Study on the photodegradation of amidosulfuron in aqueous solutions

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for personal use only and shall not be self- archived in ... Sulfonylurea herbicides are a new generation of pesticides ... (2002), they can be divided into three categories (class a–c) .... the full scan data acquisitions were made in the m/z range ... 10.00 ml of 5.00 mg L ... solutions at increasing concentration levels with the one.
Study on the photodegradation of amidosulfuron in aqueous solutions by LCMS/MS M. Benzi, E. Robotti & V. Gianotti

Environmental Science and Pollution Research ISSN 0944-1344 Environ Sci Pollut Res DOI 10.1007/s11356-013-1900-x

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Author's personal copy Environ Sci Pollut Res DOI 10.1007/s11356-013-1900-x

RESEARCH ARTICLE

Study on the photodegradation of amidosulfuron in aqueous solutions by LC-MS/MS M. Benzi & E. Robotti & V. Gianotti

Received: 30 January 2013 / Accepted: 3 June 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Sulfonylurea herbicides are extensively widespread for the protection of a variety of crops and vegetables because of their low application rates, high selectivity and low persistency in the environment; unfortunately, their low persistence does not always correspond to a lower toxicity, since new species potentially more toxic and stable than the precursor herbicides can form, owing to natural degradation processes. Here, the photodegradation of amidosulfuron in aqueous solutions was studied by high-performance liquid chromatography with diode array detection and tandem mass spectrometry to identify the degradation products in order to outline the environmental fate of the molecules generating from the simulation of one of the natural processes that can occur, i.e., photoinduced degradation. The photodegradation process results in a first order kinetic reaction with a t1/2 value of 276 h (11.5 days) and a kinetic constant of 0.0027 h−1, and three possible degradation products were identified. The results obtained are then compared to those obtained in previous works carried out in comparable experimental conditions about nicosulfuron and tribenuron-methyl, two sulfonylurea herbicides belonging to different classes, and to literature data: hypotheses on the existence of preferential degradation pathways are then drawn, in consequence of the molecular structure of the sulfonylurea pesticide. In particular, the use of Responsible editor: Hongwen Sun Electronic supplementary material The online version of this article (doi:10.1007/s11356-013-1900-x) contains supplementary material, which is available to authorized users. M. Benzi ARPA Valle d’Aosta, Località Grande Charrière 44, 11020 Saint-Christophe, AO, Italy E. Robotti : V. Gianotti (*) Department of Scienze e Innovazione Tecnologica, University of Piemonte Orientale “Amedeo Avogadro”, Viale T. Michel 11, 15121 Alessandria, Italy e-mail: [email protected]

organic solvents to obtain complete solubilization of the sample plays a fundamental role and deeply influences the degradation processes that, therefore, not always fully adhere to the actual natural photodegradation pathways. Moreover, considerations about toxicity were driven since the complete mineralisation of the sample is not reached: even when the parent pesticides are totally degraded, they are, however, transformed into other organic compounds showing, if subject to ecotoxicological tests, at least the same toxicity of the precursor herbicides. The evidence here presented suggests that, at least for the class of sulfonylurea pesticides, their professed low persistence actually does not produce any real advantage. Keywords Photodegradation . Amidosulfuron . Mass spectrometry detection . Aqueous solutions . Sulfonylurea pesticide . Liquid chromatography

Introduction Sulfonylurea herbicides are a new generation of pesticides characterised by a molecular structure comprising a sulfonylurea bridge; they are used for the protection of a variety of crops and vegetables, such as maize, rice, potatoes and sugar beet. They are amply widespread due to their low application rates, high selectivity and low persistency in the environment; about 30 different active ingredients are commercially available. The different active formulas differ for the two substituents (R1 and R2) on the sulfonylurea bridge, as indicated in Fig. 1a. According to the classification from Sarmah and Sabadie (2002), they can be divided into three categories (class a–c) based on the combination of the R1 moiety, which can be either an aliphatic, aromatic or heterocyclic group, and the R2 moiety, a triazinic or pyrimidinic system. Unfortunately, their low persistence in the environment does not always correspond to a lower toxicity, since new

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R1 -SO2NHCONH- R2 O

CH3 S

O

O N S N CH3 O H

OCH3

O C

a

Considerations are also drawn, based on toxicity studies, about the actual low persistency and complete biodegradability of this class of herbicides and their degradation products in the environment.

