Dr Pierre Hansjacob

Dr Frédéric R Leroux

Dr Morgan Donnard
email: donnard@unistra.fr

Zinc-Catalyzed Hydrocyanation of Ynamides and Photoisomerization, a General and Selective Access to E and Z Trisubstituted !-Enamidonitriles

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Introduction

For decades, acrylonitriles have been recognized for their synthetic utility in various fields such as pharmaceuticals, agrochemicals, or materials. 1 Within this family of compounds, amino-acrylonitriles have emerged over the years as versatile and valuable building blocks for the synthesis of heterocycles. However, while β-enaminonitriles (i.e. 3-amino-acrylonitriles) have been extensively studied both in terms of synthesis and application, 2 !-enaminonitriles (bearing the amino group in position 2) have been much less studied. These captodative enamines, 3 despite a real synthetic potential, suffers from a lack of versatile generic synthesis methodologies to access them. If we now consider the case of !-enamidonitriles (2amidoacrylonitriles), the observation is then accentuated by the very rare descriptions of their syntheses which are almost exclusively based on a sequence involving the formation of amido-acetonitriles followed by a condensation with aldehydes (first reported by Fleury 4 and then generalized by Takahashi 5 ) (Figure 1). 6 Notably, this approach remains mainly limited to the use of aromatic aldehydes and is therefore rather restricted in scope. Moreover, this methodology consistently suffers from a lack of stereoselectivity. Recently, during our studies on captodative enamides, 7 our group reported the first general access to tetrasubstituted !-enamidonitriles by means of a palladium-catalyzed silylcyanation of ynamides. 8 However, these overcrowded scaffolds may represent a drawback for some subsequent transformations such as Michael-type additions for instance. If we also reported the protodesilylation of these tetrasubstituted !-enamidonitriles to easily obtain the corresponding trisubstituted products, this approach requires two different synthetic steps (including two purifications) starting from the corresponding ynamides, 9 which we believe is unsatisfactory (Figure 1). In addition, depending on the substitution of the ynamides, different major stereoisomers of the C=C are obtained. For these reasons, we have decided to develop a practical and straightforward synthesis of trisubstituted !-enamidonitriles via the regio-and stereo-selective hydrocyanation 10 of ynamides using a relatively safe cyanide source (Figure 1).

Results and Discussion

Interestingly, while investigating this silylcyanation reaction, we identified the competitive direct hydrocyanation reaction (Table 1, entry 1) using TMSCN as cyanide source and leading to the E-!enamidonitrile 2a in a 22% yield. We then determined that this direct approach, [START_REF] Beletskaya | reported the direct hydrocyanation on 2 examples using TMSCN and TMSI to reach α-cyanoenamines[END_REF][START_REF] Hsung | The hydrocyanation of ynamides, using TMSCN as cyanide source, was previously attempted without success[END_REF] HCN-free and using a safe source of cyanide, could be of high interest and therefore we started our investigations to optimize the reaction conditions. First, we studied various catalysts (Table 1, entries 2-9) to evaluate their effect on different parameters such as the regio-and the stereoselectivity, the formation of the silylcyanated product 3a as well as the direct hydration of the starting ynamide 1a (a side product frequently obtained while using a Lewis acid with ynamides). Then a catalytic loading of 30 mol% was determined as the most productive both in terms of yield and selectivity (Table 1, entry 10). If other solvents appeared to be suitable for this transformation (MeCN and 1,4-dioxane), the reaction appeared to be less efficient and additionally led to a mixture of hydro-and silyl-cyanation products (Table 1, entries 12 and 13). The Ratio [a] 2a:3a:4a 11 [c] ZnBr2 (30) TMSCN -0

Yield [b] 2a (%) 1 PdCl2 (
12 [d] ZnBr2 (30) TMSCN 73:27:0 34

13 [e] ZnBr2 (30) TMSCN 82:18:0 56

14 [f] ZnBr2 (30) TMSCN 90 :10 :0 48 [g] 15 ZnBr2 (30) K4FeCN6 -0 16 ZnBr2 (30) Me2C(OH)CN 0:0:100 0 17 [h] No catalyst TMSCN 0:0:100 0 18 ZnBr2.2H2O (30) TMSCN 100:0:0 82 Using these optimized conditions (i.e. ZnBr2 30 mol%, TMSCN 2 equiv., 130°C, xylene [1M], 12 h), we then evaluated the scope and limitations of this hydrocyanation reaction (Figure 2). With pleasure, we noticed that the reaction was particularly tolerant to various substitutions on the aryl moiety of the ynamides. Thus, electron-donating or electron-withdrawing groups in the para or meta position led to the corresponding hydrocyanated products in overall good yield and full stereo-and regioselectivity (2b-d, 2f). Of note, in the case of a product bearing an electron-withdrawing group such as 2e, the yield was lower due to its relative instability.

In contrast, the stereoselectivity of the reaction seems to be particularly dependent on steric hindrance. As a representative example, the ortho-tolyl substituted substrate 1g led to the desired product 2g in a good yield of 56% but with a partially reversed E/Z selectivity (35:65). Nicely, an heteroaromatic substitution such as a 3-thiophenyl group led to the desired product 2h in 61% yield. Primary (2i) and secondary (2j) alkyl groups substituted on the alkyne gave approximately similar results with full E-selectivity and yields of 51% and 59% respectively. Due to the relative instability of the formed product 2k, the terminal ynamide 1k led stereoselectively to the desired product in 24% yield after purification. Then we had a look on the influence of the substitution at the nitrogen part of the ynamides on the course of the reaction. In terms of electron withdrawing groups, if a thiazolidinone (2l), a sultam (2m), a methoxycarbonyl (Moc) (2n) or a mesyl (Ms) (2o) group were well tolerated as the targeted products were obtained with yields of 52%, 54%, 47% and 68% respectively. Changing the alkyl chain on NMs substrates to more hindered ones such as a benzyl (2p), a cyclopropyl (2q) or a propyl (2r) decreased the yield of the transformation and even lightly eroded the stereoselectivity in the case of the benzylated substrate (1p).

Scope of the hydrocyanation of ynamides

A first hypothesis regarding the mechanism, similar to the hydrohalogenation of ynamides reported by Iwasawa et al., 13 was the hydrolysis of TMSCN by residual water in the medium leading to the in-situ formation of HCN. As the reaction did not work in presence of additional water without Zn catalyst (Table 1,entry 17), it appears that the reaction follows another pathway. A second hypothesis, was the potential in situ formation of Zn(CN)2, 14 from the reaction between TMSCN and ZnBr2, which could then undergo a carbozincation on the ynamide. Since our attempt to carry out the reaction using Zn(CN)2 as a catalyst led only to trace amounts of product (Table 1, entry 9), this possibility was quickly ruled out. Based on literature precedents, a reasonable hypothesis that could be considered is the activation of ynamides with a Lewis acid (here ZnBr2) to form the corresponding zinc keteniminium intermediate I. 15 Subsequent attack by a cyanide anion (potentially coming from TMSCN hydrolysis) on the opposite side of the metal could then take place to form a zinc vinyl intermediate II.

To determine if the hydrolysis of TMSCN was carried out by residual water in the medium, we tested the standard reaction with an addition of 1 equiv. of D2O.

In that case, no traces of the product resulting from deuterium incorporation was detected. Thus, the most reasonable hypothesis regarding the origin of water seems to be the zinc salt (ZnBr2.2H2O). Finally, the last step of the catalytic cycle could be a protodemetallation leading to the targeted product 2. This assumption is in perfect agreement with the erosion of selectivity observed in the case of congested ynamide 1g. Conversely, keteniminiums bearing an ethylbenzene substituent on the alkyne are known to cyclize readily 15,16 but we never observed the corresponding product in reactions with ynamide 1i which did not allow us to make a definitive indisputable decision on the mechanism (Figure 3). Having demonstrated the relative tolerance of this transformation leading to trisubstituted E-!-enamidonitriles with various functional groups, we then decided to study the E to Z photoisomerization 17 of compounds 2 to have a complete strategy to reach each isomer at will under mild metal-free conditions (Figure 4). Nicely, the isomerization, without additional catalyst or sensitizer in chloroform under UV LEDs (1x 52 W, 390 nm), of various selected substrates bearing substituted aryl (2a, 2d, 2e), heteroaryl (2h) or even alkyl (2i) groups as well as different nitrogen moieties such as thiazolidone (2l), sultam (2m), Moc (2n) or Ms (2o, 2q) electron withdrawing groups all led to the isomerized Z products in good yields (51-80%) (Figure 4). However, the isomerization of enamidonitrile E-2j bearing a cyclopropyl group led only, without degradation, to a 1:1 mixture of E and Z isomers. Notably, attempts to increase the ratio of isomerization of all substrates, such as the addition of a photosensitizer (riboflavin or benzophenone), change of solvent (CH3CN, DCM) or wavelength (2x 40 W, 450 nm) did not give better results. Then we decided to investigate the origin and the selectivity of the photoisomerization process. To start with, a rapid computational study of our model substrate 2a at the DFT level of theory (geometrical optimization: TPSS-D3/def2-TZVP; single point energy: PWPB95-D3/ def2-TZVP; please see SI for more details) allowed us to determine that this photoisomerization is close to thermo-neutral (E-2a⟶Z-2a, %G298 = 0.5 kcal.mol -1 ). Inspired by the enlightening recent work by Gilmour et al. on 2,2-disubstituted acrylonitriles, 18 this isomerization can be potentially explained by the difference of photo-excitability between the E and the Z isomers (Figure 5). Indeed, a UV-vis absorption spectrum of 2a pure two isomers shows: 1/ a hypsochromic shift of the wavelength of maximum absorption between E-2a (λmax = 300 nm) and Z-2a (λmax = 285 nm); 2/ a fourfold decrease of the molar absorptivity between E-2a and Z-2a (see SI). It follows that if the E-isomer can reach a triplet state in which isomerization can take place, the small portion formed of Z-isomer does not absorb enough energy to return to the triplet state and therefore does not isomerize back to the E configuration. Thus, in the course of time, according to Le Chatelier's principle, the isomerization equilibrium is shifted towards the Z isomer yet similar in energy to the E one. Interestingly, the difference of molar absorptivity between E-2a and Z-2a can potentially be rationalized thanks to the DFT geometry optimization (TPSS-D3/def2-TZVP) 19 that shows significative differences between both isomers in the dihedral angle between the olefin C=C and the aromatic ring (E-2a = 0.5° vs Z-2a = 28°) and in the less extend in the one between the olefin and the N-(CO) bond of the oxazolidinone (E-2a = 25° vs Z-2a = 36°). Consequently, these two configurations represent two very different conjugated systems (E-2a being significantly less twisted than Z-2a) which could explain the difference in light absorption observed (Figure 5). Finally, a TD-DFT (BLYP/def2-TZVP) investigation (see SI) showed that if a small difference in energy (0.9 kcal.mol -1 ) between the two isomers for the first singlet (S0→S1) excitation (E-2a = 83.8 kcal.mol -1 vs Z-2a = 84.7 ["G298 kcal.mol -1 ] (PWPB95-D3/ TPSS-D3/def2-TZVP). 19 Finally, to evaluate the synthetic potential of these building blocks, we then performed various transformations selectively involving each of its functionalities (Figure 6). After having demonstrated that our methodology could be applied on a gram scale (52% yield for E-2a), we first evaluated the synthetic possibilities on the alkene moiety of this new compound. A classical bromination with NBS led to the corresponding β-bromo-!-enamidonitrile derivative 5a in a good 71% yield but with a significant erosion of the C=C double bond configuration (E/Z = 40:60). 20 A coppercatalyzed trifluoromethylation using Togni II reagent led to the tetrasubstituted fluorinated 2-amido-acrylonitrile 6a as a single E isomer in a 46% yield. [START_REF] Wang | [END_REF] Then, we decided to use our new enamidonitriles as precursors for valuable heterocycles. First, compound E-2n bearing a Moc electron-withdrawing group on the nitrogen was treated with 4 equivalents of sodium methoxide and led to hydantoin 7n in a good 76% yield and a ratio E/Z of 60:40. 22 We finally looked at compound E-2o, bearing this time a mesyl group as electron withdrawing group on the nitrogen, that could be deprotonated 23 thanks to the use of a Grignard reagent as base to subsequently cyclize to form in 67% yield the completely original β-amino-sultam derivative E-8o with an excellent retention of the C=C double bond configuration. Nicely, the same reaction applied to the other stereoisomer Z-2o led to the stereopure β-amino-sultam Z-8o in similar yield (70%) (Figure 6).

