Groundwater quality trend and trend reversal assessment in the European Water Framework Directive context: an example with nitrates in Italy.

Groundwater resources are of utmost importance in sustaining water related ecosystems, including humans. The long-lasting impacts from anthropogenic activities require early actions, owing to the natural time lag in groundwater formation and renewal. The European Union (EU) policy, within the implementation of the Water Framework Directive (WFD), requires Member States to identify and reverse any significant and sustained upward trend in the concentration of pollutants, defining specific protection measures to be included in the River Basin Management Plans (RBMP). In Italy, official guidelines for trend and trend reversal assessment have been published recently. Statistical methods, such as the Mann-Kendall test for trend analysis and the Sen's method for estimating concentration scenarios, should be applied at the fixed terms stated by the WFD implementation cycles to identify upward trends, while the Pettitt test is proposed for the identification of trend reversal. In this paper, we present an application of a slightly modified version of the Italian Guidelines to a groundwater body in Northern Italy featuring nitrate pollution and discuss its advantages and limitations. In addition to Pettitt test, for the trend reversal analysis, we apply the Mann-Kendall test in two sections and compare the results. We conclude that this method seems more reliable than Pettitt test to identify a reversal point in quality time series. The overall procedure can be easily applied to any groundwater body defined at risk across Europe, for the assessment of the upward trends of pollutants and their reversal, even with little chemical monitoring data. Although focused on the EU legislative framework, this procedure may be relevant for a wider context, allowing to individuate upward trend as early warning for contamination processes in an integrated water resources management context.

The Reaction of Thiyl Radical with Methyl Linoleate: Completing the Picture.

Cis lipids can be converted by thiols and free radicals into trans lipids, which are therefore a valuable tell-tale for free radical activity in the cell's lipidome. Our previous studies have shown that polyunsaturated lipids are isomerized by alkanethiyl radicals (S•) in a cycle propagated by reversible double-bond addition and terminated by radical H-abstraction from the lipid. A critical flaw in this picture has long been that the reported lipid abstraction rate from radiolysis studies is faster than addition-isomerization, implying that the "cycle" must be terminating faster than it is propagating! Herein, we resolved this longstanding puzzle by combining a detailed product analysis, with reinvestigation of the time-resolved kinetics, DFT calculations of the indicated pathways, and reformulation of the radical-stasis equations. We have determined thiol-coupled products in dilute solutions arise mainly from addition to the inside position of the bisallylic group, followed by rapid intramolecular H• transfer, yielding allylic radicals (LZZ + S• ⇄ SL• → SL'•) that are slowly reduced by thiol (SL'• + SH → SL'H + S•). The first-order grow-in rate of the L-H• signal (kexp280nm) may therefore be dominated by the addition-H-translocation rather than slower direct H•-abstraction. Steady-state kinetic analysis of the new mechanism is consistent with products and the rates and trends for polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs), and mixtures, with and without physiological [O2]. Implications of this new paradigm for the thiol-ene reactivity fall in an interdisciplinary research area spanning from synthetic applications to metabolomics.

Radical-based grafting of GMA on sutures of different nature.

Irradiation of a number of different sutures largely employed in the clinical practice with either high energy electrons or with γ-rays followed by quenching with glycidyl methacrylate (GMA) conveniently led to derivatization through a radical-based process. The radicals involved were detected by means of ESR spectroscopy and were characterized on the basis of their ESR spectral parameters which were also found to be consistent with the hfs constants predicted by DFT calculations. Evidence of the GMA derivatization of the sutures was obtained via(13)C CP-MAS NMR spectroscopy, while its extent was evaluated gravimetrically.

Radiation-induced formation of purine 5',8-cyclonucleosides in isolated and cellular DNA: high stereospecificity and modulating effect of oxygen.

