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International audience; Diffuse pollution of the environment by pesticides has become a major soil threat to non-target organisms, such as earthworms for which declines have been reported. However some endogeic species are still abundant and persist in intensively cultivated fields, suggesting they become tolerant to long-term anthropogenic pressure. We thus considered the working hypothesis that populations of Aporrectodea caliginosa earthworms from conventionally managed fields developed a tolerance to pesticides compared with those from organically managed fields. To investigate this hypothesis, we studied earthworm populations of the same genetic lineage from soils that were either lowly or highly contaminated by pesticides to detect any constitutive expression of differentially expressed molecular pathways between these populations. Earthworm populations were then experimentally exposed to a fungicide-epoxiconazole-in the laboratory to identify different molecular responses when newly exposed to a pesticide. State-of-the-art omics technology (RNA sequencing) and bioinformatics were used to characterize molecular mechanisms of tolerance in a non-targeted way. Additional physiological traits (respirometry, growth, bioaccumulation) were monitored to assess tolerance at higher levels of biological organization. In the present study, we generated the de novo assembly transcriptome of A. caliginosa consisting of 64,556 contigs with N50 = 2862 pb. In total, 43,569 Gene Ontology terms were identified for 21,593 annotated sequences under the three main ontologies (biological processes, cellular components and molecular functions). Overall, we revealed that two same lineage populations of A. caliginosa earthworms, inhabiting similar pedo-climatic environment, have distinct gene expression pathways after they long-lived in differently managed agricultural soils with a contrasted pesticide exposure history for more than 22 years. The main difference was observed regarding metabolism, with upregulated pathways linked to proteolytic activities and the mitochondrial respiratory chain in the highly exposed population. This study improves our understanding of the long-term impact of chronic exposure of soil engineers to pesticide residues.

International audience; Climate change increases average temperatures and the occurrence of extreme weather events, in turn accentuating the risk of organism exposure to temperature stress. When thermal conditions become stressful, the sensitivity of insects toward insecticides can be exacerbated. Likewise, exposure of insects to insecticides can subsequently influence their ability to handle stressful temperatures. Here, we investigated the effects of constant temperature and daily heat spikes, in presence/absence of insecticide treatment (cyfluthrin), on the condition (impairment of mobility) and thermal tolerance to cold (-6 °C) and heat (42.5 °C) of the terrestrial beetle Alphitobius diaperinus. The responses of insects from four populations (three farm-collected populations, one laboratory population) to different durations of extreme temperature exposure were compared. The results showed that the laboratory population was generally more sensitive to extreme cold and heat temperatures, with less than 50% of adults recovering after an exposure at -6 or +42.5 °C for 3h. Significant differences in the level of thermal tolerance were also found among insects from poultry farms. Cyfluthrin exposure incurred detrimental effects to insects’ condition in all but one population. For two out of the four populations, mobility impairment was increased when adults were exposed to daily heat spikes (6 h per day at 38 °C) and cyfluthrin simultaneously, compared to cyfluthrin exposure at constant temperatures; yet, no significant interaction between the two stressors was found. Finally, using one farm collected population, effects of pre-exposure to cyfluthrin on extreme temperature tolerance provided another example of the toxicant-induced climate sensitivity in insects.

International audience; Primary consumers in freshwater ecosystems, such as the zooplankton organism Daphnia magna, are highly affected by cyanobacteria, both as they may use it as a food source but also by cyanobacterial metabolites present in the water. Here, we investigate the impacts of cyanobacterial metabolites focussing on the environmental realistic scenario of the naturally released mixture without crushing cyanobacterial cells or their uptake as food. Therefore, D. magna were exposed to two concentrations of cell free cyanobacterial spent medium from Microcystis aeruginosa PCC 7806 to represent higher and lower ecologically-relevant concentrations of cyanobacterial metabolites. Including microcystin-LR, 11 metabolites have been detected of which 5 were quantified. Hypothesising concentration and time dependent negative impact, survival, gene expression marking digestion and metabolism, oxidative stress response, cell cycle and molting as well as activities of detoxification and antioxidant enzymes were followed for 7 days. D. magna suffered from oxidative stress as both catalase and glutathione S-transferase enzyme activities significantly decreased, suggesting enzyme exhaustibility after 3 and 7 days. Moreover, gene-expressions of the 4 stress markers (glutathione S-transferase, glutathione peroxidase, catalase and thioredoxin) were merely downregulated after 7 days of exposure. Energy allocation (expression of glyceraldehyde-3-phosphate dehydrogenase) was increased after 3 days but decreased as well after 7 days exposure. Cell cycle was impacted time dependently but differently by the two concentrations, along with an increasing downregulation of myosin heavy chain responsible for cell arrangement and muscular movements. Deregulation of nuclear hormone receptor genes indicate that D. magna hormonal steering including molting seemed impaired despite no detection of microviridin J in the extracts. As a consequence of all those responses and presumably of more than investigated molecular and physiological changes, D. magna survival was impaired over time, in a concentration dependent manner. Our results confirm that besides microcystin-LR, other secondary metabolites contribute to negative impact on D. magna survival and stress response.

International audience; This study aimed to assess how bioturbation by freshwater benthic macroinvertebrates with different biological traits alone or in combination could modify trace elements (TE) fate between sediment and water, and if water TE concentration and animal TE content impair their body stores. Three macroinvertebrate species were exposed to TE contaminated sediment for 7 days: the omnivorous Echinogammarus berilloni (Amphipoda), the sediment feeding Tubifex tubifex (Oligochaeta) and the filter feeding Pisidium sp. (Bivalvia). Treatments were one without invertebrates (control), two with amphipods or mussels alone, and the combinations amphipod-mussel, and amphipod-mussel-worms. Water TE concentration increased significantly in 2 or 3 species mesocosms, concerning mainly Rare Earth Elements, Cr, U and Pb, known to be associated to the colloidal phase. By contrast, water soluble TE were not affected by animals. For both, amphipods and mussels, TE body content increased with the number of coexisting species. For amphipods, this increase concerned both, soluble and colloid-associated TE, possibly due to intense contact and feeding from sediment and predation on tubificids. TE bioaccumulation in mussel was less important and characterized by soluble TE, with water filtration as most plausible uptake route. Protein, triglyceride and Whole Body Energy Budget increased in amphipods with the number of coexisting species (probably by feeding on mussels’ feces and tubificids) whereas triglycerides declined in mussels (presumably filtration was disturbed by amphipods). This study highlights interspecific interactions as key drivers explaining both: TE bioturbation, depending on their water solubility or colloidal association, and the exposure/contamination of species through another species activity.