Conclusion

In summary, we have developed a direct, general, and selective access to trisubstituted E-!-enamidonitriles. This strategy is based on the first stereo and regioselective hydrocyanation of ynamides and is catalyzed by abundant, unexpensive and low toxicity zinc(II) bromide. The reaction is tolerant to various substitutions but the stereoselectivity appears sensitive to the steric hindrance in the case of high congestion around the alkyne. We have then shown that Z-!-enamidonitriles can be subsequently obtained by simple catalyst-free photoisomerization under a 390 nm irradiation (LEDs). Although close to thermo-neutral, this isomerization could be rationalized, thanks to a concordant combination of experimental and computational studies by DFT, by the difference in excitability between the two isomers shifting the equilibrium towards the Z configuration. Remarkably, if the reaction is efficient with aromatic substituted substrates, it is almost as effective with alkyl substituted ones. Finally, the synthetic utility of these original compounds has been explored thanks to various post-transformations including heterocycle syntheses.

Experimental Section

General procedure for the hydrocyanation of ynamides (1) to form 2amidoacrylonitriles (2)

A 2 mL sealed was charged with ynamide 1 (0.1-0.8 mmol, 1 eq) and zinc bromide (30 mol%) under Ar. Xylene (1M) was first added, and then TMSCN (0.4-1 mmol, 2 eq). The rubber septum was replaced by a septumcap under argon. The mixture was stirred in a pre-heated oil bath at 130 °C for 7-17h. After complete consumption of starting ynamide indicated by TLC, the reaction mixture was cooled to room temperature, filtrated over a pad of celite with dichloromethane, evaporated and directly purified by flash column chromatography over silica gel to give the desired hydrocyanated compound 2.

General procedure for the photoisomerization of E-2 to Z-2

A quartz-NMR tube was charged with hydrocyanated compound E-2 and dissolved in CDCl3 (0.1-0.2 M) under Ar. The tube was capped and placed 6 cm away from a purple Kessil PR160L UV-LED (1x 52 W, 390 nm, average intensity: 352mW/cm 2 ) and irradiated for 60 h. E/Z ratio was determined by 1 H NMR of the crude mixture. Purification of the reaction mixture by flash column chromatography over silica gel gave the desired hydrocyanated compound Z-2.

Figure 1 .

 1 Figure 1. Comparison of strategies to reach trisubstituted !-enamidonitriles.

  [a] Determined by 1 H NMR of the crude mixture; [b] Isolated yield; [c] DMF as solvent; [d] MeCN as solvent; [e] 1,4-dioxane as solvent; [f] at 110 °C; [g] incomplete conversion after 12h (55%); [h] reaction performed in the presence of 1 equiv. of water.

Figure 2 .

 2 Figure 2. Zinc catalyzed E-selective hydrocyanation of ynamides 1 and X-ray analysis of 2a.

Figure 3 .

 3 Figure 3. Tentative mechanism of the hydrocyanation.

Figure 4 .

 4 Figure4.Catalyst-free contra-or neutral-thermodynamic photoisomerization of E-2 to Z-2.

  Photoisomerization

Figure 5 .

 5 Figure 5. Potential rationalization for the thermo-neutral photoisomerization of E-2a to Z-2a through 3 2a (structures minimized by DFT (TPSS-D3/def2-TZVP)).["G298 kcal.mol -1 ] (PWPB95-D3/ TPSS-D3/def2-TZVP).19

Figure 6 .

 6 Figure 6. Follow-up chemistry.

, entry 14) and

  the use of other sources of cyanide such as K4FeCN6 or acetone cyanhydrine did not yield to the targeted acrylonitrile

	reaction proved to be less productive at lower temperatures
	(Table 1

2a (Table 1, entries 15 and 16). Finally

	, we
	performed two control reactions. The first one, without catalyst,
	led only to the hydration of the ynamide (Table 1, entry 17). The
	second one was performed using our optimized conditions with
	ZnBr2.2H2O as the catalyst. Since ZnBr2 is highly hygroscopic and
	considering that the reagent was not stored or weighed in a
	glovebox, it is realistic to consider the hydrated salt as the real
	catalyst of the reaction. In this case, the reaction gave similar
	results to those obtained with ZnBr2 (82% yield, full
	stereoselectivity; Table 1, entry 18).

Table 1 .

 1 Optimization of the reaction conditions.

	Entry	Cat.	CN
		(mol%)	source

Acknowledgements

The authors thank the CNRS and the University of Strasbourg for financial support. PH is grateful to the French ministry for research and higher education for a PhD fellowship. Lilian Wisson (PhD student in our team at the University of Strasbourg) is thanked for UV/vis measurements. The authors thank Dr. E. Wasielewski and Mr. M. Chessé, respective managers of the NMR and Analytical platforms and facilities of LIMA (UMR7042 CNRS-Unistra-UHA), who contributed, by their technical and scientific support, to the achievement of this research project. We thank Nathalie Gruber (Uni. Strasbourg) and Corinne Bailly (Uni. Strasbourg) for X-Ray analyses. The authors would like to acknowledge the High-Performance Computing Center of the University of Strasbourg for supporting this work by providing scientific support and access to computing resources. Part of the computing resources were funded by the Equipex Equip@ Meso project (Programme Investissements d'Avenir) and the CPER Alsacalcul/Big Data. Keywords: Ynamide • Hydrocyanation • Acrylonitrile • Enamide • Photoisomerization


Hugo Olvera-Vargas

Sébastien Leroy

Michael Rivard

Nihal Oturan

Mehmet Oturan

Didier Buisson
email: buisson@mnhn.fr

Microbial biotransformation of furosemide for environmental risk assessment: identification of metabolites and toxicological evaluation

Keywords: Biodegradation, oxidation, degradation pathway, environmental transformation products, ecotoxicology

Some widely prescribed drugs are sparsely metabolized, and end up in the environment. They can thus be a focal point of ecotoxicity, either themselves or their environmental transformation products. In this context, we present a study concerning furosemide, a diuretic, which is mainly excreted unchanged. We investigated its biotransformation by two environmental fungi, Aspergillus candidus and Cunninghamella echinulata. The assessment of its ecotoxicity and that of its metabolites was performed using the Microtox test (ISO 11348-3) with Vibrio fischeri marine bacteria. Three metabolites were identified by means of HPLC-MS and 1 H/ 13 C NMR analysis: saluamine, a known pyridinium derivative and a hydroxy-ketone product, the latter having not been previously described. This hydroxy-ketone metabolite was obtained with C. echinulata and was further slowly transformed into saluamine. The pyridinium derivative was obtained in low amount with both strains.

Metabolites, excepting saluamine, exhibited higher toxicity than furosemide, being the pyridinium structure the one with the most elevated toxic levels (EC50=34.406.84 mg L -1 ). These results demonstrate that biotic environmental transformation products may present a higher environmental risk than the starting drug, hence highlighting the importance of boosting toxicological risk assessment related to the impact of pharmaceutical waste.

Introduction

Pharmaceuticals, which are substances designed to possess biological effects, are able to affect any function of the body of man or animals and they are used in diagnosis, prevention or cure of diseases. They are produced and used in large tonnage, and their presence in the environment as hazardous contaminants is now well recognized [START_REF] Agerstrand | Improving environmental risk assessment of human pharmaceuticals[END_REF][START_REF] Khetan | Human pharmaceuticals in the aquatic environment: A challenge to green chemistry[END_REF]. Several reports have shown their presence in effluents of sewage treatment plants (STPs) [START_REF] Loos | EU-wide monitoring survey on emerging polar organic contaminants in wastewater treatment plant effluents[END_REF]. Indeed, most pharmaceuticals enter municipal STPs via domestic and hospital sewages or through industrial discharges, part of them escaping degradation (Al [START_REF] Aukidy | A framework for the assessment of the environmental risk posed by pharmaceuticals originating from hospital effluents[END_REF][START_REF] Frederic | Pharmaceuticals in hospital wastewater: Their ecotoxicity and contribution to the environmental hazard of the effluent[END_REF]. They have been unequivocally detected in surface, ground and in some cases, even in drinking water. Despite their low concentrations levels, their continuous release into the environment, makes them pseudo-persistent and hence a focal point of toxicity risk [START_REF] Fent | Ecotoxicology of human pharmaceuticals[END_REF].

