The retinoid metabolism of Gammarus fossarum is disrupted by exogenous all-trans retinoic acid, citral, and methoprene but not by the technical formulation of glyphosate.

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Development of a method for the simultaneous determination of multi-class pesticides in earthworms by liquid chromatography coupled to tandem electrospray mass spectrometry.

Agricultural intensification, and in particular the use of pesticides, leads over the years to a loss of biodiversity and a decline of ecosystem services in cultivated zones and agricultural landscapes. Among the animal communities involved in the functioning of agro-ecosystems, earthworms are ubiquitous and recognized as indicators of land uses and cultural practices. However, little data is available on the levels of pesticides in such organisms in natura, which would allow estimating their actual exposure and the potentially resulting impacts. Thus, the objective of this study was to develop a sensitive analytical methodology to detect and quantify 27 currently used pesticides in earthworms (Allolobophora chlorotica). A modified QuEChERS extraction was implemented on individual earthworms. This step was followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The whole analytical method was validated on spiked earthworm blank samples, with regard to linearity (from 1 to 100 method limit of quantification, r2 > 0.95), intra-day precision (relative standard deviation (RSD) < 15%), inter-day precision (RSD < 20%), recoveries (mainly in the range 70-110%), and limits of detection and of quantification (inferior to 5 ng/g for most of the pesticides). The developed method was successfully applied to determine the concentrations of pesticides in nine individuals collected in natura. Up to five of the selected pesticides have been detected in one individual. Graphical abstract.

Phenotypic defects in newborn Gammarus fossarum (Amphipoda) following embryonic exposure to fenoxycarb.

During morphogenesis numerous morphogenetic factors ensure the production of a target phenotype. By disrupting these processes, a toxic exposure during this period could cause an increase of phenotypic defects. In the present study, embryos of the freshwater amphipod Gammarus fossarum were exposed throughout the embryogenesis to increasing concentrations of fenoxycarb (0, 0.5µgL-1, 5µgL-1 and 50µgL-1), a growth regulator insecticide analog of the insect juvenile hormone. In addition, to identify morphogenesis' sensitive period, embryos were exposed during either early or late embryonic development to 5µgL-1 of fenoxycarb. In newborn individuals from exposed embryos, three phenotypes were investigated: i) eye pigmentation, ii) length of the antenna and gnathopod of both left and right sides and iii) midgut tissue state. Developmental homeostasis was assessed by measuring fluctuating asymmetry and inter-individual variance of both the antenna and gnathopod. Exposure to 5µgL-1 and 50µgL-1 fenoxycarb throughout the embryonic development induced a delayed hatching and altered appendages size. Moreover, exposure to 5µgL-1 throughout the embryogenesis and during the gastrulation phase impaired eye pigmentation, while exposure to 50µgL-1 resulted in increased tissue damages of the midgut. No significant increase of fluctuating asymmetry was observed in exposed individuals, neither for the antenna nor for the gnathopod. These results demonstrate that fenoxycarb can alter embryonic development of G. fossarum without disrupting developmental homeostasis.

Rapid analysis of diclofenac and some of its transformation products in the three-spined stickleback, Gasterosteus aculeatus, by liquid chromatography-tandem mass spectrometry.