N

b

N H

N

Experimental OCH3

Fig. 1 General sulfonylurea herbicides structure with the R1 and R2 moieties evidenced (a) and analyte structure (b)

species, potentially more toxic and stable than the precursor herbicides, can form, owing to natural degradation processes (Andersen et al. 2001; Benzi et al. 2011; Livanainen and Heinonen-Tanski 1991; Martins and Mermoud 1999; Martins et al. 2001; Metzger et al. 1996; Polati et al. 2008). Generally, natural degradation is reported in the literature as due to hydrolysis (Olmedo et al. 1997; Perreau et al. 2007; Sarmah and Sabadie 2002), microbial processes (Bossi et al. 1999; Saha and Kulshrestha 2002; Wei et al. 1998) and, in particular, for this class of herbicides, photolysis, which can be considered as a degradation pathway alternative to chemical hydrolysis (Morrica et al. 2004). In the literature, many works dealing with the degradation of sulfonylurea pesticides are present, but the experimental conditions adopted are quite heterogeneous: some authors evaluate only hydrolysis (Sarmah and Sabadie 2002); others use different solvents to dissolve the pesticides, with the risk of influencing the degradation pathways (Braschi et al. 2000; Morrica et al. 2004; Sabadie 2002; Scrano et al. 1999; Vulliet et al. 2004); and finally, some other authors use titania as catalyst in the degradation experiments (Maurino et al. 1999; Sleiman et al. 2006; Vulliet et al. 2002, 2003). The wide range of experimental settings usually explored hampers the comparison of the results obtained for different pesticides, since the observed photoproducts, as well as the photodegradation pathways, strongly depend on the experimental conditions (Vulliet et al. 2004). In this perspective, comparison of the literature that results to the identification of the general preferential degradation pathways for the class of sulfonylurea pesticides is almost impracticable. Here, the photodegradation of a sulfonylurea herbicide from class a, amidosulfuron, is considered (Fig. 1b). Then, the results are compared to those previously obtained in our laboratories for two sulfonylurea pesticides belonging to different classes: tribenuron-methyl (class b) (Polati et al. 2008) and nicosulfuron (class c) (Benzi et al. 2011). Since all the three classes of sulfonylurea pesticides are explored by studies performed in identical experimental conditions, considerations are driven about the effect played by different substituents on the sulfonylurea bridge on the environmental fate of this class of herbicides. The results obtained are compared to, and when possible, confirmed by, literature data.

Reagents Amidosulfuron 97.5 % w/w (AMIDO) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Stock solutions were prepared at 5.00 mg L−1 in ultrapure water and in acetonitrile at 100.00 mg L−1. Gradient grade acetonitrile (99.8 % w/w) and glacial acetic acid (99.8 % w/w) were purchased from Merck (Darmstadt, Germany), and ultrapure water was produced by a Millipore Milli-Q system (Milford, MA, USA). Apparatus High-performance liquid chromatography with diode array detection and tandem mass spectrometry (HPLC-DADMS/MS) analyses were carried out by a Thermo Finnigan Mass Spectra System (Finnigan, San Jose, CA, USA) equipped with a Degasser SCM1000, a SpectraSYSTEM P4000 gradient pump and a SpectraSYSTEM AS3000 autosampler, interfaced by the module SN4000 to a SpectraSYSTEM UV6000LP diode array detector (190– 700 nm) and to a Finnigan LCQ Duo mass spectrometer detector equipped with electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) ion sources and with an ion trap analyser. A CoFoMeGra solar box 3000e (Milan, Italy) was used to simulate sunlight irradiation. UV–vis analyses were performed by a Jasco V550 spectrophotometer (Tokyo, Japan) equipped with Spectra Manager for Windows 95/NT Version 1.53.00 by Jasco Corporation. HPLC-DAD-MS/MS conditions The chromatographic conditions were adapted from a method developed for the separation of sulfonylurea herbicides by Polati et al. (2006). The stationary phase was an endcapped LiChrospher column RP-18 (5 μm, 250×4 mm i.d.) with a 15×4-mm Lichrospher RP-18 (5 μm) guard precolumn (Merck, Darmstadt, Germany). The mobile phase was a mixture 55/45 % v/v of acetonitrile and acidified water (0.05 % v/v of acetic acid) at a flow rate of 0.5 ml min−1. Diode array detector acquisitions were performed in the range 200–600 nm. Mass spectrometry conditions for both ESI and APCI analyses are reported in Supplementary Information 1 and

