Adriana Wigh

Olivier Geffard

Khedidja Abbaci

Adeline François

Patrice Noury

Alexandre Bergé

Emmanuelle Vulliet

Bruno Domenjoud

Adriana Gonzalez-Ospina

Sylvie Bony

Adeline Francois

Alain Devaux
email: alain.devaux@entpe.fr

Gammarus fossarum as a sensitive tool to reveal residual toxicity of treated wastewater effluents

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

To date, wastewater treatment plants (WWTPs) continuously discharge micropollutants of concern, which can have biological adverse effects on non-target organisms in receiving ecosystems [START_REF] Oehlmann | Bisphenol A induces superfeminization in the ramshorn snail Marisa cornuarietis (Gastropoda: Prosobranchia) at environmentally relevant concentrations[END_REF][START_REF] Besse | Caged Gammarus fossarum (Crustacea) as a robust tool for the characterization of bioavailable contamination levels in continental waters: towards the determination of threshold values[END_REF][START_REF] Morrissey | Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: a review[END_REF][START_REF] Petrie | A review on emerging contaminants in wastewaters and the environment: current knowledge, understudied areas and recommendations for future monitoring[END_REF]. WWTPs were designed and are being constantly improved to reduce efficiently the most part of contaminants, but conventional biological WWTP treatments are not effective to remove some biologically refractory micropollutants [START_REF] Schwarzenbach | The challenge of micropollutants in aquatic systems[END_REF][START_REF] Wick | Fate of beta blockers and psychoactive drugs in conventional wastewater treatment[END_REF]. The Directive 2013/39/EU (European Commission, 2013) supports wastewater treatment plant authorities to develop and use new treatment technologies to reduce the potential hazard of treated effluents towards the aquatic environment. Since effluents should be shown as non-toxic for aquatic flora and fauna before entering receiving waters, standardized toxicity tests using algae, daphnids or fish are required by the European Commission (2000) to evaluate residual toxicity in treated effluents. Effluents can contain many micropollutants at very low concentrations after treatment and it has been shown that standardized bioassays might underestimate effects or might not be sensitive enough to reveal residual toxicity [START_REF] Kümmerer | The presence of pharmaceuticals in the environment due to human usepresent knowledge and future challenges[END_REF][START_REF] Wigh | Proposal to optimize ecotoxicological evaluation of wastewater treated by conventional biological and ozonation processes[END_REF]. Although concentrations of residual compounds are generally in the ng/L range, they still might impair organism fitness under chronic exposure and so the long-term ecological state [START_REF] Fent | Ecotoxicology of human pharmaceuticals[END_REF][START_REF] Newman | Ecotoxicology: A Comprehensive Treatment[END_REF]. [START_REF] Berger | Field data reveal low critical chemical concentrations for river benthic invertebrates[END_REF] showed that chemicals were likely to induce effects in the environment at concentrations much lower than those based on laboratory experiments. Moreover, extrapolating single compound toxicity, generally assessed in the laboratory, to field-situations may underestimate the actual impact, because of possible pollutant interactions due to the chemical complexity of effluents and thus of receiving water bodies [START_REF] Silva | Something from "Nothing"-eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects[END_REF]. Therefore, special attention must be paid for approaches on sensitive species and bioassays allowing long-term exposure and the measurement of relevant endpoints related to the fitness of organisms.

Gammarids as detritivorous species play an important role in the trophic food chain of the aquatic environment. They decompose organic matter and serve as prey for amphibians, insects, flatworms, other crustaceans such as crabs and crayfish, and fish [START_REF] Macneil | The validity of the Gammarus: Asellus ratio as an index of organic pollution: abiotic and biotic influences[END_REF]. Gammarids have been shown as very sensitive to pollution, in particular from wastewaters [START_REF] Peschke | Invertebrates as indicators for chemical stress in sewage-influenced stream systems: toxic and endocrine effects in gammarids and reactions at the community level in two tributaries of Lake Constance, Schussen and Argen[END_REF][START_REF] Schirling | Endocrine effects in Gammarus fossarum (Amphipoda): influence of wastewater effluents, temporal variability, and spatial aspects on natural populations[END_REF][START_REF] Schneider | Impact of an estrogenic sewage treatment plant effluent on life-history traits of the freshwater amphipod Gammarus pulex[END_REF]. They have been used in various studies for toxicity evaluation of river waters and effluents through in situ exposure or mesocosm study, by measuring toxicity endpoints such as reproduction, growth impairment and genotoxicity [START_REF] Bundschuh | Population response to ozone application in wastewater: an on-site microcosm study with Gammarus fossarum (Crustacea: Amphipoda)[END_REF]Lacaze et al., 2011c;[START_REF] Coulaud | Linking feeding inhibition with reproductive impairment in Gammarus confirms the ecological relevance of feeding assays in environmental monitoring[END_REF]. Alterations in reproduction endpoints such as molting cycle, fertility and fecundity may be caused by various pollutants in the effluents and can result in population dynamics impairment [START_REF] Mazurova | Chronic toxicity of contaminated sediments on reproduction and histopathology of the crustacean Gammarus fossarum and relationship with the chemical contamination and in vitro effects[END_REF][START_REF] Coulaud | Linking feeding inhibition with reproductive impairment in Gammarus confirms the ecological relevance of feeding assays in environmental monitoring[END_REF][START_REF] Schneider | Impact of an estrogenic sewage treatment plant effluent on life-history traits of the freshwater amphipod Gammarus pulex[END_REF]. Pollutant genotoxicity can lead to organism fitness impairment in particular when affecting gametes. Hence it has been used in many studies as a sensitive sub-lethal endpoint to assess the toxicity of environmental samples [START_REF] Stalter | Comparative toxicity assessment of ozone and activated carbon treated sewage effluents using an in vivo test battery[END_REF][START_REF] Devaux | Reproduction impairment following paternal g e n o t o x i ne x p o s u r ei nb r o w nt r o u t( Salmo trutta) and Arctic charr (Salvelinus alpinus)[END_REF]Lacaze et al., 2011c;[START_REF] Magdeburg | Evaluating the efficiency of advanced wastewater treatment: target analysis of organic contaminants and (geno-) toxicity assessment tell a different story[END_REF]. Spermatozoa of Gammarus fossarum were found to be the most sensitive cell type for genotoxicity evaluation compared to hemocytes and oocytes (Lacaze et al., 2011a).

Effluents contain a range of pesticides known to block acetylcholinesterase activity (AChE) in chemical synapses leading to neurotoxic effects due to an overactivation of postsynaptic acetylcholine receptors.

Measurement of AChE inhibition has been successfully used as a biomarker for the presence of neurotoxic compounds, in particular in gammarids [START_REF] Fulton | Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects[END_REF]Xuereb et al., 2009a). The detoxification enzyme glutathione S-transferase activity (GST activity) has been shown to increase with oxidative stress in organisms exposed to a large array of contaminants such as nitro compounds, organophosphates and organochlorines, possibly present in treated wastewaters [START_REF] Hyne | Invertebrate biomarkers: links to toxicosis that predict population decline[END_REF][START_REF] Karaouzas | Bioassays and biochemical biomarkers for assessing olive mill and citrus processing wastewater toxicity[END_REF][START_REF] Turja | Cumulative effects of exposure to cyanobacteria bloom extracts and benzo[a]pyrene on antioxidant defence biomarkers in Gammarus oceanicus (Crustacea: Amphipoda)[END_REF]. Moreover, treated effluents contain various bacteria and parasites that can enhance some adverse effects of pollutants towards organisms. Phenol-oxidase is an important enzyme for arthropod immune defense by triggering encapsulation and melanin deposition to prevent microbial growth [START_REF] Sugumaran | Comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects[END_REF]. A strong synergistic toxic effect was shown in Enallagma cyathigerum larvae exposed to the organophosphate insecticide chlorpyrifos (acetylcholinesterase inhibitor) and to the non-pathogenic bacterium Escherichia coli [START_REF] Janssens | Exposure to a widespread non-pathogenic bacterium magnifies sublethal pesticide effects in the damselfly Enallagma cyathigerum: from the suborganismal level to fitness-related traits[END_REF].

The aim of the present study was to evaluate the residual chronic toxicity of two differently treated WWTP effluents, by measuring endpoints regarding genotoxicity, neurotoxicity, biotransformation, reproduction and immune defense in gammarid Gammarus fossarum caged in effluent output.

Materials and methods

Gammarid sampling and acclimatization

The sampling site La Tour du Pin (Isère, France. 45°33′54″N5°26′40″ E) is located in an unpolluted upstream part of the Bourbre River, where the freshwater amphipod Gammarus fossarum is found at a very high population density (Lacaze et al., 2011a). Gammarids (about 8 mm in size) were collected and let to acclimatize for 12 days in the laboratory in a mixture of well and osmosed waters in standardized conditions: temperature of 12 ± 1 °C, pH 7.6 ± 0.2 and a conductivity of 600 ± 50 μS/cm. Oxygen saturation (N90%) was ensured with an aeration pump. Photoperiod was maintained at 16 h light/8 h dark. Gammarids were fed ad libitum on black alder leaves (Alnus glutinosa) and supplied once a week with freeze-dried Tubifex worms.

Pilot plant description

In situ exposure of gammarids was conducted in a pilot wastewater treatment plant in Bellecombe (Haute-Savoie, France). The capacity of the two pilot treatment lines was 60 L/h with 23 h of hydraulic contact time. The activated sludge biological treatments were operated in order to achieve a full nitrification at minimal sludge age. The concentration of sludge in the aeration tanks was 2-3 g/L. The treatment lines were fed with a mixture of raw urban and hospital wastewater at a ratio of 50% v/v with average total suspended solids (TSS), chemical oxygen demand (COD) and N-NH 4 concentration of 139 mg/L, 614 mg/L and 41 mg/L, respectively. Gammarids were exposed to an effluent treated with conventional activated sludge (CAS) and to an effluent treated with conventional activated sludge combined with the ozonation of the mixed liquor in the recirculation loop (CASO 3 loop ). The specific transferred ozone dose, expressed per volume of feeding effluent was 9.4 mg of ozone per liter (Fig. 1).