Once in the environment, pharmaceuticals undergo conversion processes that can induce the formation of a great variety of transformation products (TPs). They can be metabolized by different microorganisms (biotic transformations) and/or undergo photolysis, hydrolysis or chemical oxidation (abiotic transformations). In like manner, drugs can be transformed during sewage and drinking water treatment, resulting in the generation of different biologically and chemically transformed products [START_REF] Fatta-Kassinos | Transformation products of pharmaceuticals in surface waters and wastewater formed during photolysis and advanced oxidation processes -degradation, elucidation of byproducts and assessment of their biological potency[END_REF]. These TPs may have similar activity to the parent compound, or may be biologically more active and/or more toxic [START_REF] Dirany | Study of the toxicity of sulfamethoxazole and its degradation products in water by a bioluminescence method during application of the electro-Fenton treatment[END_REF]; [START_REF] Khetan | Human pharmaceuticals in the aquatic environment: A challenge to green chemistry[END_REF][START_REF] Morais | An uncertainty and sensitivity analysis applied to the prioritisation of pharmaceuticals as surface water contaminants from wastewater treatment plant direct emissions[END_REF]. Additionally, they are commonly present as complex mixtures, whose combined toxicity may exceed that of each isolated compounds [START_REF] Backhaus | Screening level mixture risk assessment of pharmaceuticals in STP effluents[END_REF].

Consequently, the presence of pharmaceuticals and their TPs in environmental water sources represent a substantial toxicological risk for living organisms and humans [START_REF] Celiz | Pharmaceutical Metabolites in the Environment: Analytical Challenges and Ecological Risks[END_REF][START_REF] De | Occurrence of pharmaceuticals in a water supply system and related human health risk assessment[END_REF].

Determining the toxicity and ecotoxicity of these persistent micropollutants and their TPs is thus fundamental and a prerequisite for thorough risk assessment aiming at a comprehensive protection of the environment.

Microorganisms possess multiple-enzyme systems allowing them to metabolize xenobiotics into a wide range of transformation products. Accordingly, several active microbial strains, especially fungi, have been used as a competitive and ecologically effective approach for the preparation of diverse metabolites for different purposes, including drug metabolism studies [START_REF] Azerad | Microbial models for drug metabolism[END_REF][START_REF] Marvalin | Microbial production of phase I and phase II metabolites of propranolol[END_REF]. Aspergillus species have been widely used for the preparation of several potentially bioactive derivatives, whence evidencing its ability to biotransform a variety of chemicals [START_REF] Adelin | Biotransformation of natural compounds. Oxido-reduction of Sch-642305 by Aspergillus ochraceus ATCC 1009[END_REF][START_REF] Arakawa | Unusual biotransformation products of the sesquiterpene lactone budlein A by Aspergillus species[END_REF][START_REF] Parshikov | Biotransformations of organic compounds mediated by cultures of Aspergillus niger[END_REF].

Similarly, Cunninghamella species, a filamentous fungus found in soil and plant material, particularly at Mediterranean and subtropical zones, are able to perform biotransformations using both, phase I (oxidative) and phase II (conjugative) bio-mechanisms, imitating mammalian metabolism. In fact, they have P450 monooxygenase systems comparable to those in mammals and enzymes conducting phase II metabolism of drugs [START_REF] Asha | Cunninghamella--a microbial model for drug metabolism studies--a review[END_REF][START_REF] Felczak | Quinoline biodegradation by filamentous fungus Cunninghamella elegans and adaptive modifications of the fungal membrane composition[END_REF]. In this way, microbial metabolism of xenobiotics is widely used as a model for mammalian metabolism studies. Paradoxically, the use of isolated wild strains in the investigation of the fate of drugs in the environment is not widespread [START_REF] Murphy | Microbial degradation of fluorinated drugs: biochemical pathways, impacts on the environment and potential applications[END_REF], in spite of the potentiality of biotransformations as a very useful tool for the preparation of drugs' transformation metabolites, aiming to strengthen and support predictive modelling and analysis on ecotoxicity and risk assessment. Furosemide (FRSM), a widely prescribed loop diuretic, has been unambiguously detected in aquatic environments [START_REF] Besse | Human pharmaceuticals in surface waters implementation of a prioritization methodology and application to the French situation[END_REF][START_REF] Mendoza | Pharmaceuticals and iodinated contrast media in a hospital wastewater: A case study to analyse their presence and characterise their environmental risk and hazard[END_REF][START_REF] Oliveira | Characterization of Pharmaceuticals and Personal Care products in hospital effluent and waste water influent/effluent by direct-injection LC-MS-MS[END_REF] and it has been classified by the European Medicine Agency as one of the highest risk pharmaceuticals based on its exposure criteria [START_REF] Huschek | Environmental risk assessment of medicinal products for human use according to European Commission recommendations[END_REF]. It has been reported to cause side effects on some species, mainly hepatotoxicity and ototoxicity. Furthermore, its carcinogenic and genotoxic potential has also been suggested [START_REF] Bucher | Toxicology and carcinogenicity studies of diuretics in F344 rats and B6C3F1 mice. 2. Furosemide[END_REF][START_REF] Mondal | Furosemide-induced genotoxicity and cytotoxicity in the hepatocytes, but weak genotoxicity in the bone marrow cells of mice[END_REF]. These toxicological effects have been associated with the formation of toxic metabolites, which are capable of disrupting cellular functions by binding relevant cell macromolecules. A metabolite, under the form of an electrophilic epoxide, has been reported to be highly cytotoxic to liver hepatocytes (Williams et al. 2007). Moreover, FRSM alone or in mixture with other pharmaceuticals was reported to show estrogenic activity, mixtures having synergistic effects [START_REF] Fent | Ecotoxicology of human pharmaceuticals[END_REF]. Recently, we reported the microbial biotransformation of FRSM by selected bacteria and fungi, which were able to metabolize FRSM into saluamine (SLMN), a pyridinium derivative (PRDN) and another product. The pyridinium derivative was reported to be more toxic than the parent drug [START_REF] Laurencé | Anticipating the fate and impact of organic environmental contaminants: A new approach applied to the pharmaceutical furosemide[END_REF]. Moreover, both metabolites, SLMN and PRDN, were as well detected during electrochemical oxidation of the drug by electro-Fenton (EF) process [START_REF] Olvera-Vargas | Electro-Oxidation of the Pharmaceutical Furosemide: Kinetics, Mechanism, and By-Products[END_REF], whence suggesting their high probability to be generated as environmental transformation products.

The significant progress on the ongoing regulation concerning pharmaceutical residues witnesses the increasing preoccupancy about this environmental problem. In fact, three pharmaceuticals have been included in the "watch list" of emerging pollutants within the European Water Framework Directive (Directive 2013/39/EU) [START_REF] Kuster | Pharmaceuticals in the environment: scientific evidence of risks and its regulation[END_REF][START_REF] Ribeiro | An overview on the advanced oxidation processes applied for the treatment of water pollutants defined in the recently launched Directive 2013/39/EU[END_REF]. However, data related to toxicological effects of medicines and their TPs in the environment is still scarce and sometimes ambiguous, thereby, it is of primordial importance to reinforce environmental risk assessment through ecotoxicological approaches aiming at stablishing the most appropriate strategies for environmental management and monitoring regarding pharmaceutical waste. The present work investigates the bioconversion of FRSM by the environmental fungi Aspergillus candidus and Cunninghamella echinulate for toxicological evaluation. Three main metabolites were formed: SLMN, PRDN derivative, and a hydroxy-ketone (HK), the latter being described for the first time. A mechanistic pathway for the metabolism of FRSM by Cunninghamella echinulata is proposed and ecotoxicological assays using V.

fischeri bacteria revealed that PRDN and HK metabolites possess higher toxicological levels than FRSM itself.

Materials and Methods

Chemicals and analysis methods

Furosemide (purity > 98%), trifluoroacetic acid and formic acid were of reagent grade from Fluka. PRDN was synthetized as described [START_REF] Laurencé | Preparative access to transformation products (TPs) of furosemide: a versatile application of anodic oxidation[END_REF]. Gradient grade HiPersSolv acetonitrile from VWR was used.

HPLC analysis was performed on Gilson system (pump 305, pump 306, gradient dynamic mixer 811B and autoinjector 234) with a Supelcosil LC-18, 3μm (75 mm × 4.6 mm) column. The system was controlled and the results were analyzed by Unipoint Gilson software. Column was eluted with an appropriate solvent system at a flow of 1.0 mL/min: (solvent A: water, 0.1% trifluoroacetic acid; solvent B: 50% water/ 50% acetonitrile, 0.1% trifluoroacetic acid) 0-5 min (isocratic 90/10), 5-8 min (gradient up to 50/50), 8-15 min (gradient up to 20/80), 15-20 min (isocratic 20/80). The detection was at 240 nm using an UV detector.

LC-MS analyses were performed on Nexera UHPLC chromatographer coupled with a MS2020 simple quadrupole mass spectrometer from Shimadzu, using atmospheric pressure chemical ionization (APCI). 1 µL of sample solutions were injected through a Nexera autosampler. A Kinetex column, 2.7µm, C18 100 Å, 100 x 2.1 mm, from Phenomenex, was used, and a 10 min program was developed for these analyses using a binary A/B solvent system at a flow rate of 0.5 mL/min (solvent A: methanol, 0.01% formic acid; solvent B: water, 0.01% formic acid): 0-2 min (isocratic 5/95), 2-7 min (gradient up to 80/20), 7-8 min (isocratic 80/20). Eluted compounds were detected between m/z 50 and 600. Optimal MS settings were fixed as follows: Drying gas: nitrogen, 15 L min -1 ; nebulizing gas: nitrogen, 1.5 L min -1 ; Qarray RF: 60 V; Qarray DC: 0 V; DL voltage and temperature: 0 V and 250 °C; Detector voltage: 1.1 kV; APCI interface voltage: +4.5 kV; Event time: 200 ms.

Preparative HPLC was performed on an Agilent system, using an Agilent PrepHT XDB-C18 column (21.2 x 150 mm; 5 µm; USA). Sample was injected and separated using the same gradient program for HPLC analysis. 3 main peaks were obtained and collected in separated fractions.

Mass spectra data were recorded using an electrospray time of flight mass spectrometer (ESI-TOF-MS) operating in positive and negative modes (QSTAR Pulsar I of Applied Biosys-tems). NMR experiments were recorded on Bruker Avance III HD 400 MHz spectrometers (Wissembourg, France) equipped with a BBFO Plus Smartprobe.

Microorganisms and culture conditions

Fungal strains were obtained from the American Type Culture Collection (Cunninghamella echinulata ATCC 9245, and Aspergillus candidus ATCC 20023). Cultures were maintained on agar slants (containing in g L -1 : bactopeptones 5, yeast extract 5, malt extract 5, glucose 20 and agar 20) and stored at 4 • C. Liquid culture media containing (g L -1 ) glucose 16, yeast extract 4, malt extract 10 and soybean peptones 5 (YMS medium) were sterilized without glucose at 120 • C for 20 min. Separately sterilized glucose solution (40%) was added afterwards. Flasks (100 mL) containing 50 mL of YMS culture medium were inoculated with glycerol suspension of microorganisms and incubated at 30 • C and 200 rpm (orbital shaker) for 60 h.