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the full scan data acquisitions were made in the m/z range 50–1,000, recording 3 microscans and with an inlet time of 50 ms. High purity nitrogen gas was used as nebuliser, and helium (>99.999 %) was used as quenching agent. Degradation and UV–vis spectroscopy conditions The photodegradation studies were performed by a solar box equipped with a xenon lamp set at 260 W m−2 at a temperature of 30 °C and equipped with an outdoor UV filter. These conditions were the result of a study, carried out in our laboratory, that compared experiments performed on solutions irradiated with sunlight and with the solar box (Polati et al. 2008). The results presented in the previous study showed that sunlight and solar box irradiation at the selected conditions produce similar results (Polati et al. 2008): the solar box is used instead of sunlight to provide a constant, continuous and more reproducible irradiation and temperature. The irradiance level and temperature were chosen to provide an irradiation and a temperature comparable to those naturally occurring during summer at a latitude corresponding to Northern Italy: the values were, therefore, derived by the average irradiance and temperature recorded in the period from May to September by the meteorological station present at our department. Herbicide solution (10.00 ml of 5.00 mg L−1) in ultrapure water were introduced in quartz cells and exposed to solar box irradiation for fixed exposure times (0, 1, 2, 5, 7, 10, 12, 15, 18, 20, 25, 28 and 34 days). The xenon lamp emission covers the entire solar spectrum from 290 to 800 nm. Evaluation of the contribution of temperature and hydrolysis to degradation: experimental conditions An evaluation of the contribution of temperature and hydrolysis to degradation was also carried out. To this purpose, 10.00 ml of 5.00 mg L−1 herbicide solution in ultrapure water were introduced in quartz cells protected by an aluminium foil and put (1) in the dark and at a temperature of 21±1 °C (condition A) to evaluate if hydrolysis contributes to degradation and (2) in the dark and at a temperature of 30±1 and 70±1 °C (conditions B and C, respectively) to evaluate if an increase of temperature contributes to degradation. The contribution of hydrolysis and temperature to degradation was evaluated in terms of the possible presence of degradation products in the solutions treated by conditions A, B and C. The temperature corresponding to condition C was selected since it corresponds to a temperature above twofold that corresponding to the real conditions adopted in the study; moreover, 70 °C represents an extreme condition usually never reached in real situations. Temperature control was obtained by a thermostatic bath. The evolution of degradation due to hydrolysis and temperature was tested at the same