Effluent characterization:

CASO 3l o o p effluent and CAS effluent exhibited very similar physicochemical characteristics (Table 1).

Gammarid exposure

Sufficient oxygenation (90% oxygen saturation) of exposed gammarids in the outflow tanks was provided by an aeration device. Gammarids were exposed from June 3rd to June 12th 2015 (10 days exposure). Three replicates, constituted each of 7 couples (each constituted of a guarding male and of a female at the last stage of reproductive cycle i.e. with hatched juveniles in marsupium and visible gonads) were devoted to genotoxicity and reproduction assessment for both controls and exposed groups [START_REF] Geffard | Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment[END_REF]. Additionally, 4 replicates with 20 males each were used for all other biomarker measurements. Organisms were caged in perforated polypropylene tubes (5 × 12 cm) containing black alder leaves as a food source, and fixed on holders in a perforated closed bucket, placed in the middle of a tank receiving the treated effluent.

After ten days of exposure, organisms were brought back to the laboratory for endpoints measurement.

Control groups

In the laboratory, two control groups were conducted, the number of replicates and animals being the same than for exposed groups (see above). Control 1 was kept in well water at 18 ± 1 °C, pH 7.6 ± 0.2 and 600 ± 50 μS/cm. The temperature was adapted to in situ exposure conditions since molting cycle is temperature dependent and sexual maturity increases with temperature [START_REF] Glazier | Abundance, body composition and reproductive output of Gammarus minus (Crustacea: Amphipoda) in ten cold springs differing in pH and ionic content[END_REF][START_REF] Pöckl | Life history and reproductive capacity of Gammarus fossarum and G. roeseli (Crustacea: Amphipoda) under naturally fluctuating water temperatures: a simulation study[END_REF]. A second control group (control 2) was kept in well water at 18 ± 1 °C, pH 7.6 ± 0.2 and at a conductivity of 1700 ± 50 μS/cm comparable to that of effluents, to check for possible toxic effects on gammarids due to osmoregulation impairment [START_REF] Cornet | Variation in immune defence among populations of Gammarus pulex (Crustacea: Amphipoda)[END_REF]. Water conductivity for control 2 was adjusted by adding 60 mg/L CaCl 2 , 2H 2 O, 900 mg/L NaCl, 40 mg/L NA 2 HPO 4 ,2 H 2 O, 30 mg/L MgCL 2 , 40 mg/L KCl. Oxygen saturation was ensured with an aeration pump and photoperiod was maintained at 16 h light/8 h dark.

Reproduction and embryo development

According to [START_REF] Geffard | Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment[END_REF], female molting stages, number of oocytes in ovaries and developmental stages of embryos found in the marsupium were evaluated to determine the impact on reproduction. Gammarus fossarum undergoes six molting stages, which are defined as A and B for the post-molt stage, C1 and C2 for the inter-molt stage and D1 and D2 for the pre-molt stage (Fig. 2).

Maturation of new oocytes occurs concurrently with embryonic development during the molting cycle. In a normal undisrupted cycle, each molting stage is characterized by one embryonic stage (Fig. 2). In post-molt (A-B) females, newly fertilized embryos are present in the marsupium pouch (stage 1). Embryos in stage 2 have a comma shape and are present in molt-stage C1 females. Embryonic development stage 3 corresponds to stage C2 female and the embryo developed a cephalothorax and segmented appendages. At stage 4, the eye and appendages are fully developed and the embryos at this stage occur in molting stage D1 females. When females switch to molting stage D2, the fully developed embryos (stage 5) hatch. The mean number of oocytes normalized by female size decreases with the molting cycle, and from molting stage C2 it stays stable and normally comparable to the number of embryos in the marsupium. The number of oocytes can be an indicator of the embryos produced in the marsupium for the next reproductive/molting cycle. C2 and D1 stages are the longest lasting ones where possibly occurring embryonic developmental abnormalities are at best observed. Number of oocytes and number of embryos and embryo developmental stages are determined in these two molting stages C2 and D1. For determining embryonic development stages and probable anomalies, embryos were released from the marsupium with fine forceps, placed on a slide, immersed in a drop of water and observed under a binocular light microscope. The number of embryos per female normalized according to female size (Ne), embryonic stages and developmental abnormalities in embryos were measured. Observed abnormalities were edema, irregular cleavages and necrosis, enlargement or non-specific malformations as illustrated in Lacaze et al. (2011c) and were quantified as an overall abnormality percentage.

Genotoxicity in sperm

The genotoxic potential of the effluents was determined by the assessment of the primary DNA damage level in spermatozoa using the comet assay carried out according to [START_REF] Lacaze | Genotoxicity assessment in the amphipod Gammarus fossarum by use of the alkaline comet assay[END_REF].Attheendof exposure, 10 mature males per treatment which have been exposed in precopula stage were chosen. The cephalon was cut off with microscissors and caeca were removed from the thorax. Then, the dorsal and ventral cuticle were excised and the gonads, mostly surrounded by orange lipid drops, were collected and dilacerated into 200 μL microtube containing 20 μL cold phosphate-buffer-saline (PBS) and stored on ice until comet assay processing. Since the dissection took some time and in order to prevent cell degradation, comet assay was carried out as soon as 3 sperm samples were prepared. Each cell suspension was equally mixed (50% V/V) with 37 °C heated low melting point agarose gel (1%) and 40 μL of this mixture were laid on a frosted microscope slide pre-coated with 0.8% agarose (sperm from each male provided 2 gels per slide) and then covered with a cover-slide. After 10 min at 4 °C, the cover-slides were gently removed. Slides were covered with the lysis solution (2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris-HCl, 1% Triton X-100 and 10% DMSO) and kept for 18 h at 4 °C in the dark. Afterwards, slides were placed in the electrophoresis tank filled with the electrophoresis solution (300 mM NaOH, 1 mM EDTA) and left for 40 min for DNA unwinding. Then, electrophoresis was conducted at 0.7 V/cm and 300 mA for 24 min. The negatively charged DNA migrates through the gel to the anode resulting in a so-called comet tail when DNA strand breaks occur. After electrophoresis, slides were washed 3 times for 5 min with a neutralization buffer (0.4 mM Tris-HCl) and dehydrated in 100% ethanol for 20 min and let dry at room temperature. For DNA damage scoring, slides were coated with 25 μL of a 0.02% ethidium bromide solution. Each slide was examined through a fluorescence microscope (Axioskop 40, Zeiss Ltd.). Image analysis was performed with the Comet 4.0 software (Perceptive Instruments Ltd.). For each exposed gammarid, tail % intensity (TI, % DNA in comet tail) of 2 × 50 randomly selected cells was measured. However, some males did not provide enough spermatozoa, thus the total number of gels scored for each treatment varied.

Neurotoxicity, immune defense and detoxification activity

For acetylcholinesterase (AChE), glutathione s-transferase (GST) and phenol oxidase (PO) activity measurement, in toto homogenates of five male gammarids were prepared (15 to 20 mg per male, resulting in a total mass of 80 to 100 mg). Gammarus fossarum whole bodies were homogenized in 1:10 (w:v) ice-cold phosphate buffer (100 mM pH 7.8) containing 0.1% Triton X-100, with an Ultra-Turrax® T25 basic (24,000 rpm for 35 s). Then, homogenates were centrifuged at 4 °C for 15 min at 9000g and the supernatant (post-mitochondrial fraction or S9) was conserved at 4 °C until enzymatic measurements. All enzymatic reactions were followed at room temperature in 96-well microplates.

A decrease in acetylcholinesterase activity was assigned as a biomarker of neurotoxicity [START_REF] Fulton | Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects[END_REF]Xuereb et al., 2009a). AChE activity was determined according to [START_REF] Ellman | A new rapid colorimetric determination of acetylcholinesterase activity[END_REF] adapted to microplates (Xuereb et al., 2009a). In a 96-well microtiter plate, 20 μL of 0.0076 M (final concentration) dithiobisnitrobenzoate, 20 μLofS9 and 330 μL of phosphate buffer (0.1 M final concentration) and 10 μL of acetylthiocholine iodide solution were added. Hydrolysis to the yellow product 5-thio-2-nitro-benzoic-acid was measured at 405 nm in a spectrophotometer (SafireR spectrofluorimeter microplate reader, TECAM; Trappes, France) every 60 s for at least 3 min. AChE activity was expressed as nmoles of substrate hydrolyzed per minute. Xuereb et al. (2009a) showed that natural variation of structural protein contents in female Gammarus fossarum related to physiological changes as the reproductive status, caused variability in AChE activity and therefore to an under-or over-estimation of the effect. According to the authors, variation in AChE activity is negligible in males with a weight between 15 and 20 mg and therefore male gammarids of this weight were used for AChE measurement in the present study. AChE activity was considered significantly inhibited when reaching a 12% lower value than that of control group. If AChE activity was significantly inhibited compared to the control, this was considered as resulting from exposure to neurotoxic compounds (Xuereb et al., 2009a).