International audience; Due to eutrophication, freshwater ecosystems frequently experience cyanobacterial blooms, many of which produce bioactive metabolites that can affect vertebrates and invertebrates life traits. Zooplankton are able to develop tolerance as a physiological response to cyanobacteria and their bioactive compounds, however, this comes with energetic cost that in turn influence Daphnia life traits and may impair populations. Vice versa, it has been suggested that Daphnia are able to reduce cyanobacterial dominance until a certain cyanobacterial density; it remains unclear whether Daphnia metabolites alone influence the physiological state and bioactive metabolites production of cyanobacteria. Hence, this study investigates mutual physiological reactions of toxic Microcystis aeruginosa PCC7806 and Daphnia magna. We hypothesize that a) the presence of D. magna will negatively affect growth, increase stress response and metabolites production in M. aeruginosa PCC7806 and b) the presence of M. aeruginosa PCC7806 will negatively affect physiological responses and life traits in D. magna. In order to test these hypotheses experiments were conducted in a specially designed co-culture chamber that allows exchange of the metabolites without direct contact. A clear mutual impact was evidenced. Cyanobacterial metabolites reduced survival of D. magna and decreased oxidative stress enzyme activity. Simultaneously, presence of D. magna did not affect photosynthetic activity. However, ROS increase and tendencies in cell density decrease were observed on the same day, suggesting possible energy allocation towards anti-oxidative stress enzymes, or other protection mechanisms against Daphnia infochemicals, as the strain managed to recover. Elevated concentration of intracellular and overall extracellular microcystin MC-LR, as well as intracellular concentrations of aerucyclamide A and D in the presence of Daphnia, indicating a potential protective or anti-grazing function. However, more research is needed to confirm these findings.

International audience; The mass development and expansion of cyanobacteria release cyanotoxins in the aquatic environment and cause serious problems for grazers such as micro-zooplankton. In contrast to aquatic ecosystems in temperate regions, impacts of cyanobacteria and their toxins on tropical micro-crustaceans are relatively understudied. In this study, acute and chronic effects of pure microcystin-LR (MC-LR), a crude extract of water bloom sample with a dominance of Microcystis aeruginosa containing microcystins (MCE) and living cells of Cylindrospermopsis curvispora were tested on a tropical micro-crustacean, Daphnia lumholtzi. The 24 h- and 48 h-LC50 values for MC-LR ranged from 247–299, and 331–409 μg MCE L−1, respectively. Exposures to 1–25 μg MCE L−1 decreased survival, fecundity, and reproduction of D. lumholtzi. The impacts of C. curvispora cells on life-history traits of D. lumholtzi were density-dependent and more severe than the impacts of dissolved microcystins. It could be that the adverse effects of C. curvispora on D. lumholtzi are linked to a combination of potential toxic metabolites, mal-nutrients, feeding and swimming interference. Daphnia lumholtzi used in this study is more sensitive to microcystins compared to Daphnia species from temperate regions. Therefore, we highly suggest using D. lumholtzi as a model species for toxicity testing and monitoring on water quality, particularly in tropical countries.

Most indigenous European and North American mussels live on or partially burrowed in sediments of lakes and slow-flowing streams and rivers. They nourish merely non-selectively by filtering suspended particles (and the bacteria colonizing them), and with this activity, they provide the ecosystem service of water clearance, particle processing and nutrient dynamics (release and biodeposition), and thus link the pelagic compartment to the benthic one by transferring material and nutrients from the water column to the sediment. Moreover, by their turbation activity, they oxygenize the upper layers of the sediment aiding bacterial degradation of organic matter.

International audience; Metal contamination is one of the major issues to the environment worldwide, yet it is poorly known how exposure to metals affects tropical species. We assessed the sensitivity of a tropical micro-crustacean Daphnia lumholtzi to three trace metals: copper (Cu), zinc (Zn) and nickel (Ni). Both, acute and chronic toxicity tests were conducted with metals dissolved in in situ water collected from two sites in the lower part of the Mekong River. In the acute toxicity test, D. lumholtzi neonates were exposed to Cu (3-30 mu g L-1), Zn (50-540 mu g L-1) or Ni (46-2356 mu g L-1) for 48 h. The values of median lethal concentrations (48 h-LC50) were 11.57-16.67 mu g Cu L-1, 179.3-280.9 mu g Zn L-1, and 1026-1516 mu g Ni L-1. In the chronic toxicity test, animals were exposed to Cu (3 and 4 mu g L-1), Zn (50 and 56 mu g L-1), and Ni (six concentrations from 5 to 302 mu g L-1) for 21 days. The concentrations of 4 mu g Cu L-1 and 6 mu g Ni L-1 enhanced the body length of D. lumholtzi but 46 mu g Ni L-1 and 50 mu g Zn L-1 resulted in a strong mortality, reduced the body length, postponed the maturation, and lowered the fecundity. The results tentatively suggest that D. lumholtzi showed a higher sensitivity to metals than related species in the temperate region. The results underscore the importance of including the local species in ecological risk assessment in important tropical ecosystems such as the Mekong River to arrive at a better conservational and management plan and regulatory policy to protect freshwater biodiversity from metal contamination. (C) 2016 Elsevier B.V. All rights reserved.

Climate change and human activities induce an increased frequency and intensity of cyanobacterial blooms which could release toxins to aquatic ecosystems. Zooplankton communities belong to the first affected organisms, but in tropical freshwater ecosystems, this issue has yet been poorly investigated. We tested two questions (i) if the tropical Daphnia lumholtzi is capable to develop tolerance to an ecologically relevant concentration of purified microcystin-LR and microcystins from cyanobacterial extract transferable to F1 and F2 generations? And (ii) would F1 and F2 generations recover if reared in toxin-free medium? To answer these questions, we conducted two full factorial mutigenerational experiments, in which D. lumholtzi was exposed to MC-LR and cyanobacterial extract at the concentration of 1 μg L−1 microcystin continuously for three generations. After each generation, each treatment was spit into two: one reared in the control (toxin free) while the other continued in the respective exposure. Fitness-related traits including survival, maturity age, body length, and fecundity of each D. lumholtzi generation were quantified. Though there were only some weak negative effects of the toxins on the first generation (F0), we found strong direct, accumulated and carried-over impacts of the toxins on life history traits of D. lumholtzi on the F1 and F2, including reductions of survival, and reproduction. The maturity age and body length showed some inconsistent patterns between generations and need further investigations. The survival, maturity age (for extract), and body length (for MC-LR) were only recovered when offspring from toxin exposed mothers were raised in clean medium for two generations. Chronic exposure to long lasting blooms, even at low density, evidently reduces survival of D. lumholtzi in tropical lakes and reservoirs with ecological consequences. Exposure to environmentally relevant concentrations of cyanobacterial toxins for 3 generations tropical Daphnia lumholtzi developed no or marginal tolerance.

International audience; Potential benefits of simulations and virtual laboratory exercises in natural sciences have been both theorised and studied recently. This study reports findings from a pilot study on student attitude, motivation and self-efficacy when using the virtual laboratory programme Labster. The programme allows interactive learning about the workflows and procedures of biological and biochemical experiments, the operation of relevant apparatuses, including the ability to adjust parameters, and the production of results. The programme was used as supplement to mandatory laboratory exercises in two undergraduate courses (i. microbiology and ii. pharmaceutical toxicology) at the University of Southern Denmark. With a theoretical basis in motivational theories, students’ (n = 73) motivation and attitude towards the virtual exercises were evaluated. After completing virtual laboratory cases, the students felt significantly more confident and comfortable operating laboratory equipment, but they did not feel more motivated to engage in virtual laboratories compared to real laboratories. Teachers observed that students were able to participate in discussions at higher levels than in previous years where the programme was not used. The study concludes that virtual laboratories have the potential to improve students’ pre-laboratory preparation. © 2016 Royal Society of Biology.