Pharmaceuticals are emerging organic contaminants ubiquitously present in the environment due to incessant input into the aquatic compartment mainly resulting from incomplete removal in wastewater treatment plants. One of the major preoccupations concerning pharmaceuticals released into surface waters is their potential for bioaccumulation in biota, possibly leading to deleterious effects on ecosystems especially as they could affect a broad variety of organisms living in or depending on the aquatic environment. Thus, the development of accurate and sensitive methods is necessary to detect these compounds in aquatic ecosystems. Considering this need, this study deals with the analytical development of a methodology to quantify traces of diclofenac together with some of its biotic and abiotic transformation products in whole-body tissue of three-spined stickleback. A simple and reliable extraction method based on a modified QuEChERS extraction is implemented on 200 mg of fish. The detection and quantification of the ten target compounds are performed using liquid chromatography-tandem mass spectrometry. The whole process was successfully validated regarding linearity, recovery, repeatability, and reproducibility. The method limits of detection and quantification do not exceed 1 ng/g. To reproduce environmental conditions, we measured the concentration of DCF and its transformation products in three-spined sticklebacks after a 6-month exposure in mesocosms at several levels of DCF ranging from 0.05 to 4.1 µg/L. The phase I metabolite 4'-hydroxydiclofenac was detected in fish samples exposed at the highest DCF concentration. Graphical abstract Analysis of diclofenac and some of its transformation products in the three-spined stickleback, Gasterosteus aculeatus, by QuEChERS extraction followed by LC-MS/MS.

Exposure and hazard of bisphenol A, S and F: a multi-biomarker approach in three-spined stickleback.

Due to the estrogenic behavior of bisphenol (BP) A, industries have developed many substitutes, such as BPS and BPF. However, due to their structural similarities, adverse effects on reproduction are currently observed in various organisms, including fish. Even if new results have shown impacts of these bisphenols on many other physiological functions, their mode of action remains unclear. In this context, we proposed to better understand the impact of BPA, BPS, and BPF on immune responses (leucocyte sub-populations, cell death, respiratory burst, lysosomal presence, and phagocytic activity) and on biomarkers of metabolic detoxification (ethoxyresorufin-O-deethylase, EROD, and glutathione S-transferase, GST) and oxidative stress (glutathione peroxidase, GPx, and lipid peroxidation with thiobarbituric acid reactive substance method, TBARS) in an adult sentinel fish species, the three-spined stickleback. In order to enhance our understanding of how biomarkers change over time, it is essential to determine the internal concentration responsible for the observed responses. Therefore, it is necessary to explore the toxicokinetics of bisphenols. Thus, sticklebacks were exposed either to 100 µg/L of BPA, BPF or BPS for 21 days, or for seven days to 10 and 100 µg/L of BPA or BPS followed by seven days of depuration. Although BPS has very different TK, due to its lower bioaccumulation compared to BPA and BPF, BPS affect oxidative stress and phagocytic activity in the same way. For those reasons, the replacement of BPA by any substitute should be made carefully in terms of risk assessment on aquatic ecosystems.

PBTK-TD model of the phagocytosis activity in three-spined stickleback exposed to BPA.

Due to the high production volume and persistence in the environment of bisphenol A (BPA) and its substitutes, realistic exposure scenarii were proposed in some species to better understand the relationship between external and internal concentrations. For example, a recent PBTK model has been developed and adapted to BPA ADME (Absorption, Distribution, Metabolization, and Excretion) processes in three-spined stickleback. These substances have an impact on organism physiology including reproductive and immune functions. In this context, physiologically-based toxicokinetic models coupled with toxicodynamics (PBTK-TD) have proven to be valuable tools to fill the knowledge gap between external exposure and effect dynamics. The aim of the current work was to explain the impact of BPA on the immune response by determining its temporality. In addition, the relationship between BPA dose and these responses was investigated using a PBTK-TD model. Two experiments were performed on stickleback to characterize their biomarker responses, (i) a short exposure (14 days) at 0, 10 and 100 µg/L, including a depuration phase (7 days), and (ii) a long exposure (21 days) at 100 µg/L to measure the immunomarker dynamic over a long period. The fish spleens were sampled to analyze immune responses of stickleback at various times of exposure and depuration: leucocyte distribution, phagocytic capacity and efficiency, lysosomal presence and leucocyte respiratory burst index. At the same date, blood, muscle, and liver were sampled to quantify BPA and their metabolites (BPA monoglucuronide and BPA monosulfate). All these data enabled the development of the indirect pharmacodynamic models (PBTK-TD) by implementing the responses of biomarkers in the existing BPA PBTK of stickleback. The results shown a high induction of phagocytosis activity by BPA in the two exposure conditions. Furthermore, the immunomarkers exhibit very different temporal dynamics. This study demonstrates the need of a thorough characterization of biomarker response for a further use in Environmental Biomonitoring.

