectively. All tested neonicotinoids affected the speed, distance traveled, duration and frequency of resting, and continuous mobility of both bee species. The results showed that in spite of the different susceptibility to compounds with cyano and nitro radicals, the behavioral variables showed different levels of commitment according to the molecule insecticide and bee species. These results contribute not only to the understanding of the effects of neonicotinoid insecticides on A. mellifera and S. postica, but also to help in the development of protocols that aim to reduce the impact of these insecticides in Neotropical environments.

Assessing predator-prey interactions in a chemically altered aquatic environment: the effects of DDT on Xenopus laevis and Culex sp. larvae interactions and behaviour Abstract Behavioural assays are used as a tool to understand ecotoxicological effects on organisms, but are often not applied in an ecologically relevant context. Assessment of the effect of chemical contaminants on behaviours relating to fitness and trophic interactions for example, requires incorporating predator-prey interactions to create impact assessments. Dichlorodiphenyltrichloroethane (DDT) is a controlled substance but is still regularly used as a form of mosquito control. There is little explicit information on the effect of DDT on animal behaviour and the consequent effects upon trophic interactions. This study uses a 3 × 2 factorial design to assess the feeding behaviour of Xenopus laevis toward Culex sp. larvae when supplied with different prey cues. We also assess the behavioural responses of mosquito larvae when supplied with no threat cue and predator threat cues when exposed to 0 µg/L, 2 µg/L and 20 µg/L DDT. There was a significant “DDT exposure” x “prey cue” interaction whereby DDT significantly decreased the foraging behaviour of X. laevis towards live prey cues, however there was no effect of DDT on X. laevis response to olfactory prey cues. Dichlorodiphenyltrichloroethane exposure caused mosquito larvae to appear hyperactive regardless of DDT concentration. Mosquito larvae anti-predator response was significantly dampened when exposed to 2 µg/L DDT, however when exposed to 20 µg/L the anti-predator responses were not impaired. Our results indicate a complex interplay in trophic interactions under DDT exposure, wherein effects are mediated depending on species and concentration. There are possible implications regarding reduced anti-predator behaviour in the prey species but also reduced foraging capacity in the predator, which could drive changes in ecosystem energy pathways. We demonstrate that in order to quantify effects of pesticides upon trophic interactions it is necessary to consider ecologically relevant behaviours of both predator and prey species.

Desmodesmus subspicatus co-cultured with microcystin producing (PCC 7806) and the non-producing (PCC 7005) strains of Microcystis aeruginosa Abstract Although microcystins (MCs) are the most commonly studied cyanotoxins, their significance to the producing organisms remains unclear. MCs are known as endotoxins, but they can be found in the surrounding environment due to cell lysis, designated as extracellular MCs. In the present study, the interactions between MC producing and the non-producing strains of Microcystis aeruginosa, PCC 7806 and PCC 7005, respectively, and a green alga, Desmodesmus subspicatus, were studied to better understand the probable ecological importance of MCs at the collapse phase of cyanobacterial blooms. We applied a dialysis co-cultivation system where M. aeruginosa was grown inside dialysis tubing for one month. Then, D. subspicatus was added to the culture system on the outside of the membrane. Consequently, the growth of D. subspicatus and MC contents were measured over a 14-day co-exposure period. The results showed that Microcystis negatively affected the green alga as the growth of D. subspicatus was significantly inhibited in co-cultivation with both the MC-producing and -deficient strains. However, the inhibitory effect of the MC-producing strain was greater and observed earlier compared to the MC-deficient strain. Thus, MCs might be considered as an assistant factor that, in combination with other secondary metabolites of Microcystis, reinforce the ability to outcompete co-existing species.

Determination of the acute toxic effects of zinc oxide nanoparticles (ZnO NPs) in total hemocytes counts of Galleria mellonella (Lepidoptera: Pyralidae) with two different methods Abstract Zinc oxide nanoparticles (ZnO NPs) are now commonly used in many consumer products (detergents, antibacterial products, protective creams). The aim of the study is to determine the ecotoxicological effects of ZnO NPs on the survival and in the total hemocyte counts of Galleria mellonella L. 1758 (Lepidoptera: Pyralidae) with two different methods (automated cell counter and hemocytometer). A toxicity test was performed to determine the lethal concentrations of ZnO NPs on larvae by force feeding method. After 24 h the treatment, LC50 was 6.03 μg/10 µl and LC99 was 12.86 μg/10 µl for force fed larvae according to probit assay. The NP that induced changes in the total hemocytes counts were counted by optical microscopy (larvae exposed to four different doses of ZnO NPs) and by automated cell counter. Analyses of total hemocyte counts of the insect were performed with four doses (0.5, 1, 2.5, 5 μg/10 µl) <LC50 at 24 h upon feeding larvae revealed that a number of hemocytes did not show significant changes in all treatments compared with control in the optical microscopy counting. The similar statistically insignificant counting results were also seen in the automated cell counting results. The percentage of the dead cells (10.01%) in the 5 μg/10 µl group was significantly higher than the control group (3.03%) and showed a statistically significant difference at 24 h in the optical microscopy count with trypan blue viability test.