GST activity was measured according to [START_REF] Habig | Glutathione S-transferase AA from rat liver[END_REF] in 96well microplates by adding 170 μL of 2.2 mM reduced glutathione (final concentration), 10 μL of 38 mM 1-chloro-2.4-dinitrobenzene (final concentration), 150 μL of 0.1 M phosphate buffer (final concentration) and 20 μL of the S9 fraction per well. GST activity was measured in a spectrophotometer (SafireR spectrofluorimeter microplate reader, TECAM; Trappes, France) at 340 nm every 30 s for at least 3 min. GST activity was expressed as nmoles of substrate hydrolyzed per minute per mg of protein. Protein content was measured according to [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Phenol oxidase activity was measured according to Janssens and Stoks (2013), adapted for 96-well microtiter plates. Eighty microliters of 5 mM phosphate buffer (final concentration) and 15 μL of S9 were added to each well. Then, 5 μL of chymotrypsin solution (5 mg/mL dissolved in ultra-pure water) were added and incubated for 5 min at room temperature to convert all pro-enzyme prophenoloxidase into phenoloxidase. Afterwards, 100 μL of 3.4-dihydroxy-L-phenylalanine (1.97 mg/mL in PBS) were added and the absorbance of the red colored dopachrome was measured in a spectrophotometer (SafireR spectrofluorimeter microplate reader, TECAM; Trappes, France) at 490 nm every 30 s during 6 min at 30 °C. The phenol oxidase activity was expressed as nmoles of substrate hydrolyzed per minute per mg of protein.

Statistical analysis

Nonparametric rank sum test was used to test effects of exposure conditions on the molting cycle, number of oocytes and of embryos produced. Concerning genotoxicity evaluation, the DNA damage was expressed as the median TI% value calculated from the number of gels containing enough spermatozoa (i.e. 50) to be properly scored for each condition. Median TI % values measured in organisms, exposed to each effluent, were compared to the corresponding control 1 and 2 or Fig. 2. Molting stages (A to D2) and corresponding embryonic developmental stages 1 to 5 (according to [START_REF] Geffard | Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment[END_REF].

between effluents using a rank sum test. Phenol oxidase, acetylcholinesterase and glutathione S-transferase activities measured in gammarids from the different exposure scenarios were compared to the corresponding control groups 1 and 2 using rank sum test. Statistical analyses were conducted with the Graph Pad Prism version 5 for windows (GraphPad software, San Diego, CA, USA).

Chemical analysis of effluents

The technical concept sought to improve biodegradation through ozone application by increasing the speed and the overall efficiency of total micropollutant degradation through ozonation of sludge in recycling loop. Emerging contaminants as low biodegradable pharmaceuticals and surfactants [START_REF] Baig | Ozone disinfection of urban wastewater at full-scale. Impact on Persistent Micropollutants[END_REF][START_REF] Li | Occurrence, sources, and fate of pharmaceuticals in aquatic environment and soil[END_REF][START_REF] Luo | A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment[END_REF] were monitored in both wastewater effluents CAS and CASO 3 loop to compare their efficiency to remove such micropollutants. The pharmaceutical compounds analysed correspond to those regularly measured in the SIPIBEL experimental site, i.e. the field observatory on hospital effluents and urban wastewater treatment located in Bellecombe where the pilot plant was settled [START_REF] Chonova | Separate treatment of hospital and urban wastewaters: a real scale comparison of effluents and their effect on microbial communities[END_REF][START_REF] Perrodin | A posteriori assessment of ecotoxicological risks linked to building a hospital[END_REF]. These compounds were selected on the basis of their high consumption in hospital and domestic environments, their potential for bioaccumulation and toxic effects. The detergents selected correspond to the most common ones used for industrial (including hospitals) and domestic activities. These micropollutants (Table 2) were dosed by using multi-residual method coupling solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry. Effluents (a 24 h-composite sample collected June 9th 2015 for each condition) were filtered with glass microfiber filter at 0.7 μm then extracted with SPE according to [START_REF] Vulliet | Survey regarding the occurrence of selected organic micropollutants in the ground waters of overseas departments[END_REF] for pharmaceuticals and [START_REF] Bergé | Development of a multiple-class analytical method based on the use of synthetic matrices for the simultaneous determination of commonly used commercial surfactants in wastewater by liquid chromatography-tandem mass spectrometry[END_REF] for surfactants. Limit of detection (LD) and of quantification (LQ) for each compound are presented in Table 2.

Results

Chemical analysis of effluents

Regarding pharmaceutical compounds, ciprofloxacin and ethinylestradiol concentrations remained in all analysed samples below the limit of quantification (Table 3). Salicylic acid, ibuprofen, paracetamol and econazole concentrations were in the same range in CAS and CASO 3 loop effluents. The anti-inflammatory drug diclofenac and ketoprofen concentrations, and the beta-blockers propranolol and atenolol concentrations were lower in CASO 3 loop effluent than in CAS effluent. A similar trend was observed for both sulfamethoxazole and carbamazepine.

The surfactants comperlan 100, incromine SB, lauryl pyridinium, texapon N 701 S and cetyl betaine were below the limit of quantification in CAS and CASO 3 l o o p effluents (Table 4). Triton X-100, benzyldodecyldimethyl ammonium chloride, benzyldimethyltetradecyl ammonium chloride, sodium 2-ethylhexyl sulfate, sodium dodecyl sulfate and LAS C11 to C13 also remained below their limit of quantification in both CAS and CASO 3l o o p effluents.

The linear alkylbenzene sulfonate LAS C10 concentration was 9.5 μg/ L after CAS and to 10.2 μg/L after CASO 3l o o p treatment. Stepanquat GA 90 concentration was similar in both treatment lines.

Reproduction and embryo development

The molting cycle is temperature-dependent according to [START_REF] Pöckl | Effects of temperature, age and body size on moulting and growth in the freshwater amphipods Gammarus fossarum and G. roeseli[END_REF] and [START_REF] Pöckl | Comparative study of mathematical models for the relationship between water temperature and brood development time of Gammarus fossarum and G. roeseli (Crustacea: Amphipoda)[END_REF]. As shown in Table 5,a f t e r 10 days of exposure at an average temperature between 18 and 19 °C, 90% of the females in the control 1 group (n = 20 females) were in molt stage C2, 5% in molt stage C1 and 5% in molt stage D1 (Table 5). Correspondingly, 90% of embryos found in the marsupium of control females were at developmental stage 3 and 10% at stage 4. One female (5%) showed a retarded molt cycle (C1) compared to the embryonic stage of embryos present in the marsupium (stage 3). In control 2 (n = 14 females), 71.4% of females were in molt stage C2 and 28.6% in molt stage C1, 100% of embryos were in developmental stage 3 and three females showed a delay in molt cycle although not statistically different compared to control 1.

After exposure to CAS effluent, 53% of females (n = 16) were at molt stage C1 and 46% at molt stage AB. After exposure to CASO 3lo o p effluent 41% of females were in molt stage C1 and 47% of females (n = 17) were at molt stage AB. The embryonic stages of embryos present in the marsupium were not determined because too many embryos presented developmental abnormalities. The molt cycle was significantly inhibited in both CAS and CASO 3lo op effluent exposed groups when compared to the control 1 (p b 0.001) as well as compared to control 2 (p b 0.01). No difference between CAS and CASO 3l o o p groups was found.

The number of embryos per female normalized according to female size (Ne) ranged between 1.2 and 4.6 with a median value of 3.6 in control 1, and 10% of all counted embryos (n = 279) showed developmental abnormalities (Fig. 3A). In control 2 (adapted conductivity), Ne was significantly decreased to a median value of 2.8 (ranging from 0 to 3.7) compared to control 1 (p b 0.01) but the percentage of embryos exhibiting developmental abnormalities (12%) was comparable to that of control 1. Ne was significantly reduced in CAS (p b 0.001) with a median value of 0.4 embryo per female (minimum 0 to maximum 1.6) compared to control 1 as well as compared to control 2 (p b 0.05). From the total number of embryos (n = 20), 90% presented developmental abnormalities. Females exposed to CASO 3 loop effluent carried between 0 and 1.97 embryos (median 0.5), which was significantly different compared to control 1 and control 2 (p b 0.001), and 96% of observed embryos (n = 28) exhibited developmental abnormalities. Ne was not significantly different in CAS or CASO 3l o o p exposed organisms.

The number of oocytes in females (normalized according to female size) at molt stage C2 or D1 (n = 19) ranged between 0 and 4.3 (median value at 2.8) in control 1 (Fig. 3B). In control 2 (n = 10 females in molt stage C2), the number of oocytes per female ranged between 0 and 3.2 (median value at 2.6) and did not differ statistically from that of control 1. In CASO 3 loop effluent exposure group, only one female was in molt stage C2 and exhibited 3.8 oocytes, whereas in CAS effluent exposure group none of the females were in C2 or D1 and therefore no oocyte number could be determined.

Genotoxicity assessment

Primary DNA damage in gammarid sperm (Fig. 4) from control 1 ranged between 0.06% and 17% TI with a median value of 7.3%, and between 2.4% and 21% (9.2% median value) in control 2, not statistically different compared to amphipods of control 1. Sperm of gammarids exposed to CAS effluent showed a slightly but significantly higher DNA damage level with a median TI value of 15.5% (minimum 2, maximum 25.5) compared to that of control 1 (p = 0.019), but not different compared to that of control 2. Tail intensity measured in sperm of gammarids exposed to CASO 3l o o p effluent (11% median TI, minimum 3.6, maximum 27.5) was not significantly different compared to those from both controls and from CAS group.

Neurotoxicity, immune defense and detoxification activity measurement

As proposed by Xuereb et al. (2009a), the acetylcholinesterase (AChE) activity was expressed as nmol/min (Fig. 5A). In male gammarids, AChE activity was the same in both controls, 10.3 nmol/min (CI 95% : [10.1, 10.5]) in control 1 and 11.2 nmol/min (CI 95% :[ 1 0 . 1 , 12.3]) in control 2. AChE activity measured in male gammarids exposed to CAS effluent (11.5 nmol/min (CI 95% : [11.2, 11.9])) was slightly increased compared to control 1 and 2 (11.8% and 2.9% increase, respectively), although not statistically different. AChE activity in gammarids exposed to CASO 3 loop effluent (10.5 nmol/min, CI 95% : [9.8, 11.3]) was not statistically different from that of both controls and of CAS group.