Time and dose-dependent impairment of liver metabolism in Gasterosteus aculeatus following exposure to diclofenac (DCF) highlighted by LC-HRMS untargeted metabolomics.

Anthropogenic chemicals as emerging contaminants, such as pharmaceuticals, increased worldwide in the environment. This study aimed to apply metabolomics-based approaches on the fish model species three-spined stickleback (Gasterosteus aculeatus) exposed to diclofenac (DCF) to identify toxicity pathways and potential biomarkers. For this purpose, males and females were exposed to a continuous flow of diclofenac solution in laboratory for 21 days, followed by 3 days of depuration, to nominal concentrations of 1 (low) and 100 µg/L (high) of DCF. A methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) was employed. Uni- and multivariate statistical analyses were combined to evaluate the modulations of the liver metabolome of G. aculeatus after exposure to DCF. The metabolomics data revealed variations both as a function of time and of the DCF concentration. We observed 2487 altered metabolites, with 1460 and 1027 specific to males and females, respectively. Some of them were significantly impaired by the experimental conditions. However, we showed that several metabolites were impacted by other factors as they were already modulated in the control individuals. The results indicated that the energy metabolism was up-modulated in females and down-modulated in males, with the presence of DCF. The antioxidant system was impacted in males, suggesting oxidative stress in the metabolism, while the immunity system was down-regulated in females following exposure. Moreover, our results revealed 1 and 4 metabolites as potential metabolic biomarkers in male and female sticklebacks, respectively. Among them, the glutaryl-carnitine and the adipoyl-carnitine were putatively identified in females, known to be implicated in the energy metabolism. These 5 metabolites showed to be promising biomarkers since they were early modulated during exposure to the stress and showed a notable trend through time.

Evaluation of the accumulation of the iodinated contrast agents diatrizoic acid and iohexol in Dreissena polymorpha mollusks.

Mollusks are very sensitive to aquatic environmental alterations and then, are important bio-indicators for monitoring the contamination of water bodies. Iodinated X-ray contrast media (ICMs) are ubiquitously present in the aquatic environment, primarily due to their high consumption for diagnosis purposes, high injection levels, low biodegradability, and low removal rates by wastewater treatment plants. Although these compounds are assumed to be of low toxicity, aquatic organisms are continuously exposed to these agents, which may result in adverse effects as ICMs can act as iodine source and disrupt the endocrine system. Thus, the evaluation of their environmental risk, especially on aquatic fauna is of great interest. To this end, we first compared the accumulation behavior, based on iodine analysis, of two ICM exhibiting different osmolality, diatrizoic acid and iohexol in Dreissena polymorpha bivalves exposed under laboratory conditions at concentrations of 0, 100, and 1000 µg/L during 4 and 7 days. This study was the first to provide information on iodine concentration in whole soft tissues and several organs in control zebra mussels. Moreover, it showed, after exposure, an increase of iodine content mainly in the digestive glands, followed by gills and gonads, highlighting that ICMs actually enter the organisms. Thus, bioaccumulation of ICMs studies were then performed, by liquid chromatography coupled to tandem mass spectrometry, on entire mollusks and digestive glands of organisms exposed at 0, 10, 100, and 1000 µg/L of both ICMs during 21 days, followed by 4 days of depuration. These first data on ICMs concentrations in zebra mussels, showed a clear accumulation of ICMs in mussels as a function of relative exposure level, as well as a rapid depuration. Osmolality did not seem to have a significant impact on the accumulation level, but a slight difference was observed on the accumulation pattern between both ICMs.

Identification, quantification and carbon stable isotopes determinations of organic acids in monofloral honeys. A powerful tool for botanical and authenticity control.