International audience; Cyanobacterial blooms can be harmful to environmental and human health due to the production of toxic secondary metabolites, known as cyanotoxins. Microcystins (MCs), one of the most widespread class of cyanotoxins in freshwater, have been found to be positively correlated with cyanobacterial biomass as well as with nitrogen and phosphorus concentrations in temperate lakes. However, in tropical water bodies,cyanobacterial density and cyanotoxin correlation to environmental factors is not fully understood. In the present study, we examined the effects of total nitrogen and total phosphorus (TP) concentrations among other environmental parameters on cyanobacterial community structure and MC concentrations in the Dau Tieng reservoir, a tropical, eutrophic water body in Southern Vietnam. Cyanobacterial biomass and MC content were monitored monthly from March 2012 to February 2013, when MCs were present in the Dau Tieng Reservoir. The highest concentrations of intracellular MCs were found in September and February when cyanobacteria biomass reached maximum values, with 2.50 and 2.13 mg MC.L–1, respectively. Principle component analysis and redundancy analysis showed that MC concentration was positively correlated with the biomass of the cyanobacterial order Chroococcales, whereas TP was the primary abiotic factor influencing cyanobacterial biomass and MC concentrations in the Dau Tieng Reservoir. In addition, Bayesian model average analysis was used to construct a prediction model of MCs using cyanobacterial biomass and environmental variables revealing a suite of useful predictive factors for MCs in the Dau Tieng Reservoir, includingwater temperature, TP and the biomass of Chroococcales.

International audience; Because earthworms sustain soil functioning and fertility, there is a need to advance the knowledge of their adaptation potential to chemicals in anthropogenic landscapes. Our hypothesis is that there is acclimation to organic chemicals (pesticides) in earthworms that durably persist under conventional farming in anthropogenic landscapes. The adaptation capability of two populations of earthworms (Aporectodea caliginosa) having a different chemical exposure history, - one originating from 20 years of organic farming (naïve population) and another from 20 years of conventional farming (pre-exposed population) - to cope with soil organic pollutant (Opus®, epoxiconazole a worldwide used fungicide) were investigated. Several complementary metabolic and energetic endpoints were followed, and cast production was assessed as a behavioural biomarker related to earthworms ecological role for the soil. Basal metabolism reflected by respiration rate was increased in both fungicide-exposed worms compared to controls. Glycogen resources were decreased in the same proportion in the two populations but more rapidly for the naïve (7 days) than for the pre-exposed population (28 days). Soluble protein and most amino-acids contents increased in the pre-exposed population only, suggesting a detoxification mechanism. Metabolomic profiles showed a cut-off between fungicide-exposed and control groups in the pre-exposed earthworms only, with an increase in most of the metabolites. Exposure to a low dose of epoxiconazole increased cast production of pre-exposed earthworms, and this resulted in an increase in pesticide disappearance. As far as we know, this is the first study which evidenced there is an acclimation to an agricultural chemical in earthworms derived from conventional farming that also relates to a change in their burrowing behaviour, and for which larger consequences for the soil ecosystem need to be addressed. This original finding is of major interest in the frame of ecosystem resilience to global changes. Whether this physiological adaptation is a general pattern of response against fungicides or other pesticides would need to be confirmed with other molecules and agricultural contexts.

International audience; Carbamazepine (CBZ), Ibuprofen (IBU) and Bezafibrate (BEZ) were tested for their potential to bioaccumulate and provoke molecular changes in the non-target organism Dreissena polymorpha. mRNA changes of enzymes and other proteins involved in the prevention from protein damage (heat shock protein 70, hsp70) and oxidative stress (superoxide dismutase, SOD; catalase, CAT; metallothionein, MT), biotransformation (pi-class glutathione S-transferase, piGST; aryl hydrocarbon receptor, AH-R), elimination (P-glycoprotein, P-gp) and reversible protein posttranslational modification (protein phosphatase 2A, PP2A) served as molecular biomarkers. Mussels were exposed in a flow-through system to increasing concentrations of the three substances (1, 10, 100 and 1000 nM). The two lower concentrations correspond to environmentally relevant concentrations detected in surface and effluent waters, respectively. Measuring tissue concentration after one, four and seven days the uptake of CBZ and IBU by the mussels could be evidenced, whereas no accumulation data could be achieved for BEZ. The bioconcentration factor was highest for mussels exposed to the lowest CBZ and IBU concentrations, with 90 and 460-fold higher tissue concentration, respectively, after seven days. CBZ was the only substance tested which caused a significant increase in gill mRNA level of hsp70 after only one day exposure, evidencing the potential of CBZ to immediately provoke a stress condition and assumingly protein damage in gills. After longer exposure, mussels displayed down-regulated mRNA levels of hsp70 and SOD in gills, as well as of MT and P-gp in the digestive gland, hinting on an inhibitory character of CBZ. In IBU exposed mussels increased oxidant stress conditions were evidenced by induced mRNA levels in the digestive gland of CAT and MT, as well as SOD after one and four days, respectively. A concentration as found at sewage treatment plant effluents provoked an increase in transcript levels of piGST, suggesting enhanced need for biotransformation of IBU or by-products derived from oxidative stress. Also exposure to an environmentally relevant BEZ concentration provoked an immediate increase in piGST transcript level in the digestive gland followed by up-regulated hsp70 after four and seven days evidencing a chronic stress condition for the mussels.

International audience; Lake Chao, China, is highly eutrophicated and experiences recurrent dense cyanobacterial blooms. Its surface water is used as drinking water resource for Hefei city, hence the potential toxicity of those cyanobacteria was of interest. Sixteen isolated strains of Microcystis aeruginosa evidenced that non-toxic, toxic and highly toxic strains coexist in the lake. Microcystin variants within one strain ranged up to 11, the concentration up to 4.799 mg g DW1. Mass spectrometry analysis confirmed desmethylated microcystin variants.

International audience; Seven strains of cyanobacteria from Tri An Reservoir, a drinking water reservoir for millions of people in Southern Vietnam, were isolated, cultivated, identified and described. They originated from the species Microcystis aeruginosa, M. botrys, M. wesenbergii, Anabaena circinalis,A. smithii, Aphanizomenon aphanizomenoides and Cylindrospermopsis raciborskii. Microcystin (MC) content in the cultures and two scum samples from Tri An Reservoir were determined by high performance liquid chromatography. Four variants of MC, MC-LR, MC-RR, MC-LA, MC-LY and one un-determined variant were detected in the scum samples but none were found in the cultures. The two variants MC-LA and MC-LY were recorded for the first time for Vietnam. Total MC concentrations in the two scum samples were 0.45 and 0.64 mg g-1 dried weight, respectively. The genus Microcystis in Tri An Reservoir was assumed to be a MC producer as the other possible MC producing species A. circinalis was usually detected in lower quantities.

The golden mussel Limnoperna fortunei was used as a biomonitor of environmental pollution in the Suquía River basin around Córdoba City (Argentina). The sampling sites along the river were chosen according to their increasing levels of pollutants (e.g. heavy metals) as well as biological oxygen demand (BOD) and chemical oxygen demand (COD). A water quality index (WQI) was constructed from the interaction of several normalized factors that affect the aquatic environment, such as the mentioned pollutants and physico-chemical characteristics of the sampling sites. Activity changes of biotransformation enzyme glutathione S-transferase (GST) and the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT), after exposure to pollutants, served as biomarkers. Membrane bound GST and antioxidant enzymes responded at the most polluted sampling site within 1 day showing increased activities lasting for 4 days. Further sampling was restricted due to no survival of the animals. Antioxidant enzymes GPx, GR and CAT were sensitive responding to the different pollution scenarios, showing good correlation to the chemical characterization.