Glutathione S-transferase (GST) activity was in average the same in all exposure groups, 8.2 nmol/min/mg (CI 95% : [7.5, 9.0]), 9.1 nmol/min/mg (CI 95% : [8.7, 9.5]), 8.1 nmol/min/mg (CI 95% :[ 7 . 4 , 8.8]) and 8.4 nmol/min/mg (CI 95% : [7.5, 9.4]), in control 1, control 2, CAS and CASO 3 loop exposed groups, respectively (Fig. 5B). Phenoloxidase (PO) activity measured in Gammarus fossarum from the Table 5 Occurrence (%) of molt stages (AB, C1, C2, D1, D2) and embryonic stages (1,2,3,4) in Gammarus fossarum. Control 1: well water at 18 ± 1 °C, pH 7.6 ± 0.2 and 600 ± 50 μS/cm; Control 2: well water at 18 ± 1 °C, pH 7.6 ± 0.2, 1700 ± 50 μS/cm; CAS: effluent treated with conventional activated sludge; CASO 3l o o p :effluent treated with ozonated conventional activated sludge (9.4 mg O 3 /L). Minus: stages were not determinable because too many embryonic abnormalities were present or females did not have embryos in marsupium. n is the number of females evaluated for molt stage.

Condition

Molt stages (%) Embryonic stages (%) different groups remained in the range of 2.3-2.8 nmol/min/mg and did not differ statistically from each other (Fig. 5C).

Discussion

AChE, glutathione-S-transferase and phenol oxidase activities were measured in encaged gammarids as early warning indicators for the presence and effect of neurotoxic compounds, of compounds interfering with detoxification processes and immune defense. No significant effect of effluent was shown whatever the exposure condition and the enzymatic activity. Since PO is involved in the clearance of many different pathogens, it can be suggested that Gammarus fossarum did not suffer from any immune stress and consequently of a possible synergistic effect with residual micropollutants when exposed to CAS or CASO 3l o o p treated effluents [START_REF] Braun | Analysis of the Drosophila host defense in domino mutant larvae, which are devoid of hemocytes[END_REF]. Gammarid exposure to CAS and CASO 3loop effluents did not result in an inhibition of AChE activity, arguing for the lack of neurotoxicity of the residual pollutant mixture in treated effluents. The same trend was observed regarding GST activity measured in gammarids exposed to CAS and CASO 3 loop effluents that remained close to the control activity. In theory, such biochemical biomarkers reflect the biochemical sublethal changes of an organism exposed to xenobiotics and can be used as early warning systems, since they can identify changes at cellular level before measurable effects are induced in organisms [START_REF] Hyne | Invertebrate biomarkers: links to toxicosis that predict population decline[END_REF][START_REF] Karaouzas | Bioassays and biochemical biomarkers for assessing olive mill and citrus processing wastewater toxicity[END_REF]. However, biomarker response depends on many factors, among them the characteristics of the pollutant mixture (on a qualitative and quantitative basis), the duration of exposure, the metabolic capacities of organisms exposed, what drives the sensitivity of the assay to reveal toxic events. However, absence of biochemical responses does not mean an absence of sublethal fitness impairing effects, such as a decrease in reproductive output or gamete genotoxicity like in the present study [START_REF] Domingues | Cholinesterase and glutathione-S-transferase activities in freshwater invertebrates as biomarkers to assess pesticide contamination[END_REF].

DNA damage is a relevant sub-lethal endpoint to assess genotoxic compounds often present in wastewaters that may impact the fitness of various aquatic organisms [START_REF] Chen | Ozonation products of triclosan in advanced wastewater treatment[END_REF][START_REF] Jha | Ecotoxicological applications and significance of the comet assay[END_REF][START_REF] Magdeburg | Evaluating the efficiency of advanced wastewater treatment: target analysis of organic contaminants and (geno-) toxicity assessment tell a different story[END_REF][START_REF] Misik | Impact of ozonation on the genotoxic activity of tertiary treated municipal wastewater[END_REF]. A reference value for DNA damage in spermatozoa of Gammarus fossarum exposed to uncontaminated rivers was determined to be in the range of 2.6-3.5% TI. During the warm period with water temperatures reaching 16 to 19 °C, reference DNA damage value ranged between 6.5 and 14.9% TI (Lacaze et al., 2011a). In the present work, recorded water temperatures were 18 °C ± 1 °C and basal level of DNA damage in control 1 and 2 was 7.3% and 9.2% TI respectively, thus in accordance with Lacaze et al. (2011a). Since DNA damage in gammarids exposed to CAS or CASO 3l o o p -treated effluent was not significantly different from control 2 (high conductivity), it cannot be excluded that the high conductivity of 1700 μS/cm may have contributed to the significant genotoxicity observed in CAS-exposed gammarids. It has to be stressed that the genotoxic effect observed remained low and was even no more significant when compared to control 2, suggesting a possible osmoregulation impairment due to the high conductivity of effluents [START_REF] Glazier | Energetics of amphipods in ion-poor waters: stress resistance is not invariably linked to low metabolic rates[END_REF][START_REF] Cornet | Variation in immune defence among populations of Gammarus pulex (Crustacea: Amphipoda)[END_REF].

Most of the target pharmaceuticals were detected below chronic toxicity thresholds with the exception of sulfamethoxazole, diclofenac and propranolol which were measured at concentrations exceeding PNEC (Predicted No Effect Concentration) according to [START_REF] Orias | Characterization of the ecotoxicity of hospital effluents: a review[END_REF]. Calculated PNEC for sulfamethoxazole is 0.59 μg/L and it was found at concentrations 19.6 and 6.8 times higher in CAS and CASO 3 loop effluents, respectively. A slight genotoxic effect of sulfamethoxazole was observed at a concentration higher than 500 mg/L in human lymphocyte culture [START_REF] Abou-Eisha | Genotoxicity studies on the antimicrobial drug sulfamethoxazole[END_REF]. Once again, it is unlikely, that sulfamethoxazole could have accounted for the slight genotoxic effect observed in sperm of CAS-exposed gammarids. Genotoxicity measured in spermatozoa of Gammarus fossarum exposed to the undiluted CAS was not very high when compared to DNA damage level monitored in Gammarus fossarum caged in a river downstream a wastewater treatment plant discharge using conventional activated sludge treatment, that reached a 21% TI value (Lacaze et al., 2011c). Moreover, the fact that effluents CAS and CASO 3l o o p would be diluted after discharge in the river, the risk of possible genotoxic effects on organisms in receiving water is likely to decrease to a large extent. In [START_REF] Stalter | Comparative toxicity assessment of ozone and activated carbon treated sewage effluents using an in vivo test battery[END_REF] and [START_REF] Cao | Evaluation of wastewater reclamation technologies based on in vitro and in vivo bioassays[END_REF], tertiary ozonation resulted in an increased DNA damage compared to the conventional activated sludge treatment, possibly related to genotoxic ozonation by-products. In the present study, no difference of genotoxic potential between the ozonated sludge treated effluent (CASO 3l o o p ) and conventional activated sludge treated effluent (CAS) was observed. This suggests that ozonation of activated sludge does not proceed with the formation of more genotoxic compounds than the reference conventional treatment or as well that the genotoxic by-products possibly formed upon ozone action are assimilated by the biological treatment.

In Lacaze et al. (2011b),30%Gammarus fossarum embryos showed developmental abnormalities when DNA damage level in parent spermatozoa exposed to the model genotoxicant methyl methanesulfonate was above 50% TI. In the present study, over 90% of embryos were malformed, although DNA damage in parent spermatozoa remained low after exposure to CAS and CASO 3 loop effluents (15.5% and 10.7% TI respectively). This indicates that embryonic developmental abnormalities must have resulted from other compounds than genotoxicants present in effluents. [START_REF] Sundelin | Malformations in embryos of the deposit-feeding amphipod Monoporeia affinis in the Baltic Sea[END_REF] found that the percentage of malformed embryos in female amphipods Monoporeia affinis from Baltic Sea increased the closer to industrial wastewater treatment plant discharges the sampling station was.