RATIONALE: The analytical composition and botanical origin of honey are basic data used to determine the quality of this foodstuff. Although proteins are used to characterise the analytical composition of honey, they can be eliminated during its ultrafiltration and, in the case of honeys not saturated with their own pollen, the use of proteins does not work well. As acidity is a well-known characteristic of honey and organic acids are present at around 0.5% in honey, we therefore investigated an alternative method to the protein-based White method, using organic acids as new internal standards instead of proteins. METHODS: The qualitative and quantitative analyses of 14 organic acids were carried out by ion chromatography with an electrochemical detector. The (13)C/(12)C isotopic ratios of the honeys, and of the organic acids extracted from them with an anion exchange resin, were determined by isotope ratio mass spectrometry. RESULTS: Gluconic acid is the predominant organic acid in honey, at between 1.8 and 12.7 g/kg. For fir honey the major acid is galacturonic acid at around 4.6 g/kg. The isotopic ratios of honeys and of their acids are strongly linked. Correlations between the δ(13)C values of the honey and the acids were significant, and better than those obtained using proteins. CONCLUSIONS: This study has provided a method to differentiate honeys from seven botanical origins, based on organic acid analysis. By combining various organic acid contents and isotopic ratio values through statistical processing by Principal Component Analysis it is possible to differentiate honey samples as a function of their botanical origin.

Combination of sugar analysis and stable isotope ratio mass spectrometry to detect the use of artificial sugars in royal jelly production.

The effects of feeding bees artificial sugars and/or proteins on the sugar compositions and (13)C isotopic measurements of royal jellies (RJs) were evaluated. The sugars fed to the bees were two C4 sugars (cane sugar and maize hydrolysate), two C3 sugars (sugar beet, cereal starch hydrolysate), and honey. The proteins fed to them were pollen, soybean, and yeast powder proteins. To evaluate the influence of the sugar and/or protein feeding over time, samples were collected during six consecutive harvests. (13)C isotopic ratio measurements of natural RJs gave values of around -25 ‰, which were also seen for RJs obtained when the bees were fed honey or C3 sugars. However, the RJs obtained when the bees were fed cane sugar or corn hydrolysate (regardless of whether they were also fed proteins) gave values of up to -17 ‰. Sugar content analysis revealed that the composition of maltose, maltotriose, sucrose, and erlose varied significantly over time in accordance with the composition of the syrup fed to the bees. When corn and cereal starch hydrolysates were fed to the bees, the maltose and maltotriose contents of the RJs increased up to 5.0 and 1.3 %, respectively, compared to the levels seen in authentic samples (i.e., samples obtained when the bees were fed natural food: honey and pollen) that were inferior to 0.2% and not detected, respectively. The sucrose and erlose contents of natural RJs were around 0.2 %, whereas those in RJs obtained when the bees were fed cane or beet sugar were as much as 4.0 and 1.3 %, respectively. The combination of sugar analysis and (13)C isotopic ratio measurements represents a very efficient analytical methodology for detecting (from early harvests onward) the use of C4 and C3 artificial sugars in the production of RJ.

The toxicokinetics of bisphenol A and its metabolites in fish elucidated by a PBTK model.

Bisphenol A (BPA) is a chemical of major concern due to its endocrine disrupting function, high production volume, and persistence in the aquatic environment. Consequently, organisms such as fish are subject to chronic exposure to BPA. However, physiologically-based toxicokinetic (PBTK) models, which are valuable tools to improve the understanding of a chemical's fate in an organism, have never been specifically adapted to model BPA toxicokinetics (TK) in fish. In our work, an existing PBTK developed for four different fish species was modified to model BPA ADME processes (absorption, distribution, metabolization and excretion). The metabolization of BPA into BPA-monoglucuronide (BPA gluc) and BPA-monosulfate (BPA sulf) and their TK in various organs was taking into account in the model. Experiments were performed to generate BPA TK data in a model species commonly used in ecotoxicology, the stickleback. The model structure had to include two sites of metabolization to simulate BPA TK accurately in stickleback organs. Thus, the fish liver may not be the only site of the metabolization of BPA: plasma or gills could also play a role in BPA metabolization. The PBTK model predictive performance evaluated on literature data in zebrafish and rainbow trout concurs with this conclusion. Finally, a calibration mixing data from the three species was compared to the calibration on stickleback data only.