Biotransformations

For time-course studies of biotransformation, biomass was filtered, suspended in citrate or phosphate buffer and substrate was added in DMF solution for a final concentration of 0.4 g L -1 . Biotransformations were performed in light. Samples were taken at regular intervals of time: 200 L of methanol were added to 800 L of biomass-containing sample, which was further stired, sonicated and centrifuged prior to injection. A control experiment without biomass addition was conducted under the same conditions. No decrease in the concentration of FRSM was observed after 24 h, showing that photolysis of the drug is not significant.

Production of hydroxy-ketone and saluamine

C. echinulata ATTC 9245 was cultured in YMS medium (2 L) as describe above. Biomass (300 g) was harvested by filtration and suspended in citrate buffer (0.5 L, 0.1 M pH 5). Furosemide was added (200 mg) in DMF solution, final concentration 0.4 g L -1 . Incubation was performed under an air atmosphere at 27 °C and 200 rpm for 72 h. Cells suspension was filtered and incubation medium was extracted with ethyl acetate (3 times).

Crude extract was re-dissolved in methanol and purified by preparative HPLC, giving SLMN (64 mg) in 42% yield and HK (34 mg) in 17% yield. HK: HRMS (ESI-TOF) M-H + : m/z calc mass for C12H14ClNO6S 349.0261 g mol -1 , found 349.0141 g mol -1 . 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.69 (t, J=4.8 Hz, 1H, N-H), 8.38 (s, 1H, H-10), 6.82 (s, 1H, H-7), 4.3 (d, J=4.8, 2H, H-5), 3.39 (t, j=6.4 Hz, 2H, H-3), 2.53 (t, J=7.3Hz, 2H, H-2), 1.68 (m, 2H, H-1). 13

Ecotoxicological tests

Toxicity tests were conducted with the photo-luminescent bacteria V. fischeri. The bioassay measures the decrease in bioluminescence induced in the cell metabolism by the presence of chemicals and it was performed in accordance with ISO 11348-3 procedure (Microtox @ ). The bacteria and the activation reagent, LCK 487 LUMISTOX, were provided by Hach Lange France SAS. A dilution series of the standard solutions of furosemide and the isolated metabolites: pyrimidium derivative, saluamine and hydroxyl-ketone, were prepared in 2% NaCl and pH was adjusted to a value of 7. Bioluminescence intensity was measured using a luminometer Berthold TM Autolumat Plus LB 953, at time zero and after 15 min of exposure to the samples at 15 °C and compared to the measured value of the control solution (NaCl 2%). Concentration of the toxicants produces a dose-response relationship from which the results as the 50% and 20% inhibition effective concentration (EC20 and EC50, respectively) were deduced by non-linear regression curves with variable Hill slope. Reported toxicity values are the average of 3 replicates of each sample with 95% confidence limits. Phenol was tested as a control and it was found to be within the Microtox @ set parameters, EC50 between 13 and 26 mg L -1 .

Results

Identification of furosemide metabolites

Biotransformation of FRSM by C. echinulata was conducted in preparative scale during 72 h. The chromatograms obtained by HPLC analysis showed the presence of 2 peaks apart from that of FRSM (Fig. 1A).

The corresponding products were purified by preparative chromatography and analyzed by LC-MS (Fig. 2). The MS spectrum in negative mode of the more polar compound M1 (Rt=4.0 min) showed a molecular ion m/z 249, corresponding to the [M-H + ] -ion (Fig. 2 B andD). Along with the 1 H NMR analysis, where protons from the furan ring were not observed, this product was identified as SLMN, whose formation by this fungus was confirmed. The MS spectrum in negative mode of the second compound M2 (Rt=5.01 min) showed a molecular ion [M-H + ] -at m/z 349 (Fig. 2 C and E) corresponding to an increase of 20 u compared to furosemide, which resulted from the addition of one oxygen and four hydrogen atoms. The presence of the chlorine atom in this metabolite was confirmed by the fact that the MS spectrum showed three peaks at m/z 349.0141, 351.0120 and 353.018 with relative intensity of 100, 30 and 10 respectively (Fig. S1). Additionally, the interpretation of the 1 H NMR and 13 C NMR spectra (Fig. S2 andS3), along with the two dimensional COSY, HSQS and HMBC spectra, allowed the unambiguous identification of the HK product with a molecular mass of 350.77 g mol -1 . The MS/MS daughter ion spectrum (negative ion) showed a fragment at m/z 305 (loss of 44 u, CO2), confirming the presence of the alkyl group. A fragmentation ion at m/z 227 (loss of 35 + 87 u), indicated the loss of chlorine and the -COCH2CH2CH2OH group, which corresponded to cleavage next to the keto group. Moreover, the MS spectrum in negative mode of the HK solubilized in methanol, revealed a protonated molecular ion m/z 363.0105 (Fig. S4), which corresponds to the ketal adduct resulting from intramolecular cyclization and addition of one molecule of methanol.

Regarding the biotransformation of FRSM by A. candidus, the chromatograms obtained in HPLC analysis during incubation also showed the presence of two peaks apart from that of FRSM (Fig. 1B), one of them having the same retention time of SLMN. The presence of the latter was confirmed by LC-MS analysis since an m/z peak of 249 in negative mode was observed (Fig. 3 C andE). Compound M3 showed a Rt of 1.2 min and an m/z peak of 329 in positive mode in LC-MS, which corresponded to PRDN (Fig. 3 B andC). Its formation was confirmed by the HRMS spectrum of the brut extract, which showed a peak at m/z 328.9975.

Effect of incubation conditions

The time courses of the bioconversion of FRSM were investigated in two different buffers, 0.1 M citrate buffer pH 5 and 0.1 M phosphate buffer pH 7. The results were illustrated in Figs. 4 and 5 for representative biotransformation. Fig. 4 shows the evolution of FRSM and its metabolites SLMN and HK in terms of the percentage of peak area, assuming that all metabolites have a similar molar absorption coefficient. The evolution of PRDN, which was formed in much lesser amounts, is given in Fig. 5. It can be seen from Fig. 4A that FRSM was almost completely metabolized by C. echinulata when incubation was performed in phosphate buffer, while during incubations with A. candidus (Fig. 4B), only 50% of FRSM was converted into SLMN as the main product. HK was the main metabolite produced by C. echinulata, however, its disappearance was observed after a long incubation time, as it was progressively transformed into SLMN. This fact was verified by incubation of purified HK with C. echinulata, which resulted in the formation of SLMN (Fig. S5). A control experiment was carried out in the absence of biomass, in which no significant decay on the concentration of HK was perceived, hence confirming that HK was not altered by abiotic factors. Moreover, an increase in the pH of the solution was observed during biotransformation (Figs. 4 and5), especially for the incubations carried out in citrate buffer.

In all cases, pH reached a value of 8 after 168 hours of incubation, which can be attributed to the progressive metabolism of citrate by the microorganism. It may be noted that this increase of pH coincided with the appearance of SLMN. Concerning PRDN (Fig. 5), this metabolite was produced by both microorganisms in all conditions, but in greater amount when using A. candidus. It is noteworthy that for both strains, PRDN appeared until the medium reached a pH value of 6 (Fig. 5) when citrate buffer was utilized (pH 5), thus suggesting that its formation is pH dependent, also taking into account that it was generated from the first stages of incubation when phosphate buffer (pH 7) was used. Finally, the influence of PRDN on FRSM biotransformation by A.

candidus was assessed. The addition of PRDN in the incubation medium was found to entail inhibition of FRSM metabolism; hence formation of SLMN did not take place. and pH buffer phosphate (----).

Ecotoxicological assessment

Toxicity evaluation was carried out by means of the V. fischeri bioluminescence acute test. Toxicity values are expressed in terms of the effective concentration (EC50 and EC20), which represents the concentration causing 50% and 20% of inhibition of luminescence, respectively (Fig. 6). These values were determined by fitting the experimental data to dose-response curves with variable Hill slope. The concentration-response curves are depicted in Fig. 7. Toxicity of FRSM and HK product are only expressed as EC20 being that they did not provoke more than 30% and 50% of luminescence inhibition, respectively, at the tested concentrations. Only the data at 15 min contact were considered, as negligible difference was observed between the other exposure times.

The most remarkable issue is that metabolites HK and PRDN presented the highest toxicity levels, with EC50 = 34.406.84 mg L -1 and EC20 = 18.943.81 mg L -1 for PRDN, and EC20 = 37.427.96 mg L -1 for HK.

Discussion

Biotransformation

Three metabolites have been observed during the biotransformations of FRSM, whose identification allowed proposing the metabolic pathway described in scheme 1. SLMN was formed by both microorganisms and it resulted from FRSM N-dealkylation, which occurs through hydroxylation in the α-position of the amino group involving monooxygenase like cytochrome P450 enzyme, followed by hydrolysis of the hemiaminal intermediate [START_REF] Hezari | Microbial models of mammalian metabolism. N-dealkylation of furosemide to yield the mammalian metabolite CSA using Cunninghamella elegans[END_REF][START_REF] Williams | The metabolism and toxicity of furosemide in the Wistar rat and CD-1 mouse: a chemical and biochemical definition of the toxicophore[END_REF]). SLMN has also been detected as stable metabolite during the incubation of FRSM in the presence of river sediments [START_REF] Li | Screening for pharmaceutical transformation products formed in river sediment by combining ultrahigh performance liquid chromatography/high resolution mass spectrometry with a rapid data-processing method[END_REF]. These finding demonstrate that the use of microorganisms for the synthesis of TPs from pharmaceuticals is a potential approach for assessing the fate of drugs in environmental organisms.