For the evaluation of reproduction impairment in Gammarus fossarum, the occurred molt stages in exposure groups were compared to the embryo stages monitored in the marsupium of female gammarids according to [START_REF] Geffard | Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment[END_REF]. Since molt cycle and reproductive cycle in Gammarus fossarum females occur concurrently [START_REF] Subramoniam | Crustacean ecdysteriods in reproduction and embryogenesis[END_REF], a molt cycle corresponds to an embryonic stage (as previously illustrated in Fig. 2) and a discrepancy between them may be interpreted as a toxic effect [START_REF] Geffard | Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment[END_REF]. Conductivity in wastewaters can vary from 50 to 1500 μS/cm in domestic wastewaters and up to 10,000 μS/cm in industrial wastewaters (Water Environmental Federation, 2007). In the present study, conductivity had no significant effect on the molt cycle or the embryonic developmental stages but females exposed to the higher conductivity control (control 2) showed a slight but significant decrease in embryo number per female compared to the control 1 with a lower conductivity around 600 μS/cm. When females were exposed to the CAS or CASO 3l o o p effluents the molt cycle was retarded and the total number of embryos and of mature oocytes were significantly reduced compared to the control. Exposure to CAS and CASO 3 loop effluent seems to have impaired the molt and the reproductive cycle in most of the exposed female gammarids. Chemical analysis showed that after CAS and CASO 3loop treatments most of surfactants were at concentrations below the limit of quantification, and only LAS C10 (a linear alkylbenzene sulfonate) and stepanquat GA 90 were detectable in the range of 7.1 to 10 μg/L. Previous studies showed that EC 50 value for Ceriodaphnia dubia immobilization after 48 h exposure to LAS was 5.96 mg/L and EC 10 for reproduction after 7 days exposure was 1.18 mg/L, being respectively 596 and 118 times higher than LAS concentration (10 μg/L) measured in CAS and CASO 3 loop effluent in the present study (OECD SIDS, 2005;[START_REF] Ivankovic | Surfactants in the environment[END_REF], therefore LAS is unlikely responsible for observed reproduction impairing effects. Since sulfamethoxazole concentration found in CAS and CASO 3 loop effluent was respectively 11 and 4 μg/L, and that chronic exposure for 30 days of Daphnia magna to 10 μg/L sulfamethoxazole did not affect reproduction nor growth [START_REF] Flaherty | Effects of pharmaceuticals on Daphnia survival, growth, and reproduction[END_REF], the high effects observed on reproductive output in both gammarid exposure groups were likely not induced by this compound. Diclofenac concentration measured in CAS and CASO 3l o o p effluents was 383 and 174 ng/L, respectively. Diclofenac EC 50 regarding immobilization of Daphnia magna after 48 h exposure was estimated at 68 mg/L [START_REF] Cleuvers | Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects[END_REF] and the lowest observed effect concentration for reproduction inhibition in Ceriodaphnia dubia was 2 mg/L diclofenac [START_REF] Santos | Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment[END_REF]. It leads to the assumption that effects observed on reproduction and growth in gammarids exposed to CAS and CASO 3lo o p effluents cannot be conceivably attributed to the low residual diclofenac concentrations. The same can be said for propranolol, recovered in CAS and CASO 3l o o p effluents at higher concentrations, respectively 2.5 and 1.9 times higher than the PNEC value estimated at around 50 ng/L, even if a seven day-exposure to propranolol was shown to alter reproduction in Ceriodaphnia dubia at a 2 fold propanolol concentration (250 μg/L) than those measured in the present study [START_REF] Huggett | Toxicity of a select beta adrenergic receptor-blocking pharmaceuticals (B-Blockers) on aquatic organisms[END_REF]. Beside the possible contribution to the observed toxicity of other single compounds, mixture effects should also be considered [START_REF] Richardson | Water analysis: emerging contaminants and current issues[END_REF]. [START_REF] Cleuvers | Mixture toxicity of the anti-inflammatory drugs diclofenac, ibuprofen, naproxen, and acetylsalicylic acid[END_REF] exposed Daphnia magna in the acute test to a mixture of non-toxic concentrations of diclofenac, ibuprofen, naproxen and acetylsalicylic acid. Exposure to the mixture (diclofenac at 17 mg/L, ibuprofen at 25.3 mg/L, naproxen at 41.6 mg/L and acetylsalicylic acid at 22 mg/L) resulted in EC 50 as high as EC 50 predicted by concentration addition. Such an additive effect, although here shown at concentrations of pharmaceuticals much higher than measured in CAS and CASO 3 loop , is commonly described, and has to be taken into account for complex mixtures as wastewaters [START_REF] Farre | Fate and toxicity of emerging pollutants, their metabolites and transformation products in the aquatic environment[END_REF].

The reproductive success of gammarids was significantly reduced after exposure to CAS or CASO 3 loop effluents and no significant difference between the two exposure groups were found, although micropollutant concentrations were different in both effluents. This is in accordance with the study of [START_REF] Chung | Genetic and life-history trait variation of the amphipod Melita plumulosa from polluted and unpolluted waterways in eastern Australia[END_REF], who found a reduction in embryo number per female in the amphipod Melita plumulosa exposed in polluted rivers compared to unpolluted ones. Additionally, organic matter content in the effluents might also have contributed to the observed toxicity since [START_REF] Englert | Effects of municipal wastewater on aquatic ecosystem structure and function in the receiving stream[END_REF] showed that wastewater negatively affected macroinvertebrate feeding as well as abundance and leaf litter decomposition. According to [START_REF] Coulaud | Linking feeding inhibition with reproductive impairment in Gammarus confirms the ecological relevance of feeding assays in environmental monitoring[END_REF],reproductive alterations as impaired molting cycle and reduced fertility and fecundity of female gammarids can significantly affect population dynamics. More than 90% of the embryos produced in the marsupium of females exposed to CAS or CASO 3l o o p effluent exhibited developmental malformations (although pharmaceuticals content in CASO 3 loop is lower than in CAS). Thus, both residual pollutant mixture and organic matter in both effluents reduced significantly the fecundity of female gammarids and affected embryo development. [START_REF] Cold | Consequences of a short pulse of pesticide exposure for survival and reproduction of Gammarus pulex[END_REF] showed that pulse exposure (1 h) to 50 ng/L of the pyrethroid insecticide esfenvalerate reduced the number of offspring in Gammarus pulex to 60% of the control and a decreased reproductive output was still observable 2 weeks later. The impairment of molt cycle, fecundity and induction of embryonic malformations in gammarids seem to be sensitive and ecologically relevant endpoints to reveal residual toxicity of treated effluents consisting of a mixture of low concentrated micropollutants since they may have potential long-term effects on exposed populations [START_REF] Jobling | Widespread sexual disruption in wild fish[END_REF][START_REF] Lewis | Reproductive consequences of paternal genotoxin exposure in marine invertebrates[END_REF][START_REF] Santos | Ecotoxicological aspects related to the presence of pharmaceuticals in the aquatic environment[END_REF].

Conclusion

This study shows that exposure of Gammarus fossarum to an effluent treated by conventional activated sludge and an effluent treated by ozonated conventional activated sludge, consisting of a mixture of micropollutants and residual organic matter, is appropriate to determine residual toxicity. No significant difference in any of the measured endpoints was observed between the two tested effluents (with and without ozonation of wastewater), although the concentrations of quantified micropollutants were not similar and generally lower for the ozonated CAS treatment. The most sensitive endpoints, which should be considered for toxicity evaluation of effluents, were the impairment of molt cycle and fecundity, the induction of embryonic malformations and to a lesser extent sperm genotoxicity.

Further experiments carried out in situ by caging gammarids directly in receiving waters for long exposure duration should improve the risk assessment of such effluent treatments.

Fig. 1 .

 1 Fig. 1. Scheme of the pilot wastewater treatment plant consisting of two parallel conventional activated sludge lines (CAS) from which the upper line received 9.4 mg O 3 /L through the recirculating sludge loop (CASO 3l o o p ). Gammarids were caged in aerated tanks placed at the end of treatment lines CAS and CASO 3l o o p .

Fig. 3 .

 3 Fig. 3. A: Number of embryos per female, normalized according to female size. Asterisks indicate significant difference compared to control 1 (Wilcoxon rank sum test). Dotted lines with asterisks indicate significant difference compared to control 2 (Wilcoxon rank sum test). B: Number of oocytes per female in molt stage C2 or D1, normalized according to female size. Boxplots illustrate median, 25% and 75% percentiles, minimum and maximum. Control 1: well water at 18 ± 1 °C, pH 7.6 ± 0.2 and 600 ± 50 μS/cm; Control 2: well water at 18 ± 1 °C, pH 7.6 ± 0.2, 1700 ± 50 μS/cm; CAS: effluent treated with conventional activated sludge; CASO 3 loop :effluent treated with ozonated conventional activated sludge (9.4 mg O 3 /L). Circle indicates that only one female was in stage C2/D1.

Fig. 4 .

 4 Fig. 4. Genotoxicity evaluation in Gammarus fossarum spermatozoa expressed as tail intensity [TI %] plotted against exposure conditions. Box-plots illustrate median, 25% and 75% percentiles, minimum and maximum. Asterisks indicate significant difference compared to control 1 (Mann-Whitney). Control 1 (n = 15): well water at 18 ± 1 °C, pH 7.6 ± 0.2 and 600 ± 50 μS/cm; Control 2 (n = 11): well water at 18 ± 1 °C, pH 7.6 ± 0.2, 1700 ± 50 μS/cm; CAS (n = 16): effluent treated with conventional activated sludge; CASO 3 loop (n = 14): effluent treated with ozonated conventional activated sludge (9.4 mg O 3 /L). n: number of gels scored.

Fig. 5 .

 5 Fig. 5. Acetylcholinesterase activity (A), glutathione S-transferase activity (B), phenoloxidase activity (C) in Gammarus fossarum plotted against exposure conditions. Control 1: well water at 18 ± 1 °C, pH 7.6 ± 0.2 and 600 ± 50 μS/cm; control 2: well water at 18 ± 1 °C, pH 7.6 ± 0.2, 1700 ± 50 μS/cm; CAS: effluent treated with conventional activated sludge; CASO 3l o o p : effluent treated with ozonated conventional activated sludge (9.4 mg O 3 /L).

Table 1

 1 Physico-chemical parameters for CASO 3loop =effluent treated with ozonated conventional activated sludge (9.4 mg O 3 /L) and CAS = effluent treated with conventional activated sludge. TSS = total suspended solid; COD = chemical oxygen demand, (n = 8).