The renaturation success of an urban stream, formally used for discharge of treated sewage waters was investigated by active biomonitoring with Dreissena polymorpha based on molecular biomarkers and compared to a semi-natural stream and laboratory controls. Response to pollution charges were analyzed by reverse transcriptase-PCR of heat-shock protein (hsp70), P-glycoprotein (P-gp), catalase (CAT) and pi class glutathione S-transferase (piGST). Hsp70 transcription was similarly induced at both sites, indicating protein damage. At the semi-natural stream CAT and P-gp were induced, indicating oxidative stress and increased discharge of pollutants, which correlated to high amounts of aluminum at this site. piGST was induced at one sampling date at the renaturated stream only, but identification of the causing pollutant was not achieved. Results confirm regeneration of the formerly sewage polluted stream, because induction of the tested biomarkers was either at or below the levels of the semi-natural stream.

Molluscs and especially bivalves are able to accumulate dinoflagelates, diatoms and cyanobacteria toxins, and, being vectors for these toxins, transfer them along food chains. The data obtained from laboratory experiments showed that bivalve molluscs are resistant to cyanobacteria toxins. In this work, we wanted to test if Mytilus galloprovincialis organs react to microcystins and other cyanobacteria compounds by inducing or decreasing its GST activity. Acclimated mussels M. galloprovincialis were exposed to the toxic Microcystis aeruginosa M13 strain. Exposure of mussels to toxins was done in three ways: living Microcystis cells, crude Microcystis extracts and pure toxins. The measurement of soluble and microsomal GST activity in the different mussel organs was done by using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB). Analysis of the GST activity of the control mussels using CDNB as a substrate showed that cytosolic activity is much more significant than microsomal. Intact M. aeruginosa cells did not induce any significant response from the mussels, showing that these animals are quite resistant to the cyanobacteria if they are intact. On the other hand, cell extracts caused an important effect in the gut, in the gills and in the labial palps, although in different ways. There was an increase in GST activity in the gut and gills of mussels exposed to Microcystis extracts, showing a response of this detoxication pathway, but in the labial palps a severe reduction in GST activity occurred. Pure MC LR+YR induced an increase in GST activity in all organs but the labial palps. The results showed that other substances apart from microcystins may cause stress to mussels and affect detoxication enzymes such as GST.

Influence of housefly maggot meal (magmeal) diets on the activities of catalase (CAT), glutathione S-transferase (GST) and glycogen concentration in liver of Tilapia Oreochromis niloticus fingerling was evaluated. Triplicate groups of fifteen fish (initial average weight 2.0+/-0.1 g) were fed eight weeks with seven test diets (in average 36% crude protein, dry matter) formulated by replacing fish meal with magmeal. Percentage body weight gain (591-724.46%), food conversion ratio (1.05-1.22) and standard growth rate (3.45-3.76) in all feeding groups were not significantly different (P

Dissolved organic matter from natural sources (DNOM) is omnipresent in aquatic ecosystems. Besides affecting bioavailability of substances including xenobiotics, it directly influences physico-chemistry of the habitat and there is increasing evidence for it is interaction with organisms. We investigated direct and interacting effects of DNOM from three sources, Lake Valkea-Kotinen, Svartberget Brook, and Lake Fuchskuhle with the herbicide paraquat on the oligochaete worm Lumbriculus variegatus . Bioavailability of paraquat to L. variegates as well as activities of antioxidative enzymes catalase (CAT) and peroxidase (POD) and biotransformation enzyme soluble glutathione S -transferase (sGST) were assessed without and in the presence of DNOM. Furthermore, metabolic heat dissipation due to the exposure was quantified. Uptake of paraquat into the worms was concentration dependently reduced by DNOM, and with differences concerning the DNOM sources. sGST and CAT responded with increased activities to DNOM (5 and 25 mg C l −1 ) and paraquat (5.0, 50, and 500 μg l −1 ) separately. Paraquat at 5.0 μg l −1 and DNOM in combination caused increased activities of sGST, especially at 5 mg C l −1 , but inhibition of CAT activities. The latter probably occurred due to saturation of the enzyme. Changes in enzyme activities were independent from the source of DNOM. Increasing DNOM concentrations raised metabolic heat dissipation in L. variegatus with maximum at 3 h of exposure. In the combined treatments, metabolic heat dissipation changed more due to the source of DNOM than due to the bioavailability of paraquat.

International audience; Freshwater gastropods are increasingly exposed to multiple stressors in the field such as the herbi- cide glyphosate in Roundup formulations and cyanobacterial blooms either producing or not producing microcystins (MCs), potentially leading to interacting effects. Here, the responses of Lymnaea stagnalis to a 21-day exposure to non-MC or MC-producing (33 ug L−1 ) Planktothrix agardhii alone or in combination with the commercial formulation RoundUp® Flash at a concentration of 1 ug L−1 glyphosate, followed by 14 days of depuration, were studied via i) accumulation of free and bound MCs in tissues, and ii) activities of anti-oxidant (catalase CAT) and biotransformation (glutathione-S-transferase GST) enzymes. During the intoxication, the cyanobacterial exposure induced an early increase of CAT activity, independently of the MC content, probably related to the production of secondary cyanobacterial metabolites. The GST activity was induced by RoundUp® Flash alone or in combination with non MC-producing cyanobacte- ria, but was inhibited by MC-producing cyanobacteria with or without RoundUp® Flash. Moreover, MC accumulation in L. stagnalis was 3.2 times increased when snails were concomitantly exposed to MC- producing cyanobacteria with RoundUp®, suggesting interacting effects of MCs on biotransformation processes. The potent inhibition of detoxication systems by MCs and RoundUp® Flash was reversible during the depuration, during which CAT and GST activities were significantly higher in snails previ- ously exposed to MC-producing cyanobacteria with or without RoundUp® Flash than in other conditions, probably related to the oxidative stress caused by accumulated MCs remaining in tissues.

Feathers from carcasses of the Lesser Flamingo (Phoeniconaias minor), which had died after ingesting cyanobacterial toxins (cyanotoxins) contained between 0.02 and 30.0 µg microcystin-LR equivalents per gram of feather according to HPLC and ELISA analysis of feather extracts. Anatoxin-a was detected less frequently in the Lesser Flamingo feathers, up to 0.8 µg anatoxin-a per gram of feather being recorded. When feathers from different body regions were analysed and compared for microcystins and anatoxin-a, wing feathers were found to contain the highest concentrations of these cyanotoxins, the order of concentration and frequency of analytical detection being wing > breast > head. Consistent with the presence of the microcystins and anatoxin-a in gut contents and the livers of the dead birds and negligible in vitro adsorption to feathers, the cyanotoxins associated with the feathers of the dead wild flamingos are inferred to be primarily of dietary origin. †Dedicated to the memory of Ekkehard and Angelika...