HK, formed by C. echinulata, resulted from the oxidation of the furan ring. Such oxidation reactions involving cytochromes P450 have been reported through the formation of a cis-enedicarbonyl intermediate. Two mechanisms have been proposed: the formation of an epoxide followed by hydrolysis, or direct formation of cisenedicarbonyl compound through the addition of the high valent iron (IV)-oxospecies to the -system of the furan ring [START_REF] Guengerich | Cytochrome P450 oxidations in the generation of reactive electrophiles: epoxidation and related reactions[END_REF]. The double bond and the aldehyde group of the resulting cis-enedicarbonyl compound can be further reduced to form HK (Scheme 1). Indeed, it has been reported that aldehydes can be either reduced into the corresponding alcohol by microorganisms or oxidized into the corresponding carboxylic acid in animals [START_REF] Lacroix | Microbial biotransformations of a synthetic immunomodulating agent, HR325[END_REF]. Interestingly, Antoine et al. detected a similar compound as one of the major biliary metabolites of FRSM in rats, an -keto-carboxylic acid derivative [START_REF] Antoine | Formation of cytotoxic protein reactive metabolites from furosemide: Biological consequences of drug metabolism[END_REF]). This keto-carboxylic acid compound was also found during the electrochemical degradation of FRSM by means of the EF process [START_REF] Olvera-Vargas | Electro-Oxidation of the Pharmaceutical Furosemide: Kinetics, Mechanism, and By-Products[END_REF].

Scheme 1. Proposed pathway for biotransformation of furosemide by C. echinulata ATCC 9245

Regarding PRDN, it was first reported by Chen et al. [START_REF] Chen | Chemical and enzymatic oxidation of furosemide: formation of pyridinium salts[END_REF], who explained its formation through intramolecular condensation of the amino group with the aldehyde of the generated cisenedicarbonyl intermediate, this intermediate being common in the formation route of both, HK and PRDN metabolites (scheme 1). In the case of C. echinulata, reduction of the aldehyde group resulted to be faster than its condensation with the amino group, hence mostly generating HK. Laurencé et al. [START_REF] Laurencé | Anticipating the fate and impact of organic environmental contaminants: A new approach applied to the pharmaceutical furosemide[END_REF]) also described the generation of this compound during both, the microbial bioconversion of FRSM and its electrochemical oxidation.

The dependency of FRSM's bioconversion on pH is an important fact being as it suggests that environmental conditions play a relevant role on the biodegradation of drugs. It is thus important to take into consideration these factors when assessing ecotoxocological and environmental risk.

Ecotoxicological aspects

Ecotoxicity assessment of furosemide and its microbial metabolites was carried out, firstly because furosemide and its metabolites have been described as having toxicological effects towards different organisms (Peterson 2013), and secondly because its occurrence in the environment with its concomitant biotic transformation [START_REF] Jakimska | Elucidation of transformation pathway of ketoprofen, ibuprofen, and furosemide in surface water and their occurrence in the aqueous environment using UHPLC-QTOF-MS[END_REF]) may degenerate into potential ecotoxic metabolites such as the PRDN derivative.

A low toxicity value was found for FRSM, EC20 = 72.29 mg L -1 , evidencing its limited acute toxicity on V. fischeri. This result slightly differs from that found by Isidori et al. [START_REF] Isidori | A multispecies study to assess the toxic and genotoxic effect of pharmaceuticals: furosemide and its photoproduct[END_REF], who reported no lethal risk for the same marine bacterium, and for the rotifers Brachionus calyciflorus. Similarly, a toxicological assessment conducted on this drug employing cnidarian Hydra vulgaris, deployed an absent toxicity at concentrations up to 1 mg L -1 [START_REF] Pascoe | Do pharmaceuticals affect freshwater invertebrates? A study with the cnidarian Hydra vulgaris[END_REF]. Likewise, in a recent study, a moderate environmental risk was reported for FRSM, which was associated principally to its persistence and bioaccumulation, rather than to its toxicity [START_REF] Mendoza | Pharmaceuticals and iodinated contrast media in a hospital wastewater: A case study to analyse their presence and characterise their environmental risk and hazard[END_REF]. Nonetheless, these results contrast with the toxicological evaluation conducted on rodents and fish, which demonstrated the elevated hazard represented by this diuretic drug. Furthermore, FRSM was recently reported to present high toxicological risk for invertebrates and moderate risk for fish, according to its Risk Quotient (RQ) [START_REF] Papageorgiou | Seasonal occurrence, removal, mass loading and environmental risk assessment of 55 pharmaceuticals and personal care products in a municipal wastewater treatment plant in Central Greece[END_REF].

On the other hand, results demonstrate that the TPs resulting from the biotransformation of FRSM are more toxic that FRSM itself, a fact that has been previously suggested by some authors as discussed below.

PRDN gave the greatest ecotoxicity values, EC50 = 34.406.84 mg L -1 . Furthermore, its inhibitory effects on fungal oxidative enzymes were demonstrated since its presence hindered FRSM's metabolism. This detrimental effect could also cause ecotoxicity as formerly described for other drugs [START_REF] Caracciolo | Pharmaceuticals in the environment: Biodegradation and effects on natural microbial communities. A review[END_REF]. Previous toxicological evaluation reports also revealed PRDN's noxious effects with an EC50 = 97346

M according to the MTT test, as well as induction of cell death by the caspase-3 assay. Moreover, its use to induce symptoms associated to Parkinson's disease for a chemical model of this neurodegenerative disorder has been recently patented [START_REF] Martens | Chemical model of a neurodegenerative disease, method for preparation and uses of same[END_REF]).

Regarding HK derivative, which has not hitherto been identified, it exhibited higher ecotoxicity than the parent drug (EC20 = 36.60 mg L -1 ), however being less toxic than PRDN. HK is a reactive intermediate resulting from the oxidation of FRSM´s furan ring, whose progressive enzymatic oxidation was found to yield SLMN.

The latter didn't show any effect towards V. fischeri bacteria, which is in agreement with a previous toxicity study on SLMN that also evidenced a lack of hazardous effects [START_REF] Laurencé | Anticipating the fate and impact of organic environmental contaminants: A new approach applied to the pharmaceutical furosemide[END_REF]). These findings suggest that toxicological effects can be attributed mainly to the highly active aromatic moieties; the furan and pyridinium rings.

Toxicity values indicate that the formed metabolites have a restricted acute toxicity on bacteria V.

fischeri even at concentration ranges above the environmental levels. Nevertheless, the identification of metabolites having higher adverse effects than the parent drug denotes the necessity of investigating not only parent compounds but also potential TPs when assessing environmental impact, mostly taking into account that these hazardous compounds represent a potential risk for aquatic life by the long-term at low-level exposure, causing more subtle effects in growth, fertility, sex ratios, or reproductive anomalies [START_REF] Khetan | Human pharmaceuticals in the aquatic environment: A challenge to green chemistry[END_REF].

Therefore, these findings point out the urgency of intensifying toxicological assessment facing the increasingly worrying ecological and health risk posed by pharmaceutical residues and their TPs.

Conclusion

It has been confirmed that the environmental fungi Cunninghamella echinulata and Aspergillus candidus are capable of metabolizing FRSM into 3 main transformation products: saluamine, a pyridinium derivative and a hydroxy-ketone product, the latter having been described for the first time. Toxicity acute essays demonstrated that pyridinium and hydroxy-ketone metabolites have higher toxicity levels towards V. fischeri bacteria than FRSM. These results highlight the capacity of different microorganisms to transform pharmaceutical residues into stable metabolites, which are highly probable to be found in environmental water sources, therefore representing an ecotoxicological risk for both, ecosystems and human beings, as some of them can be even more toxic that the departing drug. Accordingly, this integral work gives a general perspective of the fate of drugs and their pernicious effects in the environment, which is essential for assessing environmental risk with the goal of developing the most convenient strategies for environmental management, including water control. Furthermore, this study can be regarded as a tool for the assessment of biotic transformation products from pharmaceutical pollutants, in response to the urgent necessity of reinforcing the evaluation of environmental hazard. Moreover, it contributes with valuable information with respect to the effects of drugs on environmental microbial communities, which is crucial for the comprehension of the biodegradation mechanisms drugs suffer in natural environments, as well as the harmful impacts on microbes.

  C NMR (100 MHz, DMSO-d6) δ (ppm): 206.04 (C-4), 168.8 (C-12), 152.4 (C-6), 136.7 (C-8), 133.7 (C-10), 127.1 (C-9), 114.4 (C-7), 108.4 (C-11), 60.5 (C-1), 52.4 (C-5), 36.7 (C-3), 27.03 (C-2).

Figure 1 .

 1 Figure 1. HPLC chromatograms of biotransformation of FRSM by C. echinulata ATCC 9245 (A) and by A. candidus ATCC 20023 (B).

Figure 2 .Figure 3 .

 23 Figure 2. Biotransformation of furosemide by C. echinulata ATCC 9245. LC-MS ion-current chromatogram (negative ion current) of m/z 329 (A), of m/z 249 (B) and of m/z 349 (C) and masse spectra of peaks at Rt = 4 min (D) and at Rt = 5 min (E)

Figure 4 .

 4 Figure 4. Evolution of FRSM and its metabolites during biotransformation of the drug by C. echunilata ATCC 9245 (A and B) and A. candidus ATCC 20023 (C and D) in different buffers: citrate pH 5 (A and C), phosphate pH 7 (B and D). FRSM (), HK (▲), SLMN (), pH buffer citrate (----), pH buffer phosphate (----).

Figure 5 .

 5 Figure 5. Evolution of PRDN metabolite during FRSM biotransformation by C. echunilata ATCC 9245 (A) and A. candidus ATCC 20023 (B). PRDN buffer citrate (), PRDN buffer phosphate (), pH buffer citrate (----)

Figure 6 .

 6 Figure 6. EC20 () and EC50 () toxicity values (mg L -1 ) for V. fischeri luminiscnece acute tests with 95% confidence limits. NE = No Effect.

Figure 7 .

 7 Figure 7. Inhibition percentage curves for FRSM () and the prepared metabolites, PRDN () and HK (▲), fitted to nonlinear regression with variable Hill slope.

Figure captions Scheme 1 .Figure 1 .

 11 Figure captions

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Biotransformation of furosemide by C. echinulata. LC-MS ion-current chromatogram (negative ion current) of m/z 329 (A), of m/z 249 (B) and of m/z 349 (C) and masse spectra of peak at Rt = 4 min (D) and at Rt = 5 min (E) Figure 3. Biotransformation of furosemide by A. candidus. LC-MS ion-current chromatogram of m/z 329 in negative mode (A), of m/z 329 in positive mode (B) and of m/z 249 in negative mode (C) and masse spectra of peak at Rt = 6 min (D) and at Rt = 1.1 min (E) Figure 4. Time course in biotransformation of furosemide by C. echunilata ATCC 9245 (A and B) and A. candidus ATCC 20023 (C and D) in different buffers: citrate pH 5 (A and C), phosphate pH 7 (B and D). FRSM (), HK (▲), SLMN (), pH buffer citrate (----), pH buffer phosphate (----).

Figure 5 .

 5 Figure 5. Evolution of PRDN metabolite during FRSM biotransformation by C. echunilata ATCC 9245 (A) and A. candidus ATCC 20023 (B). PRDN buffer citrate (), PRDN buffer phosphate (), pH buffer citrate (----) and pH buffer phosphate (----).