			CAS	CASO 3loop
	Temperature	°C	19.2 ± 1.3	19.4 ± 1.6
	Dissolved O 2	mg/L	8.6 ± 0.2	8.4 ± 0.3
	pH		7.87 ± 0.34	7.85 ± 0.24
	Conductivity	μS/cm	1675 ± 126	1675 ± 126
	TSS	mg/L	6 ± 5	6 ± 3
	COD	mg O 2 /L	32 ± 9.9	34 ± 10.1
	N-NH 4	mg/L	b0.1	b0.1
	N-NO 2	mg/L	0.06 ± 0.02	0.14 ± 0.14

Table 2

 2 Limit of detection (LD) and of quantification (LQ) for pharmaceuticals [ng/L] and surfactants [μg/L].

	Pharmaceuticals	LD	LQ	Surfactants	LD	LQ
		ng/L	ng/L		μg/L	μg/L
	Carbamazepine	0.2	0.6	Comperlan 100	0.03 0.1
	Ciprofloxacin	11.8 35.3 Triton X-100	0.03 0.1
	Sulfamethoxazole 2.0	5.9	Incromine SB	0.07 0.2
	Salicylic acid	4.4	13.3 Stepanquat GA 90	1.67 5
	Ibuprofen	0.2	0.5	BDDAC benzyldodecyldimethyl	0.08 0.24
				ammonium chloride
	Paracetamol	4.1	12.2 BDTAC benzyldimethyltetradecyl	0.05 0.16
				ammonium chloride
	Diclofenac	1.7	5	Lauryl pyridinium	0.07 0.2
	Ketoprofen	3.1	9.3	Sodium 2-ethylhexyl sulfate	1.67 5
	Propanolol	0.2	0.6	Sodium dodecyl sulfate (SDS)	0.17 0.5
	Atenolol	1.4	4.1	LAS C10	0.67 2
	Econazole	0.4	1.1	LAS C11	0.67 2
	Ethinylestradiol	2.4	7.3	LAS C12	0.67 2
				LAS C13	0.67 2
				Texapon N 701 S	1.67 5
				Cetyl betaine	0.03 0.1

Table 3

 3 Pharmaceutical concentrations measured in CAS effluent treated with conventional activated sludge and in CASO 3 loop effluent treated with ozonated conventional activated sludge (9.4 mg O 3 /L) ± SD. LQ = limit of quantification.

	Pharmaceutical	LQ	CAS	CASO 3 loop
		[ng/L]	[ng/L]	[ng/L]
	Carbamazepine	0.6	1514 ± 45	662 ± 19
	Ciprofloxacin	35.3	bLQ	bLQ
	Sulfamethoxazole	5.9	11,560 ± 2080	4020 ± 723
	Salicylic acid	13.3	489.6 ± 0.7	668 ± 0.9
	Ibuprofen	0.5	31.2 ± 0.1	51.6 ± 0.1
	Paracetamol	12.2	31.5 ± 2.8	49.4 ± 4.4
	Diclofenac	5	383 ± 46	175 ± 21
	Ketoprofen	9.3	53 ± 2	26.9 ± 1.3
	Propanolol	0.6	125.4 ± 5.0	97.8 ± 3.9
	Atenolol	4.1	496 ± 10	168.4 ± 3.4
	Econazole	1.1	4.3 ± 0.9	3.5 ± 0.7
	Ethinylestradiol	7.3	bLQ	bLQ

Acknowledgements

The experimental pilot program was hosted into the SIPIBEL field observatory supported by Rhone-Mediterranean Corsica water agency, The Rhône Alpes Region and other partners.

Funding

This research was financially supported by the French Ministry of Economy, Industry and Digital (grant number 132906048) within the framework of the Project TRIUMPH (TReatIng Urban Micropollutants and Pharmaceuticals in wastewaters) labelled and managed by the European Eureka Cluster ACQUEAU.

Conflict of interest

The authors declare that they have no conflict of interest.


When using or contributing to a Coq project (especially one with third party dependencies), it can be difficult and annoying to install the exact set of dependencies at the versions the maintainers recommend. It is more convenient when projects provide an easy way to set everything up to be ready to code, even more so if the dependencies can be quickly fetched in pre-compiled form. Nonetheless, it is also important to preserve the flexibility for project maintainers to test their projects against alternative versions (releases and development) of the dependencies, both while developing locally and in a distant Continuous Integration service (CI). Finally, being able to test that candidate changes do not break reverse dependencies (i.e., packages that depend on the project) is essential to bring confidence to project maintainers.

We have designed the Coq Nix toolbox which relies on Nix [START_REF] Dolstra | NixOS: a purely functional Linux distribution[END_REF] to allow for better sharing of configurations and pre-compiled packages across several projects. This toolbox makes it easy to setup a project and generates CI configurations to test multiple versions of the dependencies and compatibility with reverse dependencies. It enables the use of a single command nix-shell to get the same working environment for every developer. This also saves compilation time since developers and users may download packages that have already been compiled in CI.

A short introduction to Nix and Nix Coq packages

Nix is a purely functional language and a software suite geared towards package management. Although this package manager is at the basis of the NixOS distribution, it can be used on any Linux distribution, on macOS, and even on Windows thanks to WSL.

Derivations in Nix.

The main container types in Nix are attribute sets, which are simply finite maps from strings to any object. A derivation is a distinguished attribute set containing data and instructions on how to produce some output (often binaries, libraries or documentation), which is called the realization of the derivation. NixPkgs [START_REF]The NixOS maintainers. Nix Package collection[END_REF] is one of the largest known package repositories: it gathers everything to make a Linux distribution, but also thousands of packages useful for developers in many programming languages. It is implemented as a big Nix program which produces an attribute set of (attribute sets of . . . ) derivations.

While Nix allows installing multiple package versions, unlike Docker, the files, binaries, etc. are accessible directly from the root filesystem, there is no notion of container or image, and often no need to build manually, since a single call to nix-shell in an appropriate context will handle everything automatically.

Generating and overriding a Coq library derivation. We build on the work of many Nix users, who created and maintained an attribute set called coqPackages in NixPkgs. This attribute set contains many Coq third party library derivations (and includes Coq itself). It is at the center of our toolbox.

We redesigned coqPackages to streamline the creation of derivations of Coq third party libraries and to make sure it was easy to alter.1 Indeed, we now provide a function mkCoqDerivation which builds a derivation out of a minimal input, defaulting to some standard practices in the Coq ecosystem (e.g. dependency on Coq, using Dune or GnuMake, etc). We also included a lightweight overriding mechanism which reuses the metadata from the derivation (URL or version control system, owner, repository, etc.) to alter source fetching using a single argument (usually a string denoting a version number, a branch or a pull request). Contrarily to Opam packages, there is no version resolution mechanism, a given derivation specifies exact versions for all its dependencies, and overriding allows testing alternative versions.

We describe here our design of the Coq Nix toolbox [START_REF] Cohen | Coq Nix Toolbox[END_REF]. It is a Nix program, which, given a project A, reads a set of configuration files with a predetermined location from the root A-root of the project (A-root/.nix/config.nix, A-root/.nix/*overlays/, etc), produces or alters several derivations from coqPackages according to the configuration files, and provides various commandline utilities.

After setup (cf. "Initialization and update"), running the command nix-shell provides a shell where all the dependencies of your project are available, along with the toolbox commandline utilities.

Producing the project derivation. The main feature of the toolbox is to modify or create (if necessary) the derivation of A to use the local directory A-root as a source. All existing reverse dependencies of A inside the local instance of NixPkgs will then refer to the development version (from A-root).

Lightweight modification of dependencies and reverse dependencies using bundles. Changing the version of a related package-e.g. a direct or reverse dependency-can usually be done in the configuration files by modifying a single field in A-root/.nix/config.nix. The file organizes packages by bundles. A bundle is an attribute set where each field describes a set of package versions that must be compatible with each other. The user can then call nix-shell --argstr bundle name to select which bundle to use.

Ready-to-publish NixPkgs overlays. Sometimes, giving a version number is not enough. Indeed, if compilation instructions change in an unforeseen way, it may be necessary to change the Nix code that creates a derivation in NixPkgs. We provide the directory A-root/.nix/*overlays/ so that users can write their own derivations locally, in the exact form that should be used for later publication to NixPkgs. This is the recommended method if your package is not yet in NixPkgs upon initialization.

Configuring CI for the project and reverse dependencies. From the information contained in Nix-Pkgs and each bundle declared in the configuration file, the commandline tool genNixActions computes the dependency graph of packages within coqPackages, filters out unrelated packages, and turns it into a GitHub Action workflow. This way we get CI tests for the specific set of versions each bundle declares, and in particular the project package is tested against each bundle of dependencies.

Caching and artifact reuse

The CI may optionally refer to one or several Cachix [START_REF] Kožar | [END_REF] repositories to store and pull compiled binaries and .vo files. If enabled, it means a derivation is never realized twice. And if users declare the appropriate caches locally, they can reuse (i.e., download) transparently any of the realizations compiled by the CI, without recompiling it. We also provide an experimental cachedMake commandline utility which attempts to download precompiled .vo to speed up local incremental builds.

Initialization and update The toolbox can be run directly from https://coq.inria.fr/nix/toolbox (nix-shell can run Nix files stored in arbitrary locations). One can then use the commands provided by the toolbox to generate the configuration files, update the toolbox version as well as the NixPkgs version. We also plan to make it possible to setup the toolbox through Coq-Community Templates [START_REF]Coq-Community Templates[END_REF].

A

  Nix toolbox for reproducible Coq environments, Continuous Integration and artifact reuse Cyril Cohen 1 and Théo Zimmermann 2

1 Université Côte d'Azur, Inria, France 2 Université de Paris, Inria, France

We thank Vincent Laporte for providing feedback and reviewing this refactoring.