time points chosen for the photodegradation study; after 34 days, the degradation products derived from hydrolysis and temperature were monitored up to 6 months of treatment by weekly measurements. All experiments were carried out in triplicate. No sample pretreatment was applied to prevent analyte and degradation product losses. The overall study was carried out using MilliQ water as solvent, mainly to work in experimental conditions identical to those already applied in previous works by our research group (Polati et al. 2008; Benzi et al. 2011) to make the results strictly comparable. Spectroscopic analyses were performed in the range 190–800 nm using Milli-Q water as reference solution. Chemical oxygen demand evaluation The chemical oxygen demand (COD) values were evaluated by the ISO6060:1989 regulation on the aqueous AMIDO solution at an initial concentration of 5.00 mg L−1 both photoirradiated and preserved in the dark at two different time points: at t0 and after 34 days. Analyses were carried out in triplicate. Ecotoxicological tests The acute toxicity test by Vibrio fischeri was performed on AMIDO solutions dissolved in ultrapure water at an initial concentration of 5.00 mg L−1 in two different experimental conditions: not irradiated (t0) and irradiated by the solar box for 600 min (a time greater than t1/2). Both experiments were repeated three times. V. fischeri is a bioluminescent gram-negative bacterium living in seawaters: the monitored effect consists in the decrease of bioluminescence due to the inhibition of luciferase. Luminescence is recorded after 15 min of incubation at 15 °C according to the IRSA Method n. 8030 (2003). This test ensures repeatability, accuracy and sensitivity as reported in the D.Lgs 152/2006. The toxic effect depends on the pollutant concentration and is due to the interaction between the molecular structure of the pollutant and the bioindicator or specific parts of its organism, when they are put in contact for prefixed times. Since bacterial bioluminescence is directly related to cellular respiration, a decrease in luminescence indicates cellular toxicity.

Results Solubility and calibration curves Literature reports that AMIDO solubility in water at 25.0 °C is 0.09 g L−1 (The E-Pesticide Manual, Version 3.2 2005– 06), but in our experiments, the maximum solubility of the

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analyte in ultrapure water is drastically lower. The maximum solubility of the analyte in ultrapure water was evaluated by comparison of the chromatographic peak area of aqueous solutions at increasing concentration levels with the one predicted by the calibration curve (R2 =0.9988) of the analyte built on the solutions prepared in acetonitrile (in the range 0.50–10.00 mg L−1), in which AMIDO is completely soluble: the analyte proved to be completely soluble in ultrapure water up to a concentration level of 5.00 mg L−1. The calibration curve of AMIDO in ultrapure water was, therefore, built in the concentration range 0.05–5.00 mg L−1 using the peak area of the extracted chromatogram at 240 nm from diode array detector data (Fig. 2 reports the chromatograms at different exposure times; AMIDO elutes at 14.22 min). The calibration curve was determined by three replications of each standard (standard solutions were prepared with concentrations of 0.05, 0.10, 0.50, 1.00, 2.00, 3.00 and 5.00 mg L−1). The calibration curve (y=(18.9±5.0)×103 +(230.8±2.1)×103x) was satisfactory, since the R2 value obtained was 0.9984 (Freg =12,127; Fig. 2 HPLC-DAD chromatogram extracted at 240 nm at different exposure times; AMIDO elutes at 14.2 min

Fcrit(!=0.05; ν1=1; ν2=19) =4.38) and no lack of fit was identified by ANOVA (Flof =1.84; Fcrit(!=0.05; ν1=5; ν2=14) =2.96). This calibration curve was used to quantify AMIDO during degradation and, therefore, to evaluate the extent of the degradation process. Photodegradation study The photodegradation of AMIDO was investigated by HPLC-DAD-MS/MS analyses on herbicide solutions in ultrapure water at a concentration level of 5.00 mg L−1, photoirradiated in a solar box for a maximum of 34 days. As for temperature and irradiation levels, totally aqueous solutions of the analyte were used to adhere to natural conditions and to study the direct photolysis. To obtain a first evidence of the degradation of the analyte at the experimental conditions considered, UV–vis absorbance spectra at different exposure times (from 0 to 34 days) were measured in the range from 190 to 600 nm. The UV–vis 14.2

1000000

800000

t= 0 days

4.9

600000 400000

2.9

200000 0 1000000

800000

t= 18 days

4.9

600000 5.6

400000 200000

10.7

4.3

14.2

0 1000000

800000

t= 34 days

5.2

600000

4.9

400000 200000 3.8

14.4

0

time (min)