Figure 6 .

 6 Figure 6. EC20 () and EC50 () toxicity values (mg L -1 ) for V. fischeri luminiscnece acute tests with 95% confidence limits. NE = No Effect.

Figure 7 .

 7 Figure 7. Inhibition percentage curves for FRSM () and the prepared metabolites, PRDN () and HK (▲), fitted to nonlinear regression with variable Hill slope.

Acknowledgments

The authors wish to thank L. Dubost for mass spectra and A. Deville for NMR spectra.

Funding This study was funded by European Commission through the Erasmus Mundus Joint Doctorate Programme « Environmental Technologies for Contaminated Solids, Soils and Sediments » (grant number FPA n°2010-0009), and CONACyT, Mexico.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.


J.-M Malherbe
email: jean-marie.malherbe@obspm.fr

Optical characteristics and capabilities of the successive versions of Meudon spectroheliograph (1908-2023)

Keywords: Sun, instrumentation, photosphere, chromosphere, spectroscopy, spectroheliograph, spectroheliograms

), but independently, by Hale in the USA and Deslandres in France and was dedicated to long-term surveys of the solar cycles. For that purpose, systematic observations of the CaII K and Hα lines started in Meudon observatory in 1908 and continue today, so that a huge collection of more than 100000 spectroheliograms, spanning 115 years of solar activity, was recorded. We present in this paper the optical characteristics and the capabilities of the successive versions of the instrument, from 1908 to now.

INTRODUCTION

Henri Deslandres (1853Deslandres ( -1948) ) was hired in 1889 by Admiral Ernest Mouchez (the director of Paris observatory) and was in charge of organizing a spectroscopic laboratory, in the context of the development of physical astronomy initiated by Jules Janssen at Meudon. Deslandres first built a classical spectrograph and studied the profiles of the CaII K line at 3934 Å wavelength; he succeeded to resolve the fine structure of the core in 1892. This was the starting point of the spectroheliographs in France (Malherbe, 2023a). With a classical spectrograph, monochromatic images can be produced by an output slit located in the spectrum (to select the light of a spectral line) when the input slit scans the solar surface. This is possible by moving the solar image upon the first slit with the translation of the imaging objective. The photographic spectroheliograph was invented on this basis by George Hale in Kenwood (1892) and by Henri Deslandres in Paris (1893), simultaneously but independently. The principle is explained by d' Azambuja (1920aAzambuja ( , 1920b)).

Many spectroheliographs were constructed in the world to study the photosphere (thin layer of 500 km with temperature decreasing from 6000 K to 4500 K and exhititing magnetic structures, such as sunspots and bright faculae) and the chromosphere above (layer of 2000 km thickness, with temperature increasing from 4500 K to 8000 K, revealing structures such as dark filaments and bright plages). The chromosphere requires spectroscopic means to be seen. Long series of continuous observations in the CaII K line were collected by the spectroheliographs of Kodaikanal in India, Mount Wilson in the USA, Mitaka in Japan, Sacramento Peak in the USA, Coimbra in Portugal, Meudon in France or Arcetri in Italy. They produced extended archives, some of them covering up to 10 solar cycles, which are convenient to investigate long-term solar activity. Spectroheliographs historically produced monochromatic images along solar cycles at the epoch of photographic plates. Most of them observed the CaII K and Hα Fraunhofer lines of the chromosphere (Malherbe, 2023b). They were progressively abandoned in the second half of the twentieth century for telescopes using narrow bandpass filters (such as Fabry-Pérot or Lyot filters), which are more compact and able to observe, at much higher cadence, dynamic events (such as flares). However, with modern and fast electronic detectors, there is a regain of interest for imaging spectroscopy, because digital spectroheliographs can now deliver not only monochromatic images, but also full line profiles for each pixel of the Sun.

Section 1 presents the optical capabilities of the initial and large quadruple spectroheliograph designed in 1908 by [START_REF] Deslandres | Recherches sur l'atmosphère solaire. Photographie des couches gazeuses supérieures. Etude simultanée des formes et des mouvements des vapeurs[END_REF]Lucien d'Azambuja (1884-1970), while section 2 describes the three successive versions of the modern era . Section 3 is dedicated to the SOLar Explorer (SOLEX) instrument, a collaborative project between amateurs and professional astronomers for observations with a mini (but high performance) spectroheliograph, in order to increase the number of annual observations.

-THE LARGE QUADRUPLE SPECTROHELIOGRAPH OF DESLANDRES AND D'AZAMBUJA

Deslandres moved from Paris to Meudon in 1898 and installed there the first versions of his spectroheliographs, but observations were not yet systematic and many experiments and tests were performed to improve the quality of monochromatic images. A new building (figure 1) was erected in 1906 for the installation of a large quadruple spectroheliograph, wich was built in collabotation with d'Azambuja, who was hired in 1899, and was also in charge of organizing the future service of systematic observations. Hence, daily observations of CaII K started in 1908 and were followed soon by Hα in 1909, and continue today. They were just interrupted during four years by WW1. The large spectroheliograph was horizontal and stayed in a fixed position. For that reason, it was fed by a coelostat (two flat mirror system, North of the building) as shown by figure 1. The instrument was composed of four spectroheliographs (figure 2) that could not operate simultaneously, but alternatively: 1spectroheliograph n° I: a 3-metres instrument dedicated to systematic observations in CaII K line, with three flint prisms of 60°. The beam in the CaII K chamber is indicated in violet. The core of the line (K3) or the violet wing (K1v, -1.3 Å from the line core) were selected successively. 2spectroheliograph n° II: a 3-metres instrument dedicated to systematic observations in Hα line, with a plane grating (568 grooves/mm). The beam in the Hα chamber is indicated in red. 3spectroheliograph n° III: a multi-purpose 3-metres instrument, using either the three prisms or the grating. 4spectroheliograph n° IV: a research 7-metres high dispersive instrument using either the three prisms or the grating, with an associated afocal reductor (n° IVbis). The beam in the chamber (at right) and the reductor (at left) is indicated in blue. The photographic plate can be put either in F4 (no reduction) or in f (with reduction). The different combinations are summarized by table 1. They all use a common entrance objective (4.0 m focal length), collimator (1.30 m focal length) and entrance slit F (0.030 mm = 1.55" width). I,n° III,n° II and n° IV. M3 and either O3,or O4,or O5,or O6 constitute an afocal reducer (concave mirror of 7.0 m focal length and lenses of various power) in order to decrease the image size of the combination n° IV. They form together the combination n° IVbis. M1 is a removable flat mirror used for spectros n° III and n° IV, while P' is a 30° prism to separate grating orders and parasitic light of spectro n° IV. After d'Azambuja (1930). After d'Azambuja (1930).

The prisms (13 x 15 cm², angle A = 61°, refractive index n = 1.65) and the plane grating (8 x 5.5 cm², 568 grooves/mm) are displayed in figure 3.

The set of 3 prisms, composed of flint glass, worked at the minimum deviation (d=51°/prism giving a full deviation of D = 3 d = 153°) and was used in the blue/violet part of the spectrum. In the CaII K line, for the flint, we have dn/dλ = -0.355 μ -1 , which provides the quantity dD/dλ = 6 (dn/dλ) sin(A/2) / cos((A+d)/2) = -1.88 μ -1 for the set of three prisms. The output slit in the spectrum had the width w = 0.075 mm, which corresponds to a small deviation dD = w/f (f = 3.0 m is the focal length of chamber n° I) = 2.5 10 -5 rd, from which we obtain the waveband of the slit dλ = 0.13 Å. This value is in full agreement with figure 4 and table 2, which give the dispersion of 1.7 Å/mm in the CaII K line, providing the waveband dλ = 1.7 w = 1.7 x 0.075 = 0.13 Å.

The plane grating was used in the first order and the angle between the incident and diffracted beam was 20°. The grating formula predicts a dispersion of 5.5 Å/mm for the 3.0 m chamber n° II used for Hα, in agreement with figure 4 and table 2, giving 5.8 Å/mm. The bandpass of the slit (w = 0.075 mm) is 0.43 Å.

In conclusion, the spectrographs n° I and n° II (figure 5) provided monochromatic images in CaII K and Hα with respectively 0.13 Å and 0.43 Å bandpass. (left, 1908) and Hα (right, 1909) in the frame of the systematic observations program. Courtesy Paris Observatory.

The 7.0 metres chamber n° IV, dedicated to high dispersion spectroscopy and scientific research, was much more powerful and complicated than the others. For wavelengths smaller than 5000 Å, the set of 3 prisms was used, because in this range, the refractive index varies much with the wavelength, providing a dispersion in the range 0.5 -1.5 Å/mm. But for the wavelengths above 5000 Å, the plane grating appeared better, and the dispersion was in the range 1.5 -2.0 Å/mm.

The spectrograph used a 7.0 m focal length concave mirror (40 cm diameter) for the chamber and the spectrum formed at slit F4 (figure 2). A 0.075 mm slit width corresponds there to a typical bandwidth of 0.10 Å, providing an excellent selectivity along the line profiles, so that chamber n° IV was convenient both for thin photospheric lines and broad chromospheric lines of the Fraunhofer spectrum. However, the magnification was so high (7.0/1.3 = 5.4) that the size of the solar image was 37.2 x 5.4 = 200 mm at F4, too large to record the full Sun with usual glass plates of 13 x 18 cm². For that reason, F4 was convenient only for regions.

In order to reduce the image size of the solar disk, an afocal reductor was added. It was composed of an afocal system with a second 7.0 m focal length concave mirror and a lens (chosen among four possibilities) to produce an image between 9 mm and 58 mm diameter (table 1). A 30° prism (P') was included in the optical path to reduce parasitic light and residual superimposed grating orders. In the focal plane, the output slit f had a usual width of 1 mm (because the waveband was always selected in F4).

The curvature of the spectral lines R is roughly equal to (f²/λ) D, where f is the focal length of the chamber, λ the wavelength and D the dispersion (dλ/dx in Å/mm). Curved slits at F4 were used. For this spectrograph, R was found to be (figure 7):

-with prisms: 4000 Å < λ < 5700 Å → 2.10 m < R < 2.50 m (increasing with wavelength) -with the grating: 5700 Å < λ < 8500 Å → 2.50 m > R > 2.35 m (decreasing with wavelength) Figure 7. The radius of curvature of spectral lines as a function of wavelength for the 7 metres research chamber n° IV. The radius does not vary much with the grating, but this is not the case with the prisms in the blue/violet part of the spectrum. Five 22 cm long slits were available (slits 1, 2, 3 for the blue, slit 4 for the green and red). After d'Azambuja (1930).