Ultrasound-assisted QuEChERS-based extraction using EDTA for determination of currently-used pesticides at trace levels in soil.

It is essential to monitor pesticides in soils as their presence at trace levels and their bioavailability can induce adverse effects on soil's ecosystems, animals, and human health. In this study, we developed an analytical method for the quantification of traces of multi-class pesticides in soil using liquid chromatography-tandem mass spectrometry. In this way, 31 pesticides were selected, including 12 herbicides, 9 insecticides, and 10 fungicides. Two extraction techniques were first evaluated, namely, the pressurized liquid extraction and the QuEChERS procedure. The latest one was finally selected and optimized, allowing extraction recoveries of 55 to 118%. The role of the chelating agent EDTA, which binds preferentially to soil cations that complex some pesticides, was highlighted. Coupled with liquid chromatography-tandem mass spectrometry, the procedure displayed very high sensitivity, with limits of quantification (LOQ) in the range 0.01-5.5 ng/g. A good linearity (R2 > 0.992) was observed over two orders of magnitude (LOQ-100 [Formula: see text] LOQ) with good accuracy (80-120%) for all compounds except the two pyrethroids lambda-cyhalothrin and tau-fluvalinate (accuracy comprised between 50 and 175%) and the cyclohexanedione cycloxydim (accuracy < 35%). Good repeatability and reproducibility were also achieved. The method was finally successfully applied to 12 soil samples collected from 3 land-use types. Among the 31-targeted pesticides, 24 were detected at least once, with concentration levels varying from LOQ to 722 ng/g. Many values were below 0.5 ng/g, indicating that the developed method could provide new knowledge on the extremely low residual contents of some pesticides.

Stable isotope ratio measurements of royal jelly samples for controlling production procedures: impact of sugar feeding.

The carbon and nitrogen stable ratios of royal jelly (RJ) samples from various origins are determined using an elemental analyser linked online to an isotope ratio mass spectrometer to evaluate authenticity and adulteration. The (13)C/(12)C and (15)N/(14)N stable isotope ratios are measured in more than 500 RJs (domestic, imported and derived from feeding experiments) in order to obtain isotopic measurements that take into account seasonal, botanical and geographical effects. Authenticity intervals are established for traditional beekeeping practices, without feeding, in the range -22.48 to -27.90‰ for δ(13)C. For these samples, the δ(15)N values range from -1.58 to 7.98‰, depending on the plant sources of pollen and nectar. The δ(13)C values of the commercial samples vary from -18.54 to -26.58‰. High δ(13)C values are typical of sugar cane or corn syrups which have distinctive isotopic (13)C signatures because both plants use the C4 photosynthetic cycle, in contrast to most RJs which are derived from C3 plants. These differences in the (13)C-isotopic composition allow the detection of the addition of such sugars. RJs from traditional sources and from industrial production by sugar feeding are thus successfully distinguished.

An optimized LC-HRMS untargeted metabolomics workflow for multi-matrices investigations in the three-spined stickleback.

