Long-term effects of the fungicide pyrimethanil on aquatic primary producers in macrophyte-dominated outdoor mesocosms in two European ecoregions.

Even though empirical data supporting sound ecoregion-specific ecotoxicological evaluations are still scarce, the differences of environmental (including climatic) conditions in specific ecoregions are already currently being regulated for environmental risk assessment of pesticides in Europe. To shed new light on the ecotoxicological effects of pesticides on aquatic communities across ecoregions, the model pollutant pyrimethanil (fungicide) was tested in an outdoor mesocosm study with macrophyte-dominated communities in the European "South" (Portugal) and "Centre" (Germany) regulatory zones. Phytoplankton indicators monitored over 12 months indicated a low risk of the fungicide (0.73 or 0.77 mg pyrimethanil L-1, single application) to phytoplankton functioning; as expected since exposure simulated worst-case scenarios. However, the growth of key structural macroalgae and macrophytes was affected by the fungicide and negative effects occurred, especially in the Central zone experiment. Such effects were not detected earlier than approximately nine months post single pyrimethanil application. The presence or absence of such extremely long-lasting/delayed pyrimethanil effects depended on species, competitive situation, and ecoregion-specific physico-chemical environment. The present findings suggest that a better understanding of both direct and indirect effects of fungicide pollution on aquatic flora in two European ecoregions helps to consolidate the environmental risk assessment of pesticides in specific regulatory zones.

Effects of the fungicide pyrimethanil on biofilm and organic matter processing in outdoor lentic mesocosms.

The effect of the fungicide pyrimethanil (0.7 mg L(−1)) on biofilm development and alder leaf litter decomposition in aquatic ecosystems was assessed in outdoor lentic mesocosms immediately and 274 days after pyrimethanil application. Pyrimethanil decreased ergosterol concentrations (an indicator of fungal biomass) and the abundance and richness of the macroinvertebrate community associated with decomposing leaves. However, because neither fungi nor macroinvertebrates were main factors contributing to decomposition in this particular system, organic matter processing rates were not affected. After 274 days, pyrimethanil concentration in the water column was ≤0.004 mg L(−1) but richness, biomass and composition of the invertebrate community associated with decomposing leaf-litter still showed the effect. The comparison of ergosterol (a molecule existing on both algae and fungal cell membranes), with chlorophyll (an indicator of algal biomass) associated with biofilm suggests that pyrimethanil may decrease fungal biomass and alter the relative abundance of algae and fungi on biofilm developing in control- and treated-mesocosms.

Phenotypic variation as an indicator of pesticide stress in gudgeon: Accounting for confounding factors in the wild.

The response of organisms to environmental stress is currently used in the assessment of ecosystem health. Morphological changes integrate the multiple effects of one or several stress factors upon the development of the exposed organisms. In a natural environment, many factors determine the patterns of morphological differentiation between individuals. However, few studies have sought to distinguish and measure the independent effect of these factors (genetic diversity and structure, spatial structuring of populations, physical-chemical conditions, etc.). Here we investigated the relationship between pesticide levels measured at 11 sites sampled in rivers of the Garonne river basin (SW France) and morphological changes of a freshwater fish species, the gudgeon (Gobio gobio). Each individual sampled was genotyped using 8 microsatellite markers and their phenotype characterized via 17 morphological traits. Our analysis detected a link between population genetic structure (revealed by a Bayesian method) and morphometry (linear discriminant analysis) of the studied populations. We then developed an original method based on general linear models using distance matrices, an extension of the partial Mantel test beyond 3 matrices. This method was used to test the relationship between contamination (toxicity index) and morphometry (PST of morphometric traits), taking into account (1) genetic differentiation between populations (FST), (2) geographical distances between sites, (3) site catchment area, and (4) various physical-chemical parameters for each sampling site. Upon removal of confounding effects, 3 of the 17 morphological traits studied were significantly correlated with pesticide toxicity, suggesting a response of these traits to the anthropogenic stress. These results underline the importance of taking into account the different sources of phenotypic variability between organisms when identifying the stress factors involved. The separation and quantification of the independent effect of such factors provides an interesting outlook regarding the use of these evaluation metrics as indicators of ecosystem health.

Behavioral response of juvenile rainbow trout exposed to an herbicide mixture.

Fish are capable of sensing water-borne chemicals at sub-lethal concentrations. Inadequate behavioral responses to physiological and environmental stimuli owing to adverse effects of aquatic toxicants can have serious implications for survival. In this study we exposed juvenile rainbow trout (Oncorhynchus mykiss) during 5 days to a low-concentration mixture of three co-occurring herbicides: atrazine, linuron and metolachlor, at maximum concentrations of 4.5, 4.9 and 13.4 µg L(-1), respectively. Our hypothesis was that fish behavior - swimming activity and interactions between individuals - would be modified due to exposure to the mixture. We studied these behaviors by observing fish twice-daily throughout the exposure period at 30-s intervals for 5 min, registering the vertical distribution of fish in the water column and the number of agoniztic acts between all individuals. Fish exposed to the mixture of herbicides were hypoactive and spent more time in the lower parts of the aquaria in comparison to non-exposed controls, reflecting inhibited swimming activity. Average swimming height of exposed fish decreased significantly with the number of agoniztic acts, whilst in control groups there was no significant relationship between the two behaviors. Overall, behavior of fish exposed for a short time to the herbicide mixture was altered in comparison to control-fish behavior. The behavioral endpoints chosen here were easily observed, simple to quantify, and of ecological relevance.