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spectra of AMIDO are reported in Fig. SI. The spectra show that, before irradiation (0 days), a maximum absorbance at 244 nm is present; after 14 days of photoirradiation, a decrease of this band and a contemporary increase of the band at 205 nm are observed. After 34 days of irradiation, both the analyte band at 244 nm and the one at 205 nm totally disappear; two new bands characterised by a maximum l of 223 and 258 nm, respectively, appear. The results point out that degradation occurs (the band at 244 nm decreases) and degradation products are formed (the band at 205 nm increases). It is also possible to argue that some degradation products are also present, probably as impurities, in the standard solution, since a band at 205 nm is visible also in the spectrum at 0 days of irradiation. After a long period of irradiation instead (34 days), the UV spectrum shows the appearance of new degradation species (bands at 223 and 258 nm). By the application of the same method as in Benzi et al. (2011), comparing the results obtained by UV–vis spectra recorded for the solutions kept in the dark with conditions A, B and C (as described in the “Experimental” section), we found by preliminary analyses that: 1. An appreciable contribution of temperature to the degradation is registered only at 70 °C. Figure 3 shows the comparison of the UV spectra obtained for the three solutions of AMIDO kept in the dark at 21, 30 and 70 °C for 15 days: while the spectra at 21 and 30 °C show no significant variations in absorption, the one recorded at 70 °C shows a drastic decrease of absorbance at 240 nm, showing a clear degradation of the analyte. This effect is present only after 15 days of exposure at 70 °C. It is, therefore, possible to state that photodegradation was not

0,4

0,3

affected in the conditions adopted in the present study (namely, 30 °C) by a relevant contribution of hydrolysis and temperature, but it is important to point out that a strict control of the temperature during the irradiation experiments is necessary to avoid the synergistic effect of temperature and photoirradiation. 2. The degradation process results in a first order kinetic reaction with a t1/2 value of 276 h (11.5 days) and a kinetic constant of 0.0027 h−1. Figure 4 reports the linear decrease of the concentration level of AMIDO along with time on a logarithmic scale. ESI and APCI ion sources in both positive ion (PI) and negative ion (NI) modes were used to obtain complementary information about the degradation products that can form; moreover, a study of the chromatogram acquired in total ion current (TIC) followed by the extraction of the selected ion monitoring (SIM) chromatograms of the most intense m/z signals have enabled the identification of the possible photodegradation transformation products. Figures 5 and 6 report the results obtained by this approach: six m/z values corresponding to possible degradation products were evidenced. The proposed structures were obtained using the molecular masses identified in the mass spectra from the pseudo-molecular ions according to the nitrogen rule, the UV–vis data collected by the diode array detector and the spectrophotometric analyses. When possible, the structures proposed were confirmed by MSn experiments performed by the direct infusion in the mass analyser of the solutions exposed to photodegradation. At regular time intervals (0, 1, 2, 5, 7, 10, 12, 15, 18, 20, 25, 28 and 34 days), the concentration of the analyte in the photoirradiated solution was evaluated by the area of its chromatographic peak. Since the chromatograms here presented were recorded in both PI and NI modes, all figures report the mass values expressed as u rather than in m/z values. First, it can be noticed that, at about 5 min, different coeluting peaks are present, characterised by the following mass values: 299, 331, 198 and 218 u (Fig. 5a–d). In particular, a peak with retention time of about 4.6 min (Fig. 5a) is

0,2

t1/2 = 11.5 days k = 0.0027h-1

Abs

2 T= 35°C T= 70 °C standard

0,1

1,5

In[A]

1

0,0

200

0,5 0

250

300

350

400

λ (nm)

-0,5

0

200

400

600

800

1000

-1 Irradiation time (hours)

Fig. 3 Effect of hydrolysis and temperature. UV spectra obtained for three solutions of AMIDO at 5.00 mgL−1 kept in the dark at 21±1, 35±1 and 70±1 °C, respectively, for 15 days. Spectra are shown in the range 200–400 nm

Fig. 4 Graphical representation of ln[A] (concentration of AMIDO in milligrammes per litre; y axis) versus the irradiation time (expressed in hours of exposure; x axis)

