Assessment of the Presence of Transformation Products of Certain Pharmaceutical Products (Psychotropic Family) by Suspect and Non-Targeted HRMS Screening in Wastewater Treatment Plants.

Aquatic environments are the final receptors of human emissions and are therefore contaminated by molecules, such as pharmaceuticals. After use, these compounds and their metabolites are discharged to wastewater treatment plants (WWTPs). During wastewater treatment, compounds may be eliminated or degraded into transformation products (TPs) or may be persistent. The aim of this study was to develop an analytical method based on high resolution mass spectrometry (HRMS) for the identification of six psychotropic drugs that are widely consumed in France and present in WWTPs, as well as their potential associated metabolites and TPs. Four out of six psychotropic drugs and between twenty-five and thirty-seven potential TPs were detected in wastewater, although this was based on full scan data. TPs not reported in the literature and specific to the study sites and therefore to the wastewater treatment processes were tentatively identified. For the selected drugs, most known and present TPs were identified, such as desmethylvenlafaxine or norcitalopram. Moreover, the short fragmentation study led rather to the identification of several TPs of carbamazepine as ubiquitous persistent TPs.

Toxicity of binary mixtures of pesticides to the marine microalgae Tisochrysis lutea and Skeletonema marinoi: Substance interactions and physiological impacts.

This study screened binary mixtures of pesticides for potential synergistic interaction effects on growth of the marine microalgae Tisochrysis lutea and Skeletonema marinoi. It also examined the single and combined effects of three of the most toxic substances on microalgal physiology. Single substances were first tested on each microalgal species to determine their respective EC50 and concentration-response relationships. The toxicity of six and seven binary mixtures was then evaluated in microplate experiments on the growth of T. lutea and S. marinoi, respectively, using two mixture modelling approaches: isobolograms and the MIXTOX tool, based on Concentration Addition (CA) or Independent Action (IA) models. Significant cases of antagonism (for both species) and synergism (for S. marinoi) were observed for the mixtures of isoproturon and spiroxamine, and isoproturon and metazachlor, respectively. These two mixtures, together with that of isoproturon and diuron, for which additivity was observed, were further studied for their impacts on the physiology of each species. Exposures were thus made in culture flasks at three concentrations, or concentration combinations for mixtures, selected to cause 25%, 50% and 75% growth rate inhibition. The effects of the selected pesticides singly and in combination were evaluated at three perceived effect concentrations on esterase metabolic activity, relative lipid content, cytoplasmic membrane potential and reactive oxygen species (ROS) content by flow cytometry, and on photosynthetic quantum yield (ϕ'M) by PAM-fluorescence. Isoproturon and diuron singly and in mixtures induced 20-40% decreases in ϕ'M which was in turn responsible for a significant decrease in relative lipid content for both species. Spiroxamine and metazachlor were individually responsible for an increase in relative lipid content (up to nearly 300% for metazachlor on S. marinoi), as well as cell depolarization and increased ROS content. The mixture of isoproturon and metazachlor tested on S. marinoi caused a 28-34% decrease in ϕ'M that was significantly higher than levels induced by each of substances when tested alone. This strong decrease in ϕ'M could be due to a combined effect of these substances on the photosynthetic apparatus, which is likely the cause of the synergy found for this mixture.

Demonstrating the need for chemical exposure characterisation in a microplate test system: toxicity screening of sixteen pesticides on two marine microalgae.

Pesticides used in viticulture create a potential risk for the aquatic environment due to drift during application, runoff and soil leaching. The toxicity of sixteen pesticides and one metabolite were evaluated on the growth of two marine microalgae, Tisochrysis lutea and Skeletonema marinoi, in 96-h exposure assays conducted in microplates. For each substance, concentrations of stock solutions were analytically measured and abiotic assays were performed to evaluate the chemical stability of pesticides in microplates. For two chemicals, microalgae exposures were run simultaneously in microplates and culture flasks to compare EC50 calculated from the two exposure systems. Results from chemical analyses demonstrated the low stability of hydrophobic pesticides (log KOW > 3). For such chemicals, EC50 values calculated using measured pesticide concentrations were two-fold lower than those first estimated using nominal concentrations. Photosystem II inhibitors were the most toxic herbicides, with EC50 values below 10 µg L-1 for diuron and around double this for isoproturon. Chlorpyrifos-methyl was the only insecticide to significantly affect the growth of T. lutea, with an EC50 around 400 µg L-1. All fungicides tested were significantly toxic to both species: strobilurins showed low overall toxicity, with EC50 values around 400 µg L-1, whereas quinoxyfen, and spiroxamine, showed high toxicity to both species, especially to T. lutea, with an EC50 below 1 µg L-1 measured for spiroxamine in culture flasks. This study highlights the need to perform chemical analyses for reliable toxicity assessment and discusses the advantages and disadvantages of using microplates as a toxicity screening tool.

Combined effects of antifouling biocides on the growth of three marine microalgal species.