Ali Moustafa Khalil

Chendong Xu

Vincent Delmas

Guillaume Calvez

Karine Costuas

Mohamed Haouas

Christophe Lescop

Coordination-driven supramolecular syntheses of new homo-and hetero-polymetallic Cu( i ) assemblies: solid-state and solution characterization

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

The preparation of advanced supramolecular materials motivates since two decades impressive synthetic works that have afforded manifold multifunctional systems. 1 In this field, coordination-driven supramolecular (CDS) chemistry 2 constitutes one of the most powerful synthetic approach defined, in which systems based mostly on the Pt(II) and Pd(II) square-planar metal centers have flourished 2 taking benefit of the high structural rigidity and appropriate lability of the coordination sphere of these ions. Importantly, while some of the most impressive achievements obtained in supramolecular syntheses using metal-to-ligand coordination bonds have been based on the coordination chemistry of the 'naked' Cu(I) ion with pre-designed polytopic organic ligands (defining a synthetic approach defined as metal-templated approach),

3 few examples involving the Cu(I) ion accordingly to the CDS chemistry basic principles (formation of dynamic noncovalent interactions between metal centers inserted in pre-assembled precursors and multitopic linkers having both suitable geometries to allow straightforward and selective synthesis of targeted intricate supramolecular assemblies) 2 have been reported so far. Yet, we have recently initiated the rational Scheme 1 Synthesis of derivatives B, 5b C M , 5b D, E and F M' .

introduction of pre-assembled precursors based on the Cu(I) ion in self-assembly procedures related to CDS syntheses. 4 In these cases, the typical labile, flexible and low-directional coordination sphere of this ion, initially regarded as strongly restrictive in conventional CDS chemistry, turned out to be valuable to conduct the selective preparation of original polymetallic supramolecular assemblies. In addition, several of the polymetallic derivatives obtained so far present a variety of luminescence properties due to the versatile photophysical processes and the structural flexibility lying in Cu(I)-based CDS derivatives. 4,5 In particular, the pre-assembled solid-state blue luminophore tetrametallic metallacycle A 5a (Scheme 1) allowed conducting adaptive CDS syntheses from its reaction with cyanido-based precursors, affording a helical one-

Results and discussion

The reaction of the precursor A at room temperature (RT) with NaN 3 or KN(CN) 2 in 1:2 and 1:1 ratios respectively afforded colorless solutions from which were obtained the derivatives D and E (Scheme 1) in good yields (D : 68 %; E : 69 %) as airstable colorless polycrystalline powders. Regarding the synthetic processes that can account for the formation of these new polymetallic scaffolds, the selfassembly paths can be rationalized considering that, on the one hand, the azido anions insert between the metal centers of the {[Cu 2 (µ 2 -dppm) 2 ]CN} + fragments A' (Scheme 1) affording [Cu 2 (µ 2 -dppm) 2 ](CN) 1 (N 3 ) 1 sub-units that selfassemble in the solid-state forming the 1D-CP scaffold of E.

On the other hand, the DCM anion behaves as a bended ditopic template that connects two A' moieties in tetrametallic sub-units that subsequently self-assemble forming the discrete Cu 8 derivative D. Thus, the bottom-up synthetic processes established to explain the preparation of derivatives B and C (Scheme 1) can also be applied for D and E. The multiplicity, the variety and the versatility of coordination modes associated to the DCM and the azido anions are clearly key factors insuring the selective preparation of these polymetallic Cu(I) species. The difference observed in the secondary structures of the 1D-CPs B (helicoidal) and E (zig-zag) can be assigned to the longer dimension of the azido anions (ca. 2.30 Å) versus the cyanido anions (ca. 1.15 Å) that likely induces destabilization of the helicoidal geometry type as observed in B for the benefit of the selection of the 'zig-zag' 1D-CP backbone of E.

Conversely, and quite remarkably, C M (Scheme 1) and D scaffolds present comparable architectures based on ''Cu 8 (CN) 4 '' metallacycle sub-units (Fig. 1d) in which can be inserted, respectively, one dianonic M(CN) 4 and two monoanionic DCM fragments in spite of the different geometries and charges displayed by these building-blocks. This is allowed by the significantly large extend of structural alteration that the ''Cu 8 (dpmm) 8 (CN) 4 '' metallacyclic subunit can bear when hosting these different templates while preserving its coarse geometry (Fig. 1d). This highlights a strong tendency of the CDS self-assembly processes based on the flexible adaptive {[Cu 2 (µ 2 -dppm) 2 ]CN} + pre-organized fragments A' to yield well-defined discrete and stable multimetallic multicyclic assemblies. Importantly while the crystallization experiments of the derivative D always gave selectively this compound whatever the conditions and duration of crystallization applied, we noticed in a few cases, the apparition after one week of few dark blue crystals in the crystallization experiments that initially only afforded the derivative E. Xray diffraction study 6 reveals the formation of a new discrete {Cu 9 (N 3 ) 4 (CN) 4 dppm 8 }(PF 6 ) 2 supramolecular assembly F Cu (Fig. 1c) that crystallizes in the non-centric I-43d space group of the cubic system with a [Cu 2 (µ 2 -dppm) 2 (Cu) 1/4 (CN) 1 (N 3 ) 1 ] fragment in the asymmetric unit. Similarly to D, this nonanuclear Cu(I) derivative can be first described as based on a ''Cu 8 (CN) 4 '' metallacycle sub-unit having in the case of F Cu a slightly twisted square shape (Fig. 1c,d). The backbone of the derivative F Cu results then of the connection of this unit to a rare 'Cu(N 3 ) 4 ' internal fragment bearing a highly distorted tetrahedral metal center (N-Cu(II)-N angles: 99.48( 14)° and 132.10(4)°) coordinated to four N 3 -ligands.

The assembly F Cu is dicationic since two PF 6 -anions are observed in the unit cell. An oxidation degree of +2 is therefore formally assigned to the central metal ion, while the eight metal centers of the ''Cu 8 (CN) 4 '' metallacycle subunit are endowed a +1 oxidation state. [START_REF]EPR spectra of F Cu recorded in the solid state at room temperature presented the typical pattern observed for Cu(II) metal center[END_REF] In the derivative F Cu , the azido ligands connect the central Cu(II) ion up to eight Cu(I) metal centers owing to the four peripheral Cu 2 (µ 2dppm) 2 fragments, via a µ ,1,1,3 coordination mode in which the metal centers of the Cu(I) dimers ( 4,5 So far, this hindered the observation of sharp NMR patterns that would be clearly diagnostic of the persistence of the specific molecular architectures observed in solid state structures. This situation is in marked contrast with the plethora of Pt(II) and Pd(II)-based CDS assemblies for which multinuclear NMR spectroscopies constitute a standard and routine key characterization technique that allows to probe the stability and reactivity in solution of these derivatives. 2 The same stands for the Cu(I)-based supramolecular assemblies obtained according to the metaltemplated approach in which the specific design of the multitopic assembling ligands insure a high conformational stability to the resulting derivatives.

3 Indeed, in the case of the compound D, the 31 P{ 1 H} NMR spectrum reveals two broad multiplets centered at -15.0 and -18.2 ppm which are upfield shifted compared to the signals observed for A (two broad singlets at d = -8.5 ppm and -10.5 ppm, Fig. S2). [START_REF]For experimental details[END_REF] The recorded for the derivatives C M , 5b which is in agreement with their overall similar solid-state supramolecular backbones. Yet, it does not allow to formally confirm the preservation in solution of the multimetallic multicyclic scaffolds of these derivatives since one cannot exclude that similar self-dissociation and re-organization equilibria could take place for derivatives D and C M . Conversely, and unexpectedly, the 1 H{ 31 P} NMR (Fig. 2a) and 1 H{ 63 Cu} spectra of F Zn display well-defined sharp signals as a signature of a frozen molecular system. [START_REF]For experimental details[END_REF] In particular, an intriguing pattern of two apparent multiplets (Fig. 2a) centered at +2.61 ppm and +3.48 ppm is observed, accounting for the nonequivalent methylene protons of the coordinated dppm ligands. 2D 1 H{ 31 P} J-Resolved NMR experiment (Fig. S13) revealed that these methylene multiplets are assigned to a distribution of overlapping independent doublets with average geminal 2 J H-H values of 13.1 Hz, which is confirmed by simulation of these multiplets with eight equivalent 2 J H-H

13.1 Hz doublets (Fig. 2c). This reveals the preservation of the solid state X-ray molecular structure of F Zn in solution. degree of conformational flexibility in the backbone of F Cd in solution at RT. Yet, variable temperature NMR study shows that the resolution of the 1 H{ 31 P} spectra of F Cd improves upon temperature cooling (Fig. S18), presenting a low temperature a pattern very similar to the RT 1 H{ 31 P} spectra of F Zn . The 31 P{ 1 H} NMR spectrum of F Zn (Fig. S2) shows two complex multiplets centered at -14.7 and -17.9 ppm. Crystalline samples of D display at 300 K an eye-perceived yellowish luminescence upon excitation at 365 nm (Fig. 3a). This corresponds to a broad and featureless RT emission band with an emission maximum l em at 500 nm associated with a bi-exponential emissive lifetime t 1 = 52 µs and t 2 = 18 µs and an emission quantum yield (EQY) of 13 %. Upon cooling to 80 K, the emission spectrum of derivative D presents a gradual enhancement of its large emission band.

It is associated with a moderate red-shift of the emission maximum l max down to ca. 200 K and then a slight blue-shift of the l max is observed as the temperature decreases to 80 K (l em = 512 nm at 80 K). The overall unsymmetrical shape of these spectra suggests that several radiative relaxation pathways could be operant in this system, with relative efficiencies that could be dependant of the temperature. Note moreover that the rise of a shoulder at ca. 650 nm at low temperature. The detailed study of the temperature dependence of the emission lifetime (Fig. 3a) was conducted, revealing bi-exponential decay times all along the temperature range with a short-lived component having an almost linear and moderate progression upon cooling (at 80 K, t 2 = 56 µs). Conversely the long-lived component presents a larger amplitude variation (at 80 K, t 1 = 238 µs, Fig. 3a) bearing a sigmoidal profile that resembles the temperature dependant profile typically observed in the case of thermally activated delayed fluorescence (TADF). 6 They are highly similar (Fig. 3b) and correspond to electronic transitions from the metal-dppm centered molecular orbitals (MOs) to exclusively ligand-centered MOs involving both cyanido and DCM ligands together with the phenyl rings of the dppm ligands. This demonstrates that introduction of different pseudo-halide ligands impacts not only the geometries and the self-assembling processes but also the photophysical properties of CDS Cu(I) assemblies. Conversely, derivatives E, F Zn and F Cd present only very weak luminescence in the solid-state upon UV-vis light excitation intense stretching vibrational modes typically associated with the coordinated azido ligands that are responsible of very efficient non-radiative relaxation pathways.