Author's personal copy Environ Sci Pollut Res Fig. 5 HPLC-MS chromatograms extracted from the TIC in SIM mode of the transformation products (M1–M3) (c, e, f) and the impurities identified (I1–I3) (a, b, d)

a b

I1 299 u

I2 331 u.

m/z 298 SIM ESI ( - ) I = 5.98 E5 m/z 332 SIM ESI (+) I = 9,65 E4 m/z 199 SIM ESI (+) I = 2,89 E6

c

M1 198 u

d

I3 218 u.

m/z 219 SIM APCI (+) I = 4,23 E5

e

M2 155 u

m/z 156 SIM APCI (+) I = 4,06 E6

f

M3 294 u

identified by the extraction of the m/z 298 from the PI-ESI chromatogram: the peak was already present in nondegraded solutions (t0) and increases during the first 8 days of irradiation, then decreases along with irradiation but does not disappear even after 34 days of exposure. The same behaviour characterises the signal corresponding to the fragment at m/z 332 (Fig. 5b) that, in HPLC-PI ESI chromatograms, defines a peak at retention time of about 4.8 min. For these two fragments, called I1 and I2 respectively, we suppose a double origin: in part, they were already present in the standard solution of AMIDO as impurities (they are present at t0), but they also formed during the first step of the photodegradation process (first 8 days of treatment); then, their content decreases since they are partially transformed in minor fragments. The proposed structures for I1 and I2 possibly derive from the opening of the pyrimidinic ring and were confirmed by MS/MS analyses (Fig. 5). The signal at m/z 199 evidenced in the HPLC-PI APCI chromatogram (Fig. 5c) is already present at t0 and its intensity increases along with irradiation; again, this fragment seems to be ascribed to an impurity already present in the standard solution (t0) but it also originates from the degradation of AMIDO along with the entire photodegradation experiment. This degradation product (M1) derives from the loss of R1 and part of the R2 moieties (Fig. 6). By comparing the peak areas along the photoirradiation process and the proposed molecular structures, it can be supposed that I2 is probably transformed into the M1 degradation product; in fact, when I2 starts to decrease, M1 increases.

m/z 293 SIM ESI ( - ) I = 8,73 E6

The last co-eluting signal at about 5.0 min is characterised by an m/z value of 219 (I3) and is well identified by the PI ESI chromatogram (Fig. 5d); it can be surely assigned to an impurity since the corresponding peak is already present in the AMIDO standard solution, and its area decreases along with the irradiation time. A possible degradation product (M2) was found in both the NI/PI ESI and in PI APCI chromatograms (Fig. 5e) by the extraction of the m/z values corresponding to 155 u as a peak at a retention time of about 9.7 min that is visible after 24 h of irradiation; its area increases along with photoirradiation. The structure proposed is also confirmed by the MS/MS experiment that shows the formation of the fragment at 99 u, compatible with the mentioned assignment (Fig. 6). This degradation product is acknowledged as a typical molecule that derives from the degradation of sulfonylurea herbicides by hydrolysis (Sarmah and Sabadie 2002; Benzi et al. 2011). The last possible degradation product (M3) was characterised by a signal found in NI ESI at m/z 293. This signal is visible after 48 h of photoirradiation with a retention time of about 17.2 min (Fig. 4). The structure proposed derives from the loss of the NH–CH3 and SO2–CH3 groups and the insertion of an O–CH3 group. This structure is also confirmed by MS/MS analyses, thanks to the signals at 222 and 237 u (Fig. 6). Hydrolysis does not occur significantly, since after 4 months of preservation in the dark, only 10 % of the analyte degrades and the typical hydrolysis product is formed, deriving from the breaking of the N–C bond of the ureic part of the molecule that corresponds to the M2 degradation product identified during photodegradation (Fig. 6).

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299 u I1 O

O

331 u I2

HC

NH

CH3

CH2

NH

O

O

C CH

N

N

S

N

H

H

O

CH3 O

S

CH3

198 u CH3O

R2

O

N

C

N CH3O

O

R1

O

S

N

N

S

N

H

H

O

CH3

CH3

M1

O

218 u I3 MS2

155 u M2

H4C2

C N CH2 OCH3

99 u

O

O

MS2

H S N

C

O H

294 u

NH N C N C CH3 H

OCH3 2 2 ua.m..