The toxicity of the antifouling compounds diuron, irgarol, zinc pyrithione (ZnPT), copper pyrithione (CuPT) and copper was tested on the three marine microalgae Tisochrysis lutea, Skeletonema marinoi and Tetraselmis suecica. Toxicity tests based on the inhibition of growth rate after 96-h exposure were run using microplates. Chemical analyses were performed to validate the exposure concentrations and the stability of the compounds under test conditions. Single chemicals exhibited varying toxicity depending on the species, irgarol being the most toxic chemical and Cu the least toxic. Selected binary mixtures were tested and the resulting interactions were analyzed using two distinct concentration-response surface models: one using the concentration addition (CA) model as reference and two deviating isobole models implemented in R software; the other implementing concentration-response surface models in Excel®, using both CA and independent action (IA) models as reference and three deviating models. Most mixtures of chemicals sharing the same mode of action (MoA) were correctly predicted by the CA model. For mixtures of dissimilarly acting chemicals, neither of the reference models provided better predictions than the other. Mixture of ZnPT together with Cu induced a strong synergistic effect on T. suecica while strong antagonism was observed on the two other species. The synergy was due to the transchelation of ZnPT into CuPT in the presence of Cu, CuPT being 14-fold more toxic than ZnPT for this species. The two modelling approaches are compared and the differences observed among the interaction patterns resulting from the mixtures are discussed.

The influence of natural dissolved organic matter on herbicide toxicity to marine microalgae is species-dependent.

Microalgae, which are the foundation of aquatic food webs, may be the indirect target of herbicides used for agricultural and urban applications. Microalgae also interact with other compounds from their environment, such as natural dissolved organic matter (DOM), which can itself interact with herbicides. This study aimed to evaluate the influence of natural DOM on the toxicity of three herbicides (diuron, irgarol and S-metolachlor), singly and in ternary mixtures, to two marine microalgae, Chaetoceros calcitrans and Tetraselmis suecica, in monospecific, non-axenic cultures. Effects on growth, photosynthetic efficiency (Ф'M) and relative lipid content were evaluated. The chemical environment (herbicide and nutrient concentrations, dissolved organic carbon and DOM optical properties) was also monitored to assess any changes during the experiments. The results show that, without DOM, the highest irgarol concentration (I0.5: 0.5 mg.L-1) and the strongest mixture (M2: irgarol 0.5 µg.L-1 + diuron 0.5 µg.L-1 + S-metolachlor 5.0 µg.L-1) significantly decreased all parameters for both species. Similar impacts were induced by I0.5 and M2 in C. calcitrans (around -56% for growth, -50% for relative lipid content and -28% for Ф'M), but a significantly higher toxicity of M2 was observed in T. suecica (-56% and -62% with I0.5 and M2 for growth, respectively), suggesting a possible interaction between molecules. With DOM added to the culture media, a significant inhibition of these three parameters was also observed with I0.5 and M2 for both species. Furthermore, DOM modulated herbicide toxicity, which was decreased for C. calcitrans (-51% growth at I0.5 and M2) and increased for T. suecica (-64% and -75% growth at I0.5 and M2, respectively). In addition to the direct and/or indirect (via their associated bacteria) use of molecules present in natural DOM, the characterization of the chemical environment showed that the toxic effects observed on microalgae were accompanied by modifications of DOM composition and the quantity of dissolved organic carbon excreted and/or secreted by microorganisms. This toxicity modulation in presence of DOM could be explained by (i) the modification of herbicide bioavailability, (ii) a difference in cell wall composition between the two species, and/or (iii) a higher detoxification capacity of C. calcitrans by the use of molecules contained in DOM. This study therefore demonstrated, for the first time, the major modulating role of natural DOM on the toxicity of herbicides to marine microalgae.

Microalgal sensitivity varies between a diuron-resistant strain and two wild strains when exposed to diuron and irgarol, alone and in mixtures.

A wild strain of Chaetoceros calcitrans and wild and diuron-resistant strains of Tetraselmis suecica, were exposed to the PSII inhibitor herbicides diuron and irgarol, individually and in mixtures. The effects of three concentrations of diuron and irgarol and four binary mixtures were evaluated on doubling time, relative reactive oxygen species and lipid content by flow cytometry, and on photosynthetic efficiency by pulse amplitude modulated fluorescence. In both wild strains, significant effects were observed for each molecule at the highest concentration tested: at irgarol 0.5 µg L(-1), C. calcitrans was shown to be more sensitive than T. suecica (+52% and +19% in doubling time, respectively), whereas at diuron 5 µg L(-1), T. suecica was more affected (+125% in doubling time) than C. calcitrans (+21%). Overall, irgarol had a higher toxicity at a lower concentration than diuron (no effect at diuron 0.5 µg L(-1)) for both wild strains. The strongest mixture (irgarol 0.5 µg L(-1) + diuron 5 µg L(-1)) increased doubling time by 356% for T. suecica, thus showing amplified effects when the two compounds were mixed. Sequencing of the diuron-resistant strain demonstrated a single mutation in the psbA gene coding sequence. Although resistance of this strain to diuron was confirmed with no effect at the highest diuron concentration, no resistance to irgarol was shown. In addition, the mutant strain exposed to the strongest mixture showed a 3.5-fold increase in doubling time compared with irgarol alone, thereby supporting the hypothesis of a biochemical interaction between these two compounds.