Immediate and mid-term effects of pyrimethanil toxicity on microalgae by simulating an episodic contamination.

Since pesticides can represent a threat for non-target aquatic communities, including microalgae, we looked at the effects of the fungicide pyrimethanil on the growth of the freshwater green microalgae Selenastrum capricornutum. Additionally, attenuation of the toxicity of pyrimethanil due to its dissipation in the water was assessed. Pyrimethanil-contaminated samples were taken from outdoor mesocosms one (1.4 mg L(-1) of pyrimethanil) and ten (0.78 mg L(-1) of pyrimethanil) days after pyrimethanil application. Different dilutions were prepared using both nutrient-rich culture medium (LC Oligo) and non-contaminated mesocosm samples, and cell growth inhibition was assessed. Reference mesocosm samples were also diluted with LC Oligo in order to verify how the nutrient concentration in the LC Oligo could improve cell growth. Comparing cell growth of population exposed to pyrimethanil-treated sample taken at day 1 with cells growing in reference sample and LC Oligo, the growth inhibition was 80% (± 6.5) and 95% (± 2.0), respectively. The toxicity of samples taken from contaminated mesocosms at day 10 was attenuated to 34% (± 15) (when compared with reference sample) and 88% (± 3.0) (when compared with LC Oligo), as pyrimethanil concentrations in the mesocosms decreased. In conclusion, (i) pyrimethanil can be an environmental disturber for the microalgae; (ii) the toxicity of pyrimethanil in water was reduced almost 2.4 times (when compared with the reference sample) at as short a period as 10d if assuming that pesticide entrance is not continuous; (iii) toxicity of an environmental sample could be underestimated if the sample/medium used in dilution presents different nutrient levels.

Avoidance response of Danio rerio to a fungicide in a linear contamination gradient.

The present study examines the ability of juvenile Danio rerio to avoid pyrimethanil-contaminated water. An avoidance assay system was used with a contamination gradient formed by seven compartments, through which the fish could move and choose the preferred compartment(s). Additionally, the influence of fish movements in promoting the mixing between compartments and thus disruption of the gradient over time was also examined by testing sodium chloride (NaCl) at sublethal concentrations. Samples with pyrimethanil were obtained from the commercial formulation Mythos®, which was applied to mesocosm systems. Samples of the pyrimethanil-contaminated mesocosms water were collected and a series of seven concentrations (0.2 to 1.4mgL(-1) plus a control) diluted with reference (uncontaminated) mesocosm water were added to the system to form the gradient. After 4h exposure, fish avoidance in the three highest pyrimethanil concentrations ranged from 29 to 66%. The 4h-AC50 (concentration at which 50% of the fish avoided pyrimethanil after 4h exposure) was 1.10 (confidence interval: 1.07 to 1.12)mgL(-1). However, the avoidance pattern after 12h was strongly reduced and it was not possible to calculate the AC50. This is explained by the results of the NaCl experiment, which showed that the movement of fish in the system accelerates the mixing of the solutions between compartments. As pyrimethanil can trigger avoidance response in D. rerio, this fungicide, even at non-lethal concentrations, could be considered an environmental disturber.

Preference and avoidance responses by tadpoles: the fungicide pyrimethanil as a habitat disturber.

Tadpoles of two amphibian species, the neotropical anuran Leptodactylus latrans and the North American bullfrog Lithobates catesbeianus, were used in experiments to assess their preferred spatial distribution along habitat gradients and, thus, to what extent contamination by the fungicide pyrimethanil could trigger active spatial avoidance. The tadpoles were tested in a non-confined multi-compartment static system with a pyrimethanil contamination gradient through which organisms could move freely. Two samples, with and without (reference) pyrimethanil contamination, taken from outdoor mesocosms, were assayed. Tadpoles showed to be able to detect and move to the most favorable environment by preferring compartments containing reference mesocosm water. Pyrimethanil concentrations from 0.2 to 1.4 mg L(-1) were below lethal levels, but acted as habitat disturber since spatial avoidance was triggered. Avoiders of L. latrans reached almost 50 % at 1.4 mg L(-1). The present data reinforces the hypothesis regarding the risk of plant protection products to act, not only as toxicants, but also as habitat disturber, potentially leading to avoidance-driven population decline of amphibians.

Copper-driven avoidance and mortality in temperate and tropical tadpoles.

Amphibians have experienced an accentuated population decline in the whole world due to many factors, one of them being anthropogenic contamination. The present study aimed to assess the potential effect of copper, as a worldwide and reference contaminant, on the immediate decline of exposed population due to avoidance and mortality responses in tadpoles of three species of amphibians across climatic zones: a South American species, Leptodactylus latrans, a North American species, Lithobates catesbeianus, and a European species, Pelophylax perezi. A non-forced exposure system with a copper gradient along seven compartments through which organisms could freely move was used to assess the ability of tadpoles to detect and avoid copper contamination. All species were able to avoid copper at a concentration as low as 100 µg L(-1). At the lowest (sublethal) concentrations (up to 200 µg L(-1)) avoidance played an exclusive role for the population decline, whereas at the highest concentrations (> 450 µg L(-1)) mortality was the response determining population decline. The median concentrations causing exposed population immediate decline were 93, 106 and 180 µg L(-1) for Le. latrans, Li. catesbeianus and P. perezi, respectively. Contaminants might, therefore, act as environmental disruptors both by generating low-quality habitats and by triggering avoidance of tadpoles, which could be an important response contributing to dispersion patterns, susceptibility to future stressors and decline of amphibian populations (together with mortality).