Environmental metabolomics has become a growing research field to understand biological and biochemical perturbations of organisms in response to various abiotic or biotic stresses. It focuses on the comprehensive and systematic analysis of a biologic system's metabolome. This allows the recognition of biochemical pathways impacted by a stressor, and the identification of some metabolites as biomarkers of potential perturbations occurring in a body. In this work, we describe the development and optimization of a complete reliable methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) for untargeted metabolomics studies within a fish model species, the three-spined stickleback (Gasterosteus aculeatus). We evaluated the differences and also the complementarities between four different matrices (brain, gills, liver and whole fish) to obtain metabolome information. To this end, we optimized and compared sample preparation and the analytical method, since the type and number of metabolites detected in any matrix are closely related to these latter. For the sample preparation, a solid-liquid extraction was performed on a low quantity of whole fish, liver, brain, or gills tissues using combinations of methanol/water/heptane. Based on the numbers of features observed in LC-HRMS and on the responses of analytical standards representative of different metabolites groups (amino acids, sugars…), we discuss the influence of the nature, volume, and ratio of extraction solvents, the sample weight, and the reconstitution solvent. Moreover, the analytical conditions (LC columns, pH and additive of mobile phases and ionization modes) were also optimized so as to ensure the maximum metabolome coverages. Thus, two complementary chromatographic procedures were combined in order to cover a broader range of metabolites: a reversed phase separation (RPLC) on a C18 column followed by detection with positive ionization mode (ESI+) and a hydrophilic interaction chromatography (HILIC) on a zwitterionic column followed by detection with negative ionization mode (ESI-). This work provides information on brain, gills, liver, vs the whole body contribution to the stickleback metabolome. These information would help to guide ecotoxicological and biomonitoring studies.

Does water temperature influence the distribution and elimination of perfluorinated substances in rainbow trout (Oncorhynchus mykiss)?

Perfluorinated and polyfluorinated substances (PFASs) are widely found in freshwater ecosystems because of their resistance to degradation and their ability to accumulate in aquatic organisms. While water temperature controls many physiological processes in fish, knowledge of the effects of this factor on PFAS toxicokinetic is still limited. This study presents experimental results of internal distribution and elimination rates of two perfluorinated acid compounds, namely perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) in adult rainbow trout (Oncorhynchus mykiss) exposed to three temperatures. Dietary exposure experiments were conducted at 7 °C, 11 °C, and 19 °C and liver, blood, muscle, brain, and kidney were sampled for analysis. PFOS concentrations were comparable to or exceeded those of PFHxS, while PFHxS was eliminated faster than PFOS, whatever the temperature. Internal distribution changed significantly for both substances when fish were exposed to a range of temperatures from 7 to 19 °C. Indeed, PFOS and PFHxS relative distribution increased in blood, liver, and brain while they decreased in muscle when the water temperature rose. The water temperature variation affected the elimination half-lives, depending on the substances and organs.

Comparison in the response of three European Gammarid species exposed to the growth regulator insecticide fenoxycarb.

Growth regulator insecticides with juvenoid activity can affect the development and reproduction of non-target organisms such as crustaceans. In this perspective, our previous studies revealed deleterious effects of the juvenoid fenoxycarb at 5 µg L-1 on the embryogenesis and at 50 µg L-1 on the reproductive behavior of the amphipod Gammarus fossarum. In the present study, to determine whether data generated with one amphipod species can be extended to other gammarid species, we tested the effects of a 5 µg L-1 fenoxycarb exposure on three European amphipod species: G. fossarum, Gammarus roeseli, and Echinogammarus longisetosus. We exposed individually 60 freshly fertilized females to fenoxycarb throughout the entire oogenesis/embryogenesis cycle (i.e., 19 days). In newborn individuals from exposed embryos, we measured both pigmentation and lipid reserve impairments while in exposed females, we observed reproductive behavior. At 5 µg L-1 fenoxycarb, reproductive behavior was only altered in G. fossarum. This study demonstrates the variability of the toxic response among the three gammaridae species, underlining the need for acquiring data with a broad phylogenetic representation to better predict toxic effects on freshwater ecosystems.

Exposure assessment of honeybees through study of hive matrices: analysis of selected pesticide residues in honeybees, beebread, and beeswax from French beehives by LC-MS/MS.