The present work is aimed at gaining conclusive mechanistic insights into the radiation-induced formation of the 5'R and 5'S diastereomers of both adenine and guanine 5',8-cyclo-2'-deoxyribonucleosides, with emphasis on the delineation of the inhibitory effect of O(2) in isolated and cellular DNA. The levels of purine 5',8-cyclo-2'-deoxyribonucleosides as assessed by HPLC-MS/MS were found to decrease steadily with the increase of O(2) concentration, the 5',8-cyclo-2'-deoxyguanosine being produced more efficiently than the 5',8-cyclo-2'-deoxyadenosine for low O(2) concentrations. A high stereoselectivity was observed in the intramolecular addition of the C5' radical to the C8 of the purine leading, after the creation of the C5'-C8 bond and a subsequent oxidation step, to the predominant formation of the 5'R diastereomer for both purine 5',8-cyclonucleosides. The reduced formation yield of the 4 tandem lesions in the presence of O(2) explains, at least partly, the low efficiency of radiation-induced yields of the purine 5',8-cyclo-2'-deoxyribonucleosides in cellular DNA, which are about two orders of magnitude lower than the previously reported data obtained from HPLC-MS analysis.

Hydrogen hyperfine splitting constants for phenoxyl radicals by DFT methods: regression analysis unravels hydrogen bonding effects.

DFT calculations using the B3LYP functional, medium-sized basis sets and empirical scaling of the results provide quantitative estimates of the hydrogen isotropic hyperfine splitting constants (hscs) in 2,6-di-alkyl phenoxyl radicals (1-11). Literature hscs for phenoxyl (12), 4-methylphenoxyl (13) and 4-methoxyphenoxyl (14) radicals, on the other hand, are poorly predicted by using this method. This different behaviour is explained considering that experimental hscs of 12-14 are influenced by H-bonds formed between phenoxyls and their parent phenols, usually present in large amounts in solution as radical precursors. This was confirmed experimentally by measuring the EPR spectra of 12-14 in the presence of increasing amounts of their parent phenols, and by calculating the hscs in the case of the formation of 1 : 1 and 1 : 2 complexes between these radicals and phenol. Relevance of these results to the choice of reference hscs as benchmarks for theoretical calculations and to kinetic and thermochemical determinations on unhindered phenoxyl radicals is discussed.

The reaction of spiro[indoline-naphthopyrans] with nitrogen oxides. Not a case of biradical double trapping.

The reactions of six differently substituted photochromic spiro[indoline-naphtopyrans] with .NO or .NO(2) under normal daylight conditions have been investigated by means of EPR spectroscopy along with those of three structurally related spiro[indoline-benzopyrans]. The spectra due to cyclic oxynitroxides originating from double trapping of biradicals by .NO were observed with the three latter derivatives, this finding being in agreement with previous results. Similar signals were also observed with the six former compounds, but in this case they were responsible for just a minor component of the spectra, the main spectral signals being due to hitherto unreported paramagnetic species that on the basis of their spectral parameters are identified as iminoxy radicals. DFT calculations at the B3LYP/6-31G* level carried out on a variety of radicals support this assignment.

Comparison of isoelectronic 8-HO-G and 8-NH2-G derivatives in redox processes.

8-Oxo-7,8-dihydroguanine (8-oxo-G) is the major lesion of oxidatively generated DNA damage. Despite two decades of intense study, several fundamental properties remain to be defined. Its isoelectronic 8-aminoguanine (8-NH(2)-G) has also received considerable attention from a biological point of view, although its chemistry involving redox processes remains to be discovered. We investigated the one-electron oxidation and one-electron reduction reactions of 8-oxo-G and 8-NH(2)-G derivatives. The reactions of hydrated electrons (e(aq)(-)) and azide radicals (N(3)(*)) with both derivatives were studied by pulse radiolysis techniques, and the transient absorption spectra were assigned to specific tautomers computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The protonated electron adducts of 8-NH(2)-G and 8-oxo-G showed a substantial difference in their absorption spectra, the unpaired electron being mainly delocalized in the imidazolyl ring and in the six-membered ring, respectively. On the other hand, the deprotonated forms of one-electron oxidation of 8-NH(2)-G and 8-oxo-G showed quite similar spectral characteristics. In a parallel study, the one-electron reduction of 8-azidoguanine (8-N(3)-G) afforded the same transient of one-electron oxidation of 8-NH(2)-G, which represents another example of generation of one-electron oxidized guanine derivatives under reducing conditions. Moreover, the fate of transient species was investigated by radiolytic methods coupled with product studies and allowed self- and cross-termination rate constants associated with these reactions to be estimated.