Without any reduction, the size of chamber n° IV was 7 metres; with image reduction, chamber n° IV was called n° IVbis and the length was 14 metres (figure 8). It was not possible to move very fast from chamber n° I (dedicated to CaII K) to chamber n° IV because two parts of chamber n° IV had to be dismounted when it was not used (the F4 environment, which was suspended at the ceiling, figure 5, and the enclosure of chamber n° IVbis, figure 8). On the contrary, it was very easy to switch between chambers n° I, n° II (daily observations of CaII K and Hα) and n° III, which was a multi-purpose spectrograph of 3.0 m focal length, medium dispersion as shown by figure 4, working either with the 3 prisms or the grating, and dedicated to research. The chambers n° IV and n° IVbis were intensively used by d' Azambuja (1930) for his thesis work and observations of unusual lines. For example, figure 9 shows spectroheliograms in the infrared lines of CaII (8498 Å and 8542 Å) which were compared to CaII K3 and K1v, in order to validate the initial choice of Deslandres for chromospheric imagery. This figure also shows the first spectroheliogram got worl-wide in HeI 10830 Å (filaments as well as plages, usually bright, appear dark). These observations were not reproduced later. However, daily observations in the infrared lines of CaII and HeI started in 1974 at Kitt Peak National Observatory. The CaII 8542 Å is convenient for chromospheric magnetic fields (Zeeman effect). EUV images of HeII 304 Å (80000 K temperature) are produced by EIT/SOHO since 1996 and AIA/SDO since 2010.

-THE SPECTROHELIOGRAPH OF THE MODERN ERA (1989 -TODAY)

The spectroheliographs n° III, n° IV and n° IVbis were dismantled in 1960 after the retirement of Lucien d'Azambuja (1954) and his wife Marguerite, born Roumens (1959). At the beginning of the eighties, after 70 years of continuous service, it was necessary to refurbish the optical and mechanical parts of the spectroheliographs n° I and n° II in order to prolongate systematic observations of CaII K and Hα. It took many years and the new version (figure 10) was ready on 1 st January 1989. The 1989 version was studied by Gualtiero Olivieri . The entrance objective was replaced, together with the enclosures of the spectroheliographs. Optical elements of the spectrograph were also replaced, but the overall capabilities of the instrument were saved, for compatibility reasons with the glass plates collection. Chamber n° I (left, figure 10) was no longer used, but preserved, while chamber n° II (right, figure 10) was dedicated to systematic observations of CaII K and Hα. For that purpose, the old set of 3 prisms and plane grating were replaced by a 300 grooves/mm blazed grating (b = 17°27') working at order 3 for Hα and order 5 for CaII K. The grating was fixed and, for both lines, it worked with incidence and diffraction angles of respectively 7° and 27°. The interference orders were selected by coloured filters (figure 11). The focal length of the chamber (3.0 m) did not change, but glass plates were replaced by 13 x 18 cm² film plates. The dispersion was 0.30 mm/Å and 0.50 mm/Å, respectively for Hα and CaII K. With a 0.075 mm selecting slit in the spectrum, the bandpass was 0.25 Å and 0.15 Å, respectively for Hα and CaII K. Hβ was available in order 4 but not observed, because of the chromatism of the optics which was optimized for the red and the violet.

In order to observe together prominences at the limb and filaments on the disk, a disk attenuator (figure 12) was introduced to increase the exposure time without burning the disk. It is an artificial moon of neutral density 1.0 which moves upon the solar image (37.2 mm diameter) at the focus of the entrance objective. Two moons are available, one for winter and another one for summer. Standard observations consisted of CaII K3, CaII K1v, Hα, without any attenuator, and a long exposure CaII K3 with the attenuator to reveal prominences. The 1989 version was also ready for testing a Reticon linear array of 2048 pixels (13 µ square) located in another 1.0 m focal length chamber inside the spectrograph. With a linear array, the curvature of spectral lines (radius 1.55 m for 1.0 m focal length, wavelength independent at constant angles) must be suppressed; for that purpose, the entrance slit of the spectrograph was replaced by a curved slit of radius 1.965 m, in order to compensate the curvature of the spectrum. This change introduced a small geometric distorsion in the final image with a maximum shift of 4.5" at the poles (2.2" at 45° latitude, 1.1" at 30° latitude, null at the equator). The Reticon focus was never used for regular observations, because rectangular CCD appeared soon.

The next version is the one of 2002. It was also a major release, because the photographic chambers were abandoned (but preserved for historical reasons), and the selecting slit in the spectrum was suppressed. The 2002 version uses a specific 0.90 m chamber, located inside the spectrograph (figure 13), and a back illuminated CCD from Princeton Instruments (1340 x 100 pixels, 1 MHz, 20 μ square pixels, 200000 electrons full well, 14 bits dynamic range, 6-9 electrons of readout noise, water cooled at -40°C operating temperature, ST138 controler). As the CCD was too slow, a 1340 x 5 pixels window was recorded, so that the core of line profiles was described by 5 points only. The spectral sampling was 0.22 Å and 0.13 Å, respectively for Hα and CaII K. The quantum efficiency was exceptional (figure 14) both in orders 3 and 5. Figure 20 shows an example of observations performed since July 2017 (here 28 October 2021). Up to three series of four observations are performed daily, with Hα, Hα long exposure for prominences, CaII H/K simultaneously, and CaII H/K long exposure with the disk attenuator. Each observation is a data-cube (x, y, λ), twelve cubes/day at maximum, from which classical images (slices) are derived at fixed wavelength.

Table 3 summarizes the capabilities of the modern versions of the spectroheliograph since 1989. We plan to replace the 6.5 µm sCMOS camera by a more recent model with smaller pixels (4.6 µm) without any change of optics. This will improve both the spectral sampling and spatial sampling (from 1.1" to 0.78", in comparison to the usual Meudon seeing of 2"). The optical spectral resolution, with an entrance slit of 0.03 mm (1.55") is 0.25 Å and 0.15 Å respectively for Hα and CaII H/K. With a 4.6 µm pixel, the sampling would be well optimized (accordingly to the Shannon theorem), with 0.11 Å and 0.07 Å, respectively for Hα and CaII H/K.

-A PRO/AM COLLABORATIVE PROJECT: OBSERVATIONS WITH THE SOLAR EXPLORER (SOLEX), A MINI-SPECTROHELIOGRAPH (2023)

In order to improve the temporal coverage of solar observations freely available to the international community (a maximum of 250 days/year are observed at Meudon), we have initiated a collaboration with amateur astronomers. A mini-spectroheliograph was proposed to them by Christian Buil in 2022, the SOLar Explorer (SOLEX, http://www.astrosurf.com/solex/sol-ex-presentation-en.html) together with a sophisticated processing software (INTI) written by Valérie Desnoux (http://valerie.desnoux.free.fr/inti/), with capabilities close to the Meudon instrument. Amateur data are now delivered by the BASS2000 solar archive (https://bass2000.obspm.fr/home.php) since February 2023, together with professional data. Many SOLEX are disseminated world-wide. Figure 21 shows an example of SOLEX at the focus of a Vixen apochromatic refractor (ED 81 mm / 625 mm) with focal reducer 0.67, designed for full Sun observations with ZWO ASI178. The SOLEX instrument is a compact spectrograph (figure 22) with a 4.5 mm slit (0.01 mm wide) that is compatible with equivalent focal lengths in the range 380-480 mm for full disk observations (the median focus of 430 mm provides an image of 4.0 mm on the slit). The spectrograph is produced by 3D-printers; the focal lengths of the collimator and chamber lenses are, respectively, 80 mm and 125 mm, so that the solar diameter on the detector is about 6 mm. The dispersive device is a 2400 grooves/mm grating at order 1. All optical elements are provided by Shelyak Instruments, and the standard camera is the CMOS ZWO ASI178 monochrome model (3096 x 2080 pixels) with 2.4 μ square pixels (14 bits). The quantum efficiency (figure 23) is about 50% for Hα and CaII K. However, the spectrograph can select any line in the range 3500-7500 Å. The dispersion is 0.038 mm/Å and 0.031 mm/Å, respectively for Hα and CaII K. For a focal length of 430 mm, the width of the slit corresponds to about 4.8".

The spectral resolution, sampling and miscellaneous informations are given by table 4 for a typical SOLEX designed for full Sun observations (430 mm focal length and ZWO ASI178 camera) and Meudon spectroheliograph, allowing to compare some of their main characteristics. Figure 24 shows two Hα spectra got with both instruments with the pixel sampling of 0.063 Å and 0.155 Å, respectively for SOLEX and Meudon. Two Hα images are displayed in figure 25 and demonstrate the high quality of the SOLEX instrument. The SOLEX instrument is able to observe a large variety of lines (either photospheric or chromospheric) in the range 3500-7500 Å, just by rotating the grating at order 1, as shown by figure 26. This capability is of great interest to explore other lines than the usual Hα or CaII H/K, and can be used for more sophisticated observations, such as magnetometry of the photosphere in some Zeman sensitive lines. The dispersion lies in the range 0.03-0.05 mm/Å and increases towards the red. The curvature of spectral lines also increases towards the red (the radius decreases with wavelength and lies in the range 4-13 cm). On the contrary, the grating of Meudon spectroheliograph is fixed, so that only a few lines appearing in orders 3 to 5 are theoretically observable. The line curvature does not depend of orders and is compensated by the curvature (2 m radius) of the entrance slit. In practice, the Meudon instrument is limited to orders 3 (Hα) and 5 (CaII H/K), because the objectives are optimized only for these orders and present chromatism outside. For instance, Hβ at order 4 is possible, but one has to correct the focal length variation of the imaging objective.

Figure 1 .

 1 Figure 1. The coelostat of the spectroheliograph catches the solar light and redirects it in the horizontal and Nort/South direction of the laboratory. It uses two 40 cm flat mirrors. The first one follows the Sun (rotation speed of one turn during 48 hours); the second one reflects the beam towards the 4-metres focal length entrance objective (250 mm diameter). Courtesy Paris Observatory.

Figure 2 .