Conclusions

With the preparation of the new polymetallic assemblies D, E and F M' , we demonstrate that the variety of building blocks embedded in Cu(I) CDS processes can be extended to different pseudo-halide ions giving access to a rich diversity of original intricate heteroleptic and heterometallic supramolecular scaffolds. Thus, the scope offered by CDS syntheses that be conducted using Cu(I) pre-assembled precursors is greatly enlarged since a large number of alternative ligands can be involved in these rational selfassembly processes. Moreover, we show that the derivative E, as its precursor A, can also be used as a new adaptive Cu(I) pre-assembled precursor for CDS syntheses, enlarging the possible accesses to large family of new Cu(I)-based heterometallic CDS assemblies.

Importantly, the preservation of these large complex supramolecular architectures in solution is demonstrated for the first time. This shows that, if the amplitude of the conformational flexibility of Cu(I) CDS scaffolds can be important at RT in solution inducing ill resolved and poorly informative NMR spectra, this do not imply systematic dissociative and reorganisation equilibria in solution. Such behaviour of Cu(I) CDS assemblies may be due to the high thermodynamic stability of these intricate scaffolds bearing multiple chelates and metallacycle sub-units that disfavours self-dissociative processes in solution. This study opens new perspectives for solution-route processes luminescent molecular-based devices (OLEDs, luminescent sensors, …) 10,11 based on Cu(I)

CDS assemblies whose photophysics can be modulated thanks to the combination of different pseudo-halide ligands with pre-assembled polymetallic Cu(I) precursors, a field of investigation that attracts currently a large interest for the preparation of new light-emitting devices or stimulisensitive luminescent multifunctional materials based in this cheap metal ion.

10,11

Fig. 1 .

 1 Fig. 1. a) View of the molecular X-ray structure of the derivative D; b) Molecular X-ray structure of the bimetallic repetition unit of E (top), view of the 1D zig-zag coordination polymer E (bottom); 6b c) Molecular X-ray structure of the derivative F Cu ; d) Simplified views and selected metric data of the X-ray structure of derivatives C Pt , 5b D and F Cu (Counteranions, H atoms and solvent molecules have been omitted for clarity; location of the C and N atoms of the cyanido ligands has been chosen arbitrarily, black dots represent the central points of the Cu-Cu segments of each [Cu 2 (µ 2 -dppm) 2 ] fragments).dimensional polymer B and the discrete supramolecular assemblies C M (Scheme 1). 5b These self-assembled processes

Fig

  Fig. 2. a) 1 H{ 31 P} NMR and b) 1 H{ 31 P} DOSY RT spectra of derivative F Zn in CD 2 Cl 2 ; c) Spectral decomposition of 1 H{ 31 P} NMR resonances for methylenic protons in F Zn , using 8 equal doublets with J H-H » 13.1 Hz. Black line = experimental spectrum, Red line = calculated spectrum, Blue lines = components. Chemical shifts of each individual component are indicated; d) 113 Cd NMR RT spectrum of derivative F Cd in CD 2 Cl 2 .

  Scheme 2 Suggested mechanism of adaptive selfdissociative assembling processes based on the derivative E and its reaction with M'(II) metals salts.

Fig. 3 .

 3 Fig. 3. a) Non-normalized solid-state temperature dependant emission spectra of D (l ex = 365 nm); insert: plot of the emission decay lifetime (long-lived component) against temperature (80 K to 300 K), the red line represents the fit according to the TADF Boltzmann-type equation S1; b) Isosurface representations of the electronic chargedensity difference between the first vertical singlet (S 1 ) and triplet (T 1 ) excited states, and the one of the ground state (S 0 ) of D (violet = density depletion, cyan = density increase) (± 1.10-4 e.bohr -3 ).

6

 6 These signals are observed at similar chemical shifts than the broader signals recorded for F Cd , D (Fig. S2) and C M , 5b which is in agreement with the ''Cu 8 (CN) 4 '' metallacycle sub-units featured by all these derivatives. The preservation of the solid state discrete structure of F Zn and F Cd in solution is definitively confirmed by DOSY NMR analysis (Fig. 2b and S14) which revealed that all of the proton signals correspond to the same diffusion coefficient D, and thus the same supramolecular object. The hydrodynamic radius R h as calculated using the Stokes-Einstein equation (R h =

10

  Yet, a single radiative relaxation pathway operant along the entire temperature window is quite unlikely regarding the thermal dependence of the emission spectra and a precise assignment of the relaxation processes occurring after UV-Vis excitation in derivative D would be uncertain. Attempting a fit of the thermal variation of the t 1 component to the Boltzmann-type equation S1 in SI, 10a the values of DE(S 1 -T 1 ) = 978 cm -1 , t(S 1 ) = 229 ns and t(T 1 ) = 231 µs can be eventually obtained. This set of values ranges among those determined for the other Cu(I) CDS assemblies based on the [Cu 2 (µ 2 -dppm) 2 ] 2+ fragment (including the precursor A) that were identified as TADF luminophores, 5 suggesting that a TADF process could take part in the radiative relaxation processes occurring in derivative D. Note that the calculated DE(S 1 -T 1 ) = 978 cm -1 is a priori not supported by the spectral shifts observed in the temperature dependant emission spectra, which supports the hypothesis of multiple emission processes. Interestingly, D and the structurally related derivative C Pd (Scheme 1a) share significantly similar radiative relaxation parameters despite a larger room temperature EQY for D (EQY C Pd = 2 %). 5b Quantum chemical calculations at the DFT level were performed for D. The geometry optimization 6 of the ground state S 0 reveals that the metallacycle frame is preserved in vacuum which confirms the stability of such nanosized scaffold. The first lowest singlet and triplet excited states were calculated by time dependent DFT (vertical excitation).

  After two months, the amount of crystals of F Cu did not significantly increased at the expense of those of E. It suggests that the apparition of crystals of F Cu can likely be assigned to residual traces of free Cu(I) ions in the synthesis conducted under air, that upon time are oxidized in Cu(II) ions. The characterization of F Cu reveals that equilibria in solution take place in these preparations, along which the templating action of the Cu(II) ion favors the formation of the discrete species F Cu . This motivated us to react preprepared amount of E (in suspension in CH 2 Cl 2 ) with the

	d(Cu-Cu) = 3.354(9) Å) are symmetrically bridged (d(Cu(I)-N N3 = 2.134(6) and 2.149(6) Å). adequate stoichiometric ratio of Cu(II) ions (dissolved in MeOH). As a result, a deep blue and clear solution was instantaneously obtained from which single crystals of F Cu were selectively grown after two days in a good yield (85 %), without any traces of the 1D-CP E. Therefore, similarly to A (scheme 1), E behaves as a source of heteroleptic [Cu 2 (µ 2 -dppm) 2 ](CN) 1 (N 3 ) 1 reactive fragments E' (Scheme 2) allowing the rational and selective preparation of the discrete nonametallic species F Cu . Supporting this hypothesis, the diamagnetic F Zn and F cd heterometallic assemblies were also selectively prepared in high yields (F

Zn : 91 %; F cd : 88 %) as colorless polycrystalline powder by reacting E with, respectively, Zn(II) and Cd(II) ions. Single crystal X-ray diffraction studies revealed that both derivatives are isostructural with F Cu , having therefore a M(II)(N 3 ) 4 internal fragment bearing a highly distorted tetrahedral metal center inserted in a ''Cu 8 (CN) 4 '' metallacycle sub-unit due to the coordination of four µ ,1,1,3 azido ligands. While the 1D-CP E is insoluble in CD 2 Cl 2 , the discrete derivatives D, F Cu , F Zn and F Cd are moderately (D) or readily soluble (F Cu , F Zn and F Cd ) allowing to conduct solution multinuclear NMR studies of the diamagnetic compounds D, F Zn and F Cd . In a general point of view, as a result of the lability and fluxionality of the coordination sphere of the Cu(I) ions in the Cu(I)-based CDS derivatives reported so far, rapid and reversible intramolecular conformational reorganizations dominate the dynamics of such assemblies in solution.

  Zn backbone in solution, showing that the general pattern observed at RT is preserved over the temperature range investigated. In the case of the derivative F Cd the 1 H{ 31 P} spectrum 6 exhibits broader signals than those obtained for F Zn which probably indicate a larger

	Indeed, a non-centric S 4 point group symmetry lies in the
	polycyclic solid-state backbone of F Zn and renders the four
	[Cu 2 (µ 2 -dppm) 2 ]	2+ fragments non-equivalent. Moreover, on
	each of these [Cu 2 (µ 2 -dppm) 2 ]	2+ dimers, the four methylene
	protons are symmetrically inequivalent, two of them
	pointing inward the metallacycle while the two other are
	orientated outward. Persistence in solution of such a
	molecular structure should result in the methylene protons
	signals zone in a total of sixteen signals divided into two
	groups of eight doublets embedded with geminal	2 J H(inward)-
	H(outward) coupling, which is indeed experimentally observed.
	A variable temperature	1 H{	31 P} NMR study (Fig. S17)
	confirmed the rigidity of F

(derivative F Cu is non emissive). This is very likely due to the Sup

Acknowledgements

This work was supported by the French Research Ministry and the CNRS. C.X. thanks the Chinese Scholarship Council for PhD fellowship. The ANR projects SMAC and SUPRALUM are aknowledged. K.C. and V.D. acknowledge support by the "Grand équipement national de calcul intensif (GENCI)" through HPC resources of CINES, TGCC and IDRIS (grants 2020/2021-A0080800649/A0100800649).

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.