A reevaluation of the ambident reactivity of the guanine moiety towards hydroxyl radicals.

Radically different: Contrary to previous proposals, the main reaction of the HO(*) radical with guanosine or 2'-deoxyguanosine is the hydrogen abstraction from the NH(2) moiety to give a guanyl radical. This radical, characterized by a broad band in the visible region (around 610 nm), undergoes tautomerization to the most stable isomer.

Reaction of hydrated electrons with guanine derivatives: tautomerism of intermediate species.

Here, we show that two tautomers are produced by the protonation of the guanine-electron adduct. The fate of electron adducts of a variety of substituted guanosines was investigated by radiolytic methods and addressed computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The reaction of e(aq-) with guanosine and 1-methylguanosine produces two transient species, whereas the reaction with N2-ethylguanosine and N2,N2-diethylguanosine produces only one. The two short-lived intermediates, which show a substantial difference in their UV-visible spectra, are recognized to be two purine tautomers (i.e., iminic 18 and aminic 19 forms). The tautomerization 18 --> 19 occurs with a rate constant of ca. 1.5 x 106 s(-1) , and theory suggests that it is a water-assisted process.

On the actual EPR detection of thioacyl radicals.

The direct EPR detection of thioacyl radicals has been reported only once, while thioacyl nitroxides remain an elusive species. We failed to detect the thioacyl radicals from two thioaldehydes and from phosphoryldithioformates but have obtained EPR evidence that thioacyl radicals react with 2-methyl-2-nitrosopropane to give thiocarbonyloxyaminyls rather than thioacyl nitroxides. The results of DFT calculations support this unexpected reactivity of thioacyls, while making questionable their previous EPR identification.

One-electron reduction of 8-bromo-2-aminoadenosine in the aqueous phase: radiation chemical and DFT studies of the mechanism.

Two tautomeric forms of one-electron oxidized 2-aminoadenosine (2AA) have been produced by reactions of hydrated electrons (e aq-) with 8-bromo-2-aminoadenosine (8-Br-2AA) at natural pH, whereas only one tautomer is formed by oxidation of 2AA. Tailored experiments by pulse radiolysis and time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations allowed the definition of the reaction mechanism in some detail. The electron adducts of 8-Br-2AA protonated at C8 eject Br- and produce the two short-lived tautomers (8 and 9). The first observable species decays by first-order kinetics to produce the second intermediate, which is also obtained by oxidation of 2AA by SO4*-. The rate of tautomerization (k taut = 4.5 x 104 s-1) is strongly accelerated by phosphate and is retarded in D2O (kinetic isotope effect 7). B1B95/6-31+G** calculations showed that the tautomerization is a water-assisted process. In acidic or basic solutions, the "instantaneous" formation of one-electron oxidized 2AA or its deprotonated forms has been produced by reactions of e aq- with 8-Br-2AA. gamma-Radiolysis of 8-Br-2AA in aqueous solutions followed by product studies led to the formation of 2AA as a single product.

Ring opening of the cyclobutane in a thymine dimer radical anion.

The reactions of hydrated electrons (e(aq) (-)) with thymine dimer 2 and thymidine have been investigated by radiolytic methods coupled with product studies, and addressed computationally by means of BB1K-HMDFT calculations. Pulse radiolysis revealed that one-electron reduction of the thymine dimer 2 affords the radical anion of thymidine (5) with t(1/2)<35 ns. Indeed, the theoretical study suggests that radical anion 3, in which the spin density and charge distribution are located in both thymine rings, undergoes a fast partially ionic splitting of the cyclobutane with a half-life of a few ps. This model fits with the in vivo observation of thymine dimer repair in DNA by photolyase. gamma-Radiolysis of thymine dimer 2 demonstrates that the one-electron reduction and the subsequent cleavage of the cyclobutane ring does not proceed by means of a radical chain mechanism, that is, in this model reaction the T(-)* is unable to transfer an electron to the thymine dimer 2.

Tautomerism in the guanyl radical.