 2 Figure 2. The optical combinations of the quadruple spectroheliograph. The entrance objective (Op, 4.0 m focal length), slit (F) and collimator (O, 1.3 m focal length) are common to the four chambers n° I (violet beam), n° II (red beam), n° III and n° IV (blue beam). (t, m) are couples of velocity transformers and motors driving the 13 x 18 cm² glass plates. R and P are the disperser devices (grating or 3 prisms). L is a plane-cylindrical lens (astigmatism corrector of prisms). F1, F2, F3, F4, f are slits located in the spectrum. O1, O'1, O2 are chamber lenses (3.0 m focal length) while M2 is a concave mirror (7.0 m focal length) respectively for combinations n° I, n° III, n° II and n° IV. M3 and either O3, or O4, or O5, or O6 constitute an afocal reducer (concave mirror of 7.0 m focal length and lenses of various power) in order to decrease the image size of the combination n° IV. They form together the combination n° IVbis. M1 is a removable flat mirror used for spectros n° III and n° IV, while P' is a 30° prism to separate grating orders and parasitic light of spectro n° IV. After d'Azambuja (1930).

Figure 3 .

 3 Figure 3. The plane grating (568 grooves/mm), flint prisms (60°) and photographic plate support. Courtesy Paris Observatory.

Figure 5 .

 5 Figure 5. Spectroheliographs n° I (3 prisms) and n° II (plane grating) providing alternatively spectroheliograms in the CaII K and Hα lines. F1 and F2 are the 12 cm output slits in the spectrum, in the focal plane of the chambers. (t, m) are couples of velocity transformers and motors which synchronize the translations of the 4.0 m focal length imaging objective and the photographic plates of the chambers. Courtesy Paris Observatory.

Figure 6

 6 Figure6shows two of the first spectroheliograms obtained in CaII K with chamber n° I (1908) and in Hα with chamber n° II (1909). Systematic observations started in 1908 and continue today, with an interruption during WW1.

Figure 6 .

 6 Figure 6. Two examples of the first spectroheliograms obtained in CaII K (left, 1908) and Hα (right, 1909) in the frame of the systematic observations program. Courtesy Paris Observatory.

Figure 8 .

 8 Figure 8. The 2 x 7.0 meters chambers n° IV and n° IVbis. M1 is a flat mirror; M2 is the 7.0 m concave mirror of chamber n° IV, forming the spectrum on the slit F4 (image size 200 mm). M3 is the 7.0 m concave mirror of chamber n° IVbis; it forms an afocal system with one lens near prism P' to reduce the image size and focuses the output spectrum on the exit slit f. The distance between M2 and M3 is 14 m. The couple (t, m) is the motorized support of the photographic plate. Courtesy Paris Observatory.

Figure 9 .

 9 Figure 9. Spectroheliograms in special lines made with chambers n° IV and n° IVbis. HeI 10830 Å(left, after d'Azambuja & d'Azambuja, 1938) and the two CaII infrared lines(right, after d'Azambuja, 1930).

Figure 10 .

 10 Figure 10. The 1989 version of the spectroheliograph. Courtesy Paris Observatory.

Figure 11 .

 11 Figure 11. Bandpass of selecting order filters (wavelength in abscissa, in nanometres; transmission in ordinates, in %). Courtesy Paris Observatory.

Figure 12 .

 12 Figure 12. Transmission of the disk attenuator (wavelength in abscissa, in nanometres; transmission in ordinates, in %). Courtesy Paris Observatory.

Figure 13 .

 13 Figure 13. The numerical versions of the spectroheliograph. Bottom: 2002 version with CCD; top: 2018 version with scientific CMOS. Courtesy Paris Observatory.

Figure 14 .

 14 Figure 14. The quantum efficiency of the 2002 back illuminated CCD detector with VIS/AR + Lumogen coating. CaII K and Hα are respectively observed in orders 5 and 3. Hβ is visible in order 4. Courtesy Paris Observatory.

Figure 15

 15 Figure 15 shows an example of daily observations made since 1989, on film plates until 2001 and with the CCD after. Hα, CaII K3, CaII K1v and a long exposure CaII K3 for prominences were done two or three

Figure 16 .

 16 Figure 16. Full spectrum from 3900 Å to 6800 Å and interference orders. Courtesy Paris Observatory.

Figure 17 .

 17 Figure 17. Windows which are selected in the observed spectrum to record the line profiles of Hα (top) and CaII K and H (bottom). CaII K and H are recorded simultaneously. Courtesy Paris Observatory.

  Figure 18. Quantum efficiency (in %) of the CIS2020 sCMOS detector as a function of wavelength. Courtesy Paris Observatory and PCO.

Figure 19 .

 19 Figure 19. Evolution of the spectral sampling of the line profiles of Hα, CaII K and H. 1909-2001: one point (squares). 2002-2017: 5 points (triangles). 2018-now: 40 to 100 points (red crosses). The CaII H collection is new: it started in July 2017. CaII K and H are observed simultaneously. Courtesy Paris Observatory.

Figure 20 .

 20 Figure 20. Example of daily observations since July 2017. Top: Hα line (40 points in the profile), the selected images are the blue continuum, the blue wing and the line core. Centre: CaII H line (100 points in the profile), the selected images are the blue wing H1v and line core H3 (short and long exposure). Bottom: the same for CaII K line with K1v and K3. 28 October 2021. Courtesy Paris Observatory.

Figure 21 .

 21 Figure 21. Example of SOLEX instrument. Courtesy Paris Observatory.

Figure 22 .

 22 Figure 22. The SOLEX optical design. Courtesy Christian Buil.

Figure 23 .

 23 Figure 23. The IMX178 quantum efficiency (%). Courtesy ZWO.

Figure 24 .

 24 Figure 24. Spectra of the Hα line got with SOLEX (top, 0.063 Å sampling) and Meudon spectroheliograph (bottom, 0.155 Å sampling). The noise on the top image is due to the presence of dense clouds during the observational test reducing considerably the photometry. Courtesy Paris Observatory.

Figure 25 .

 25 Figure 25. Spectroheliograms in the centre of the Hα line got with SOLEX (left) and Meudon spectroheliograph (right) on 9 March 2023. For SOLEX: courtesy Eric Bernard, Saint Germier Observatory (Gers, France). For Meudon: courtesy Paris Observatory.

Figure 26 .

 26 Figure 26. Left: the spectral domain that can be explored with SOLEX or with Meudon spectroheliograph (which is restricted to discrete wavelengths in orders 3 to 5). Right: dispersion (mm/Å) and line curvature radius (cm) for SOLEX. Hα and CaII H/K lines are indicated by the coloured bars. Courtesy Paris Observatory.

Table 1 . The optical combinations of the quadruple spectroheliograph of figure 2. After d'Azambuja (1930).

 1

	Chamber	Optical Path (figure 2)	Length	Image	Spectral line
			(m)	diameter (mm)
	For systematic observations
	n° I	P+O1+F1	3.0	86	CaII K
	n° II	R+O2+F2	3.0	86	Hα
	For research observations
	n° III	(P or R)+M1+O'1+F3	3.0	86	multi-purpose
	n° IV	(P or R)+M1+M2+F4	7.0	205	High resolution
	n° IVbis (n° IV with	(P or R)+M1+M2+F4+M3+P' 2 x 7.0
	image reduction γ)	with O1 (γ = 0.29)		58.5	High spectral
		or O2 (γ = 0.19)		39.5	resolution
		or O3 (γ = 0.09)		19.5	with image
		or O4 (γ = 0.04)		9.0	reduction

Table 2 .

 2 Characteristics

	n° I	3 prisms	3.0	3900-4100	3934	1.7		86.5
	n° II	grating	3.0	3600-9000	3934		5.7	86.5
					5184		5.8	87.5
					6563		5.9	89.5
	n° III	prisms/grating 3.0	3600-9000	3934	1.7	4.8	86.5
					5184	4.6	4.4	87.5
					6563	9.4	4.0	89.5
	n° IV	prisms/grating 7.0	3600-9000	3934	0.7	2.0	206.0
					5184	2.0	1.9	205.5
					6563	4.1	1.8	205.0

of the four spectrographs in 1909.

After d'Azambuja (1930)

.

Spectrograph Spectrograph Focal Spectral Dispersion (Å/mm) Image number type length (m) range (Å) at λ (Å) with prisms or grating diameter (mm) Figure 4. The dispersion in Å/mm of the quadruple spectroheliograph. Three 3-metres chambers: n° I: 3 prisms, for CaII K systematic observations. n° II: plane grating, for Hα systematic observations. n° III: 3 prisms or plane grating, multi-purpose, medium dispersion, for research. The 7-metres chamber: n° IV: 3 prisms or plane grating, high dispersion for research observations.

Table 3 .

 3 Features of the modern versions of Meudon spectroheliograph. BW = bandwidth of monochromatic images. The focal length of the collimator is 1.30 m. The right column indicates the number of simultaneous wavelengths recorded along the line profiles (for electronic detectors only).

	Date	Chamber	Hα BW CaII K BW Hα dispersion CaII K dispersion Wavelengths
		(m)	(Å)	(Å)	(Å/mm)	(Å/mm)	(pixels)
	1989	3.0	0.25	0.15	3.2	1.9	1
		slit 75 μm, FILM
	2002	0.90	0.22	0.13	12.5	7.5	5
		CCD 20 μm
	2018	0.40	0.155 0.093	28.3	14.3	> 100
		sCMOS 6.5 μm
	2024 ?	0.40	0.114 0.068	28.3	14.3	> 150
		sCMOS 4.6 μm

Table 4 .

 4 Main characteristics of SOLEX and the 2017 version of Meudon spectroheliograph (SHG). Collimator (dia./focal length) 0.025/0.08 m 0.13/1.3 m Chamber (dia./focal length) 0.025/0.125 m 0.08/0.4 m Old SHG: 3.0 m, 0.9 m DETECTOR Type CMOS Cooled sCMOS Old SHG: Glass, film, CCD

	SOLEX	Meudon SHG	Remarks

The last version appeared in July 2017. It was also an important event, because for the first time, full line profiles were recorded for each solar pixel, providing (x, y, λ) FITS data-cubes. The classical images are slices of the cube. This version uses a specific 0.40 m chamber, located inside the spectrograph (figure 13), and a scientific CMOS array (PCO camera, Fairchild CIS2020 sensor, 2048 x 2048 pixels, 400 MHz, 6.5 μ square pixels, 30000 electrons full well, 16 bits dynamic range, 1.4 electrons of readout noise, water cooled at 5°C operating temperature). We record 40 points along the Hα line profile (6 Å wide range), and 100 points along the CaII H and CaII K line profiles (9 Å wide range) which are observed simultaneously (figure 16 and figure 17). The spectral sampling is 0.155 Å and 0.093 Å, respectively for Hα and CaII H/K. The quantum efficiency is excellent (figure 18) in orders 3, 4 (Hydrogen lines) and moderate in order 5 (CaII). The wavelength sampling of spectral lines is reported in figure 19. The spatial sampling is now 1.1"/pixel.