Apiculture and pollination services are seriously threatened by bee weakening and losses phenomena. In this context, a survey was performed on samples from beehives across French areas during the 2012-2016 growing seasons, primarily taken from symptomatic colonies. A total of 488 honeybees, beebread, and wax were analyzed for the presence of pesticide residues. A total of 13 analytes including neonicotinoids and pyrethroids insecticides together with some of their metabolites and the fungicide boscalid were screened within samples. Methodologies based on efficient modified quick, easy, cheap, effective, rugged, and safe extractions followed by an LC-MS/MS quantification were implemented for each matrix. Thirty-eight percent of the 125 bee samples, 61% of the 87 wax samples, and 77% of the 276 beebread samples contained at least one of the targeted pesticides. Beebread was the most contaminated matrix with an average of two pesticide detections by positive sample and a maximum of seven compounds for a sample. Neonicotinoids and boscalid were the most often detected pesticides, whatever the matrix. The comparison of neonicotinoid detections in samples collected before and after the partial neonicotinoid ban in France displays a decrease in the frequency of detections for contamination levels lower than 1 ng/g in beebread. For higher levels and other matrices, no tendency can be drawn.

Fenoxycarb exposure disrupted the reproductive success of the amphipod Gammarus fossarum with limited effects on the lipid profile.

Insect growth regulator insecticides mimic the action of hormones on the growth and development of insect pests. However, they can affect the development of non-target arthropods. In the present study, we tested the effects of the growth regulator insecticide fenoxycarb on several endpoints in the freshwater crustacean Gammarus fossarum (Amphipoda). Females carrying embryos in their open brood pouch were exposed to 50 µg L-1 fenoxycarb throughout the entire oogenesis (i.e. 21 days). After exposure, newborn individuals from exposed embryos were removed from the maternal open brood pouch for lipidomic analysis, while males were added to assess the reproductive success. After fertilization, the lipid profile, energy reserve content (lipids, proteins and glycogen), and activity of phenoloxidase - an enzyme involved in the immune response - were measured in females. No significant effect of fenoxycarb exposure was observed on the lipid profile of both newborn individuals and females, while reproductive success was severely impaired in exposed females. Particularly, precopulatory behavior was significantly reduced and fertilized eggs were unviable. This study highlighted the deleterious effects of the insect growth regulator fenoxycarb on gammarid reproduction, which could have severe repercussions on population dynamics.

Determination of a new index of sexual maturity (ISM) in zebra mussel using flow cytometry: interest in ecotoxicology.

The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

Determination of carbamazepine and 12 degradation products in various compartments of an outdoor aquatic mesocosm by reliable analytical methods based on liquid chromatography-tandem mass spectrometry.

The aims of this work are to develop suitable analytical methods to determine the widely used anticonvulsant carbamazepine and 12 of its degradation/transformation products in water, sediment, fish (Gasterosteus aculeatus) and mollusc (Dreissena polymorpha). Protocols based on solid phase extraction for water, pressurized-liquid extraction for sediments and QuEChERS (quick easy cheap efficient rugged and safe) extraction for both organisms followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are developed, validated and finally applied to samples collected during a 6-month experiment in outdoor mesocosms. Very low detection limits are reached, allowing environmentally realistic doses (namely, 0.05, 0.5 and 5 µg/L nominal concentrations) to be employed. The results indicate several metabolites and/or transformation products in each compartment investigated, with concentrations sometimes being greater than that of the parent carbamazepine. Biotic degradation of carbamazepine is demonstrated in water, leading to 10,11-dihydrocarbamazepine and 10,11-epoxycarbamazepine. In sediment, the degradation results in the formation of acridine, and 2- and 3-hydroxycarbamazepine. Finally, in both organisms, a moderate bioaccumulation is observed together with a metabolization leading to 10,11-epoxycarbamazepine in fish and 2-hydroxycarbamazepine in mollusc. Acridone is also present in fish. This study provides new and interesting data, helping to elucidate how chronic exposure to carbamazepine at relevant concentrations may affect impact freshwater ecosystems.