Despite a few decades of intense study, a full description of tautomers of one-electron-oxidized guanine remains to be achieved. Here we show that two of these tautomers are produced by the protonation of an 8-haloguanine electron adduct. The rate constants for the reactions of hydrated electrons (e(aq)(-)) with a variety of 8-substituted guanine derivatives have been measured by a pulse radiolysis technique and correlated with both inductive and resonance components of the substituents. The fate of electron adducts was investigated by radiolytic methods coupled with product studies and addressed computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The reaction of e(aq)(-) with 8-haloguanosine or 8-halo-2'-deoxyguanosine produces the first observable transient species that decay unimolecularly (k = 1 x 10(5) s(-)(1) at 22 degrees C) to give the one-electron oxidized guanosine or 2'-deoxyguanosine. Theory suggests that the electron adducts of 8-bromoguanine derivatives protonated at C8 form a pi-complex, with the Br atom situated above the molecular plane, that is prompt to eject Br(-). The two short-lived intermediates, which show a substantial difference in their absorption spectra, are recognized to be the two purine tautomers (i.e., iminic 7 and aminic 3 forms). The spin density distributions of the two tautomers are quite different at the O6 and N10 positions, whereas they are very similar at the N3, C5, and C8 positions. The resonance structures of the two tautomers are discussed in some detail. B1B95/6-31+G calculations show also that the tautomerization from the iminic (7) to the aminic (3) arrangement is a water-assisted process.

Chemical radiation studies of 8-bromo-2'-deoxyinosine and 8-bromoinosine in aqueous solutions.

The reactions of hydrated electrons (e(aq) (-)) with 8-bromo-2'-deoxyinosine (8) and 8-bromoinosine (12) have been investigated by radiolytic methods coupled with product studies and have been addressed computationally by means of BB1K-HMDFT calculations. Pulse radiolysis revealed that one-electron reductive cleavage of the C--Br bond gives the C8 radical 9 or 13 followed by a fast radical translocation to the sugar moiety. Selective generation of a C5' radical occurs in the 2'-deoxyribo derivative, whereas in the ribo analogue the reaction is partitioned between the C5' and C2' positions with similar rates. Both C5' radicals undergo cyclizations, 10-->11 and 14-->15, with rate constants of 1.4 x 10(5) and of 1.3 x 10(4) s(-1), respectively. The redox properties of radicals 10 and 11 have also been investigated. A synthetically useful photoreaction has also been developed as a one-pot procedure that allows the conversion of 8 to 5',8-cyclo-2'-deoxyinosine in a high yield and a diastereoisomeric ratio (5'R)/(5'S) of 4:1. The present results are compared with data previously obtained for 8-bromoadenine and 8-bromoguanine nucleosides. Theory suggests that the behavior of 8-bromopurine derivatives with respect to solvated electrons can be attributed to differences in the energy gap between the pi*- and sigma*-radical anions.

The kinetics of Z/E isomerization of methyl oleate catalyzed by photogenerated thiyl radicals.

The reaction of beta-mercaptoethanol with methyl oleate in tertbutyl alcohol was investigated under photochemical conditions. The time-dependent Z/E isomerization of the unsaturated moiety as well as the thiol-adduct formation were analyzed on the basis of radical generation rates and established rate constants. This provides precise room-temperature rate constants for the reversible thiyl radical addition to both Z and E isomers. The rate constants for HOCH2CH2S* addition to the Z and E forms were found to be 1.6 x 10(5) and 2.9 x 10(5) M(-1) S(-1), respectively. For the beta-elimination of the thiyl radical from the alkyl radical adduct, the rate constants were 1.7 x 10(7) and 1.6 x 10(8) S(-1), the faster associated with the formation of the E isomer. DFT-BB1K calculations on 2-butene assign this large preference of fragmentation for the E isomer to a lower activation barrier for the formation of the E transition state from the equilibrium radical adduct structure.

Redox and spin-trapping properties of phosphoryldithioacetates.

The redox properties of ethyl diethoxyphosphoryldithioacetate (1), methyl diethoxyphosphorylfluorodithioacetate (2) and methyl diethoxyphosphoryldifluorodithioacetate (3) were measured by cyclic voltammetry. Addition of free radicals to the three dithioesters led to the detection of the ESR spectra of the spin adducts only with the last two compounds. Based on the reduction potentials of the three dithioacetates, this may be explained by capto-dative stabilization of the adducts. On the basis of DFT calculations, the temperature dependent variations of the spectral patterns exhibited by the radicals were attributed to restricted rotation about the Calpha-SMe bond.