The famous population of Lesser Flamingos (Phoeniconaias minor) at the saline-alkaline Rift Valley lakes of East Africa is periodically subjected to massive dieoffs. Toxic cyanobacteria may contribute to the deaths of the flamingos. One of the potential sources of the toxins are cyanobacterial mats in hot springs. Here we focus on the dominant taxa of cyanobacterial mats in hot springs on the shores of the alkaline lakes Bogoria and Magadi (Kenya), Manyara and Natron (Tanzania), and in the Hell’s Gate National Park (Kenya) and their potential toxicity.

The uptake and accumulation of microcystin-RR (MC-RR) in fishwas investigated under laboratory conditions and in wild fish. Jenynsia multidentata and Corydoras paleatus were exposed for 24 h to 50 µg/L MC-RR dissolved in water. After exposure, liver, gill, brain, intestine, gall bladder, blood and muscle were analyzed for MC-RR by HPLC and analysis confirmed by LC-ESI-TOF–MS spectrometry. Furthermore, wild individuals of Odontesthes bonariensis were sampled from the eutrophic, cyanobacteria-containing San Roque reservoir, and analyzed for the presence of MC-RR in liver, gill, intestine, and muscle. MC-RR was found in liver, gills, and muscle of all exposed and wild fish, while in C. paleatus MC-RR was also present in the intestine. Moreover, we found presence of MC-RR in brain of J. multidentata. Results indicate that MC-RR uptake might occur at two different organs: intestine and gills, through either feeding (including drinking) or respiratory activities. This suggests that MC-RR is taken into the blood stream after absorption, and distributed to different tissues. The liver showed the major bioaccumulation of MC-RR in both experimentally exposed and wild individuals, with muscle of wild fish showing relative high amounts of this toxin in comparison with those exposed in the laboratory; thoughMC-RRwas present in muscle of fish exposed for 24 h. The amount of MC-RR in muscle of O. bonariensis exceeded the value suggested by WHO to be safe, thus causing a health risk to persons consuming fish as a result of chronic exposure to microcystin. Gills also showed bioaccumulation of MC-RR, raising questions on the mechanism involved in the possible uptake of MC-RR through gills as well as on its accumulation in this organ. Although MC-LR has been reported in brain of fish, this is the first report confirming the presence of MC-RR in this organ, which means that both toxins are able to cross the blood–brain barrier. These findings also raise questions on the probable neurotoxicity of microcystins. Fil: Cazenave, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto Nacional de Limnología. Universidad Nacional del Litoral. Instituto Nacional de Limnología; Argentina Fil: Wunderlin, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina Fil: Bistoni, Maria de Los Angeles. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Cátedra de Diversidad Animal I; Argentina Fil: Amé, María Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Krause, Eberhard. Institute of Molecular Pharmacology; Alemania Fil: Pflugmacher, Stephan. Leibniz Institute of Freshwater Ecology and Inland Fisheries; Alemania Fil: Wiegand, Claudia. Leibniz Institute of Freshwater Ecology and Inland Fisheries; Alemania

(2005). Contribution of toxic cyanobacteria to massive deaths of Lesser Flamingos at saline-alkaline lakes of Kenya. SIL Proceedings, 1922-2010: Vol. 29, No. 2, pp. 783-786.

Der Artikel enthalt folgende Kapitel: Einleitung DHM: Interaktionen mit Licht und als Nahrstoffquelle Interaktionen von DHM mit Nahrstoffen, Metallen und Biopolymeren Huminstoffe und Interaktionen mit Organismen: Direkte Effekte Huminstoffe und Interaktionen mit Organismen: Indirekte Chemikalien-Effekte Okosystemare Bedeutung Schlussfolgerungen Literatur Keywords: Alge; Bakterien; Binnengewasser; Biopolymer; Cyanotoxin; Daphnia magna; DHM; dissolved humic material; Eisen; Fisch; Freiwasser; Huminstoff; Makrophyt; Nahrungsnetz; Nematode; Phosphor; Pilz; Plankton; Stickstoff; Xenobiotika

For decades frequent mass mortalities of Lesser Flamingos (Phoeniconaias minor Geoffroy) have been observed at alkaline-saline Kenyan Rift Valley lakes. To estimate the potential influence of toxic cyanobacteria on these mass deaths, the phytoplankton communities were investigated in Lakes Bogoria, Nakuru and Elmenteita. Cyanobacterial toxins were analyzed both in the phytoplankton from the three lakes and in isolated monocyanobacterial strains of Arthrospira fussiformis, Anabaenopsis abjjatae, Spirulina subsalsa and Phormidium terebriformis. Lake Bogoria was dominated by the cyanobacterium A. fusiformis. In L. Nakuru and L. Elmenteita the phytoplankton mainly consisted of A. fusiformis, A. abjatae and Anabaenopsis arnoldii, and in L. Nakuru an unknown Anabaena sp. was also found. Furthermore, this is the first time A. abijatae and the unknown Anabaena sp. have been found in Kenyan lakes. Phytoplankton wet weight biomass was found to be high, reaching 777 mg L -1 in L. Bogoria, 104 mg L -1 in L. Nakuru and 202 mg L -1 in L. Elmenteita. Using HPLC, the cyanobacterial hepatotoxins microgstin-LR, -RR -γR, -LF and -LA and the neurotoxin anatoxin-a were detected in phytoplankton samples from L. Bogoria and L. Nakuru. Total microcystin concentrations amounted to 155 μg microcystin-LR equivalents g -1 DW in L. Bogoria, and 4593 μg microcystin-LR equivalents g -1 DW in L. Nakuru, with anatoxin-a concentrations at 9 μg g -1 DW in L. Bogoria and 223 μg g -1 DW in L. Nakuru. In L. Elmenteita phytoplankton, no cyanobacterial toxins were found. A. fusiformis was identified as one source of the toxins. The isolated strain of A. fusiformis from L. Bogoria was found to produce both microcystin-YR (15.0 μg g -1 DW) and anatoxin-a (10.4 μg g -1 DW), whilst the A. fusiformis strain from L. Nakuru was found to produce anatoxin-a (0.14 μg g -1 DW). Since A. fusiformis mass developments are characteristic of alkaline-saline lakes, health risks to wildlife, especial the Arthrospira-consuming Lesser Flamingo, may be expected.