Water effect on the o-h dissociation enthalpy of para-substituted phenols: a DFT study.

The effect of water on the O-H bond dissociation enthalpy (BDE) of para-substituted phenols has been investigated by means of DFT calculations. It is shown that the experimental BDE values are fairly well-reproduced by simple B3LYP/6-31G* calculations carried out on the phenol/phenoxyl-water complexes taking into account only hydrogen-bonding (HB) interactions of water molecules with molecular sites (HB model). On the contrary, the BDE values computed with the polarizable continuum model (PCM/B3LYP/6-31G*)8 are overestimated by about 3-4 kcal/mol. Discrepancy between theory and experiment increases using the PCM method in addition to the HB model. Calculations show that, in general, the HB interaction with water molecules decreases the BDE of phenols bearing electron-releasing groups while increasing the BDE of phenols bearing electron-withdrawing substituents. This opposite effect is explained by considering the resonance structures with charge separation both in phenols and in phenoxyl radicals. With electron donors, the phenoxyl radical is preferentially stabilized by the HB acceptor interaction with two water molecules, while with electron acceptors the phenol is preferentially stabilized by the HB donor interaction with one water molecule.

Selective generation and reactivity of 5'-adenosinyl and 2'-adenosinyl radicals.

The reaction of hydrated electrons (e(-)(aq) with 8-bromoadenosine 7 has been investigated by radiolytic methods coupled with product studies. Pulse radiolysis revealed that one-electron reductive cleavage of the C-Br bond gives the C8 radical 8 followed by a fast radical translocation to the sugar moiety. The reaction is partitioned between C5' and C2' positions in a 60:40 ratio leading to 5'-adenosinyl radical 9 and 2'-adenosinyl radical 11. This radical translocation from C8 to different sites of the sugar moiety has also been addressed computationally by means of DFT B3LYP calculations. In addition, ketone 21 was prepared and photolyzed providing an independent generation of C2' radical 11. Both C5' and C2' radicals undergo unimolecular reactions. Radical 9 attacks adenine with a rate constant of 1.0 x 10(4) s(-1) and gives the aromatic aminyl radical 10, whereas C2' radical 11 liberates adenine with a rate constant of 1.1 x 10(5) s(-1).

A critical evaluation of the factors determining the effect of intramolecular hydrogen bonding on the O-H bond dissociation enthalpy of catechol and of flavonoid antioxidants.

New experimental results on the determination of the bond dissociation enthalpy (BDE) value of 3,5-di-tert-butylcatechol, a model compound for flavonoid antioxidants, by the EPR radical equilibration technique are reported. By measurement of the equilibrium constant for the reaction between 3,5-di-tert-butylcatechol and the 2,6-di-tert-butyl-4-methylphenoxyl radical, in UV irradiated isooctane solutions at different temperatures, it has been shown that the thermodynamic parameters for this reaction are DeltaH degrees = -2.8+/-0.1 kcal mol(-1) and DeltaS degrees = +1.3+/-0.2 cal mol(-1) K(-1). This demonstrates that the entropic variations in the hydrogen exchange reaction between phenols and the corresponding phenoxyl radicals are also negligible when one of the reacting species is a polyphenol and that the EPR radical equilibration technique also allows the determination of the Obond;H BDEs in intramolecularly hydrogen-bonded polyphenols. The BDE of 3,5-di-tert-butylcatechol (78.2 kcal mol(-1)) was determined to be identical to that of alpha-tocopherol. Through use of the group additivity rule, this piece of data was also used to calculate the strength of the intramolecular hydrogen bond between the hydroxyl proton and the oxygen radical centre in the corresponding semiquinone radical (5.6 kcal mol(-1)), which is responsible both for the excellent antioxidant properties of catechols and for the BDE of catechol (81.8 kcal mol(-1)). These values are in poor agreement with those predicted by DFT calculations reported in the literature (9.5 kcal mol(-1) and 77.6 kcal mol(-1), respectively). Extensive theoretical calculations indicate that the BDE of catechol is reproduced well (81.6 kcal mol(-1)) by use of diffuse functions on oxygen and the CCSD method.