Humic substances are the main component of dissolved organic matter in all aquatic ecosystems, comprising a variety of molecular structures and functional groups. They bind organic pollutants and metals, thereby decreasing the bioavailability and consequently the toxicity of these substances in most instances. Recent studies also describe direct interactions and effects on organisms. Current studies also show that in some cases mitigation effects are stronger at lower concentrations of natural organic matter (NOM) and that toxicity increases with increased NOM concentrations. We hypothetise that at higher concentrations the mitigating effects are overlayed by direct effects of NOM themselves, thus these aspects were investigated in combination. So, on the one hand, this study demonstrates direct effects on toxicological parameters and activities of transformation enzyme systems of Daphnia magna , provoked by two NOM and one synthetic humic-like substance, HS1500. On the other hand, capacities of NOM and synthetic HS1500 to mitigate effects of the insecticide cypermethrin were investigated. Taken together, mitigation effects were overlayed by direct own effects of the NOM and HS1500. The NOM used were isolates from Suwannee River (XAD, Spring 2000) and from streams of the Svartberget forest (reverse osmosis, Spring 2000). The HS1500 was synthetically produced by radicalic autoxidation of hydroquinones. Suwannee River NOM at concentrations between 50 and 100 mg/l, and HS1500 (10–50 mg/l), but not Svartberget NOM increased immobility and lethality of the daphnids. All elevated the activity of the soluble glutathione S -transferase from 0.5 mg/l (HS1500), 1.0 mg/l (Suwannee River NOM) and 10 mg/l (Svartberget NOM) onwards, the microsomal glutathione S -transferase did not react. The glutathione peroxidase tended to increase. In the single exposure, the insecticide cypermethrin increased all toxicological parameters, elevated soluble and tendentially microsomal glutathione S -transferase activity between 0.001 and 1.0 μg/l and tendentially increased glutathione peroxidase. In contrast to that, 50 mg/l of the NOM and HS1500 did not mitigate toxicological effects. HS1500 in combination with cypermethrin even increased immobility, compared to cypermethrin alone. Increase of the NOM or HS1500 concentrations in combination with cypermethrin did not increase mitigation as seen in the activity of soluble glutathione S -transferases, activities of microsomal glutathione S -transferase and glutathione peroxidase even decreased, relative to control, which can be a hint of enzyme disfunction or further damages in the cell. An increase of concentration did not increase mitigation. Mitigation was higher at lower NOM or HS1500 concentration, probably as a consequence of the direct effects caused by themselves. Consequently, direct effects are relevant for analysing the mitigation qualities.

The effect of two strains of Cylindrospermopsis raciborskii on the survivorship, somatic growth, and detoxification processes of juvenile Daphnia magna were investigated. Both strains of C. raciborskii (and also Ankistrodesmus falcatus, used as the control) were given to newborn D. magna at equivalent biovolumes. The survival curves for D. magna subjected to the two C. raciborskii treatments differed from those of the starved and fed treatments. After 48 h of exposure, the percentage of D. magna surviving after exposure to Cylin-A (a cylindrospermopsin-producing strain isolated from Australia) and Cylin-P (a non-cylindrospermopsin-producing strain isolated from Portugal) was 10.00% and 93.33%, respectively. The strain that produces cylindrospermopsin caused the greatest toxic effect in juvenile D. magna. Statistically significant differences in D. magna body size between the four treatments (Cylin-A, Cylin-P, A. falcatus, and starved) were detected after 48 h of exposure. The juvenile D. magna that received the two C. raciborskii treatments showed an increase in size (relative to their size at T0) of 2.54% and 38.14%, respectively. These values were statistically significantly different than those of the A. falcatus–fed control (55.54%) and the starved control (11.47%). In both C. raciborskii treatments there was a tendency for increased GST enzyme activities after 24 h of exposure. Cylindrospermopsin was detected (HPLC-MS/MS) in D. magna tissues after 24 and 48 h (0.025 and 0.02 ng animal−1, respectively). The results of this study indicate that C. raciborskii can affect the fitness and growth potential of juvenile D. magna. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 453–459, 2004.

An funf Rift-Valley-Seen in Kenia wird gezeigt, welchen Gefahren diese einmaligen Habitate ausgesetzt sind. Als Parameter zur Gewasserbewertung wurde die Biodiversitat des Phytoplanktons herangezogen. Besonders die Cyanobakterienpopulationen haben erheblichen Einfluss auf das okologische Gleichgewicht der Seen und die Nutzbarkeit des Wassers. Die Untersuchungsgewasser reprasentieren hochinteressante Biodiversitats- und Salinitatsgradienten. Der Baringosee ist starken Verlandungs- und zunehmend auch Versalzungsproblemen ausgesetzt, daruber hinaus wurden Microcystine nachgewiesen. An den alkalinen Seen Bogoria und Nakuru ist seit zwei Jahrzehnten eine erhohte Sterberate des Kleinen Flamingos beobachtet worden. Bisherige Befunde weisen auf verschiedene Ursachen hin: Pestizide, Schwermetalle, bakterielle Krankheiten sowie Leber- und Nervengifte aus der Gruppe der Cyanotoxine. Der Naivashasee als Suswasser leidet unter erheblicher Ubernutzung und Degradation, was sich auch an seiner ehemaligen Bucht, dem Oloidiensee, durch einen deutlichen Anstieg des Salzgehaltes und einer Verarmung des Phytoplanktons zeigt.

Cyanobacterial mats at hot springs on the shore of the alkaline Lake Bogoria, Kenya, were investigated regarding species community and cyanobacterial toxin content. The hepatotoxins microcystin-LR, -RR, -LF and -YR, and the neurotoxin anatoxin-a were present. The mats were dominated by Phormidium terebriformis, Oscillatoria willei, Spirulina subsalsa and Synechococcus bigranulatus. The concentration of microcystins in mat samples, ranged from 221 to 845 Wg microcystin-LR equivalents g 31 DW of mat. Anatoxin-a concentrations ranged from 10 to 18 W gg 31 DW of mat. A contribution of the cyanobacterial toxins from the hot spring mats to the mass mortalities of Lesser Flamingos is suggested by: (a), the presence of hot spring cyanobacterial cells and cell fragments, and high concentrations of the cyanobacterial hepato- and neurotoxins in flamingo stomach contents and faecal pellets; (b), observations of neurological signs of bird poisoning at the lake. Cyanobacterial toxins in stomach contents, intestine and fecal pellets were 0.196 W gg 31 fresh weight (FW) for the microcystins and 4.34 W gg 31 FW for anatoxin-a. Intoxication with cyanobacterial toxins could occur by uptake of detached cyanobacterial cells from the mats, as the flamingos need to drink fresh or brackish water, and to wash their feathers daily, which they do in the vicinity of the hot springs, where salinity is lower than in the main body of water of the lake. @ 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The toxicity and metabolism of the cyanobacterial toxins microcystin-LR (MCLR), Dhb-microcystin-HtyR and nodularin were investigated in the cysts, nauplii and adults of the brine shrimp Artemia salina. The presence of the phase II detoxication system glutathione S-transferase (sGST) in these stages was shown using different substrates. Exposure of adult A. salina to the toxins led to an elevation of GST activity in vivo. All three toxins were conjugated to glutathione via GST, which has been shown as an initial step of microcystin and nodularin detoxication.

Environmental Pollution, in press; International audience; Organisms are regularly subjected to abiotic stressors related to increasing anthropogenic activities, including chemicals and climatic changes that induce major stresses. Based on various key taxa involved in ecosystem functioning (photosynthetic microorganisms, plants, invertebrates), we review how organisms respond and adapt to chemical- and temperature-induced stresses from molecular to population level. Using field-realistic studies, our integrative analysis aims to compare i) how molecular and physiological mechanisms related to protection, repair and energy allocation can impact life history traits of stressed organisms, and ii) to what extent trait responses influence individual and population responses. Common response mechanisms are evident at molecular and cellular scales but become rather difficult to define at higher levels due to evolutionary distance and environmental complexity. We provide new insights into the understanding of the impact of molecular and cellular responses on individual and population dynamics and assess the potential related effects on communities and ecosystem functioning.

The commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) was rapidly taken up by the emergent reed plant Phragmites australis with clear distribution in the different cormus parts of the plant. Highest uptake was detected in the stem, followed by the rhizome. Enzyme extracts of the rhizome system, the stem, and the leaf revealed the presence of soluble glutathione S-transferases (sGST) measured with the model substrate 1-chloro-2,4-dinitrobenzene. A significant elevation of sGST activity in the rhizome and stem parts of P. australis was detected after a 24-h exposure to 0.5 microg/L MC-LR. Rhizome, stem, and leaf tissues were also able to conjugate several microcystin toxins. However, no conjugation, either chemical nor enzymatic, was detected using the related cyanobacterial toxin nodularin as substrate. Highest glutathione S-transferase activity for the toxin substrates was detected in the pkat/mg range in the stem of P. australis. For MC-LR, a complete metabolism from the formation of a glutathione conjugate to the degradation of a cysteine conjugate in all cormus parts of the plant is reported. The stepwise degradation of the MC-LR-glutathione conjugate to a gamma-glutamylcysteine and a cysteine conjugate was demonstrated by comparison with chemically formed reference compounds and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This is the first evidence for the uptake and metabolism of cyanobacterial toxins by an emergent aquatic macrophyte.

In an aquatic ecosystem, during cyanobacterial bloom lysis, a mixture of toxins and other cyanobacterial and bacterial components will be present in the water, acting on aquatic organisms. Most of the research into toxic effects of cyanobacteria has involved the use of purified toxins. In this study, the "real-life" situation of a cyanobacterial lysis event was investigated. For this purpose, intact cells from a natural cyanobacterial bloom from Lake Muggelsee, Berlin, were taken and the cells were broken by repeated freeze/thaw cycles. This crude extract was used to expose several aquatic organisms ranging from microalgae (Scenedesmus armatus), macrophyte (Ceratophyllum demersum), invertebrate (Chaoborus crystallinus) up to fish eggs (Danio rerio) to look at several physiological parameters such as detoxication enzyme activity and, in the case of the microalgae and the macrophyte, also the effect on activity of photosynthesis. In all the tests, the cyanobacterial crude extract caused stronger effects than the pure cyanobacterial toxins used in equivalent concentrations.

Lepidopterans generally can successfully defend themselves against a variety of parasites or parasitoids. One mechanism they use is to encapsulate the invader in many layers of hemocytes. For encapsulation to occur, the hemocytes must attach to the foreign material, spread, and adhere to each other. The molecules that mediate these processes are not known. One method to identify proteins potentially necessary for adhesion, spreading, and, thus, encapsulation is to use monoclonal antibodies that interfere with these functions. In this paper, we report that a monoclonal antibody against Manduca sexta plasmatocytes effectively inhibited encapsulation of synthetic beads in vitro and in vivo. Furthermore, it inhibited plasmatocyte spreading in vitro. Other anti-hemocyte antibodies did not have these effects. The plasmatocyte-specific monoclonal antibody, mAb MS13, recognized a protein of approximately 90,000 daltons as indicated by Western blot analysis of hemocyte lysate proteins. The epitope recognized by mAb MS13 was present on the exterior surface of plasmatocytes. Using indirect immunohistochemistry with hemocyte-specific antibodies, we also determined that during encapsulation plasmatocytes were the first cells bound to latex beads and later layers consisted of both plasmatocytes and granular cells. Arch.

The effects of cyanotoxins on fish have been studied mainly in adults, rather than in early life stages which could be more sensitive or, in view of their immobility, more readily affected. The uptake of microcystin-LR by different early life stages of the zebra fish (Danio rerio) was investigated using 14C-labelled microcystin-LR. The effects on the activity of the detoxication enzymes, microsomal and soluble glutathione S-transferases (GST), and glutathione peroxidase (GP-X) were examined. There was a detectable uptake of microcystin from the first day of embryonic development up to 5 day old larvae. On average, an absorption of 0.5 ng microcystin for eggs and eleuthero-embryos was calculated over the entire exposure time. Because of the differences in volume of the eggs and eleuthero-embryos, there was an increase in the microcystin-LR concentration between these stages. In the eggs, approximately 25% of the medium concentration was found, and in eleuthero-embryos an equilibrium between fish and medium was reached. The activity of the detoxication enzymes differed during ontogenesis, but the effects of activation and suppression of these enzymes were similar at all stages. Minor activation of the soluble GST was found and a marked activation of GP-X was evident. The reaction of the microsomal GST was not so obvious. These results showed that there was an uptake of microcystin-LR by early life stages of the zebra fish and that the detoxication system reacted to this toxin, possibly indicating the ability of the organism to metabolize microcystin-LR to a less harmful compound. Chronic toxic effects, such as reduction in growth, in such early life stages when organogenesis is not finished and hence the microcystin-LR affects not one single target organ but the whole organism, might be due to the increased energy demand of these detoxication processes. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 89–95, 1999

This study investigates if acclimatization to residual pesticide contamination in agricultural soils is reflected in detoxification, antioxidant enzyme activities and energy budget of earthworms. Five fields within a joint agricultural area exhibited different chemical and farming histories from conventional cultivation to organic pasture. Soil multiresidual pesticide analysis revealed up to 9 molecules including atrazine up to 2.4 ng g -1 dry soil. Exposure history of endogeic Aporrectodea caliginosa and Allolobophora chlorotica modified their responses to pesticides. In the field, activities of soluble glutathione-S-transferases (sGST) and catalase increased with soil pesticide contamination in A. caliginosa. Pesticide stress was reflected in depletion of energy reserves in A. chlorotica. Acute exposure of pre-adapted and naïve A. caliginosa to pesticides (fungicide Opus ®, 0.1 μg active ingredient epoxiconazole g-1 dry soil, RoundUp Flash®, 2.5 μg active ingredient glyphosate g-1 dry soil, and their mixture), revealed that environmental pre-exposure accelerated activation of the detoxification enzyme sGST towards epoxiconazole.

International audience; Cyanobacterial toxins and pesticides regularly impact freshwaters. Microcystin-LR is one of the most toxic and common cyanobacterial toxins whereas glyphosate is the active ingredient of a widely use herbicide. As filter feeders, freshwater mussels are particularly exposed. Like many native bivalve species, Unio pictorum suffers from a continuous decline in Europe. In order to get a deeper insight of its response to contaminants, U. pictorum was exposed to either 10 μg L(-1) of microcystin-LR or 10 μg L(-1) of glyphosate or a mixture of both. Proteins of the digestive glands were extracted and analyzed by DIGE. Gel analysis revealed 103 spots with statistical variations, and the response seems to be less toward glyphosate than to microcystin-LR. Specific spots have variations only when exposed to the mixture, showing that there is an interaction of both contaminants in the responses triggered. The proteins of 30 spots have been identified. They belong mostly to the cytoskeleton family, but proteins of the oxidative pathway, detoxification, and energetic metabolism were affected either by glyphosate or microcystin-LR or by the mixture. These results demonstrate the importance to study contaminants at low concentrations representative of those found in the field and that multicontaminations can lead to different response pathways.

Cyanobacterial blooms affect aquatic ecosystems due to their capability of producing cyanotoxins (e.g., microcystins, MC; cylindrospermopsin, CYN) and other bioactive compounds. Filter feeding zooplankton are amongst the first organisms affected and research has mainly focused on their interactions with toxic cyanobacteria. We investigated oxidative stress, biotransformation and energetic responses of Daphnia magna after exposure to cyanobacterial extracts and behavioral changes of the animals exposed to living cells of non-MC and non-CYN producing strains. Enzyme and energetic responses were measured in time kinetic experiments, using extracts of Microcystis botrys, M. wesenbergii, Aphanizomenon aphanizomenoides, Cylindrospermopsis raciborskii, corresponding to 10 mg DW L−1. In behavioral experiments, D. magna was exposed to living cells of Microcystis aeruginosa, A. aphanizomenoides, Dolichospermum circinalis, C. raciborskii, at the density of 400 000 cells mL−1 using a Daphnia toximeter equipment. Despite not producing MC or CYN, some cyanobacterial extracts caused significant increase of biotransformation enzyme, especially catalase, activities from the exposed D. magna after a longer incubation. Total carbohydrates and glycogen contents were increased but the activity of one of the involved enzymes, the malate dehydrogenase, was not changed. Animals' behavior (e.g., swimming, position within water column) was altered in exposures to cultures of C. raciborskii and D. circinalis. These physiological and behavioral alterations indicate stress, which may impair overall performance of zooplankton at the environmental realistic chronic exposure scenario.

Humic substances comprise the majority of organic matter in freshwater ecosystems and were thought to be inert or refractory, except for photolytic degradation. However, evidence is increasing that humic substances interact with aquatic organisms similarly to weak anthropogenic chemicals with nonspecific and specific effects. One specific effect is a hormonelike effect, namely, modulation of the number of offspring, which was first described with the nematode Caenorhabditis elegans. Yet a hormonelike effect is not restricted to only the nematode. With the ornamental swordtail fish, Xiphophorus helleri, and the South African clawed frog, Xenopus laevis, we present phenomenological evidence that slight feminization occurred when these vertebrate species were exposed to a synthetic humic substance, a condensation product of polyphenols. The slight feminization was dose dependent.

International audience

SUMMARYAnthropogenic and climate factors increase the frequency of problematic cyanobacterial blooms in freshwater. Among other toxins, some cyanobacteria produce microcystins (MCs), which inhibit phosphatases type 1 and type 2A and provokes oxidative stress. Toxic cyanobacteria affect the growth, survival and reproduction of zooplankton, particularly those from the genus Daphnia, which have a central position in pelagic food webs. However, one possibility to ameliorate effects is to biotransform MC via glutathione S transferase (GST) to a less toxic glutathione conjugate. This process was hypothesised to underlie the ability of Daphnia to withstand MC and to explain the enhanced tolerance of the offspring from mothers exposed to toxic cyanobacteria. Thus we conducted multigenerational experiments with D. magna, exposing the parental generation to MC for 1 or 7 days and determining the enzyme-mediated tolerance to MC in their offspring by assessing the acute effect of MC on biotransformation and antioxidant and metabolism enzymes, and through 21 day chronic tests on toxicity and growth. Seven days of exposure of the parental generation to MC induced higher activity of GST and malate dehydrogenase in the offspring and enabled them to increment the catalase activity when challenged with MC, whereas 1 day of exposure of the parental generation did not. Offspring from non-exposed and 1-day-exposed mothers suffered decreased survival when exposed to MC compared with offspring from 7-day-exposed mothers; survival was correlated with the elevated activity of GST, malate dehydrogenase and catalase, suggesting maternal transfer of activation factors. However, increased survival occurred at the expense of individual growth. These results suggest that transgenerational effects are provoked by MC in D. magna, which may explain the observed acquirement of enhanced tolerance over generations.

In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10(-11), 10(-10), 10(-9), and 10(-8)M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10(-9) and 10(-8)M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) β in both genders. This decrease was persistent after further treatment until NF 66 in the 10(-8)M LNG treatment. Expression of follicle-stimulating hormone (FSH) β was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10(-9) and 10(-8)M significantly increased FSHβ mRNA levels in males. In NF 66 females, 10(-9)M LNG treatment increased FSHβ gene expression, whereas a decrease was observed in NF 66 males exposed to 10(-8)M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10(-8)M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10(-8)M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHβ expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.

International audience; Freshwater mussels such as the invasive Dreissena polymorpha and the indigenous Unio tumidus nourish by high filtration rates and may accumulate cyanobacteria and their toxins during cyanobacterial blooms. Physiological adaptations to cyanotoxins enable organisms to endure cyanobacteria lblooms but may differ between species. Biotransformation and excretion capacities for cyanobacteria and anthropogenic pollutants have been demonstrated for Dreissena polymorpha but less for unionid species. This study compares the activities of biotransformation (glutathioneS-transferase, GST) and antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) in Dreissena polymorpha to Unio tumidus in response to cyanotoxin exposure (10 mg L 1 and 50 mg L 1 microcystin-LR, respectively, total microcystin from a cyanobacterial crude extract) for 24h and 7 d exposure duration. Enzyme activities in Dreissena polymorpha were measured in the whole mussel tissue, digestive gland and in gills and in Unio tumidus in the digestive gland, gills, mantle, foot as well as in the remaining tissue. The sGST was elevated for the entire exposure period in the whole mussel tissue of Dreissena polymorpha but despite higher basal activities in digestive gland and gills of Unio tumidus, it was rather inhibited or unaltered in most of their tissues. Elevated SOD activity indicated oxidative stress response in Dreissena polymorpha, but not in Unio tumidus. The CAT activity was barely affected in both species, rather inhibited in Unio tumidus, despite again higher basal activities in digestive gland and remaining tissue. Compared to the indigenous Unio tumidus, the investigated biotransformation and oxidative stress combating enzymes respond stronger in the invasive Dreissena polymorpha.

International audience; Synthetic gestagens, including levonorgestrel (LNG), are active compounds in contraceptives, and several studies report their occurrence in surface waters. However, information about endocrine-disrupting effects in nontarget organisms is scarce. The present study investigated effects of LNG exposure on thyroid hormone–dependent metamorphosis of Xenopus laevis. Premetamorphic X. laevis tadpoles at Nieuwkoop and Faber (NF) stage 48 were exposed in a flow-through culture system to four LNG concentrations (10211, 10210, 1029, and 1028M) over the period of metamorphosis. At NF 58 and 66, tadpoles were examined sex specifically. Developmental time and organismal responses were recorded and correlated with molecular and histopathological endpoints. Exposure to 1028M LNG caused an inhibition of metamorphosis resulting in developmental arrest at early climax stages as giant tadpoles or tailed frogs. In brain-pituitary tissue of NF 58 tadpoles, gene expression of thyroid-stimulating hormone (b-subunit; TSHb), TH receptor b (TRb), and deiodinase type 3 (D3) was not changed. Instead, prolactin (PRL) messenger RNA (mRNA) was significantly increased by 1029M LNG in females and by 1028M LNG in both sexes. In NF 66 tadpoles, mRNAlevels of TSHb mRNA were significantly increased in the 1029 and 1028M LNG treatment groups indicating a hypothyroid state. No changes of TRb, D3, and PRL gene expression were detected. Histopathological evaluation of thyroid gland sections revealed no typical sign of hypothyroidism but rather an inactivated appearance of the thyroid. In conclusion, our data demonstrate for the first time a completely new aspect of thyroid system disruption caused by synthetic gestagens in developing amphibians.