M3

OH

O

MS2

OCH3

S

N

C

H

H H

N C N C

H O CH3 H H

C

CH2

237u.m.a.

Fig. 6 Proposed structures and MS/MS characterisation of the transformation products of amidosulfuron photodegradation

For what concerns the impurity already present in the standard and pointed out so far, the standard was checked and its presence was confirmed. The COD values obtained from the AMIDO solutions that underwent photoirradiation for 34 days and were preserved in the dark for the same time confirmed that a complete mineralisation is not achieved, since about 22 and 93 % of the initial oxidizable species remain, respectively. In addition, ecotoxicological tests using V. fischeri were performed according to Benzi et al. (2011); the I% values obtained indicate the percentage of inhibition induced by the chemicals on the activity of the organisms: I% ranging between 0 and 20 corresponds to a non-toxic sample, while the sample is weakly toxic for values between 20 and 50 and toxic for values >50. The I% values obtained were 16.90±0.40 for the solution preserved in the dark and 40.50±0.70 for the photoirradiated solution; the results indicate that the pesticide solution at the initial concentration seems not particularly toxic but reaches a weakly toxic level after irradiation.

Discussion It is common opinion that different photodegradation processes can occur within the sulfonylurea family, depending on the chemical structure of the two chromophoric groups of the molecule under examination (Morrica et al. 2004). In addition, the experimental conditions adopted (temperature, pH, solvent, etc.) can deeply influence the degradation pathway: this makes difficult the comparison of the results obtained by different research groups, usually exploiting heterogeneous experimental approaches, to draw common and general considerations on the fragmentation patterns of this class of herbicides. Here, the photodegradation of AMIDO was studied by the same experimental strategy already applied by our research group on nicosulfuron (NICO) (Benzi et al. 2011) and tribenuron-methyl (TRIBE) (Polati et al. 2008). Since the three herbicides investigated cover all the possible classes of sulfonylurea pesticide structures (Sarmah and Sabadie 2002), it is possible to draw considerations on the effect played by different

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substituents on the photodegradation products obtained by comparing the results obtained in the three studies. The molecular structures of the three pesticides differ especially for the R1 moiety (Table 1) that is aliphatic for AMIDO, aromatic for TRIBE and heterocyclic for NICO. The degradation products that preserve the R1 moiety regard only NICO and TRIBE (N5 and T2) and are characterised by a cyclic structure resulting from the breaking of the same N–C bond of the sulfonylureic bridge associated to the loss of the R2 group. AMIDO does not form this kind of degradation product, maybe owing to the presence in the structure of an aliphatic moiety that is characterised by a lower stability. The R2 portion is the same pyrimidinic group in AMIDO and NICO, while it is a triazinic group in TRIBE; in our studies, a part from the principal hydrolysis products (A1, N1 and T1), only NICO and AMIDO show degradation products with R2 in the final structure (A2, N2 and N4), whereas no degradation products of this kind are formed for TRIBE: this behaviour could be ascribed to a larger reactivity of the triazinic group with respect to the pyrimidinic group. One of the degradation products formed is identical for AMIDO and NICO (A2 and N2) and derives from the

loss of the R1 group by the breaking of the S–N bond and the insertion of a –OCH3 group; another typical degradation product is formed by the C–N bonds, the breaking of the R2 ring and the retention of R1 for NICO (N3) and loss of R1 for AMIDO (A3). Finally, a comparison of our results with those reported in the literature in similar works was performed with the aim of point out if preferential degradation pathways exist. A study performed by Braschi et al. (2000) concerns primisulfuron (a class a sulfonylurea herbicide) degraded in several ways, including hydrolysis and photolysis. In the hydrolysis experiment, the typical degradation product that derives from the breaking of the N–C bond of the ureic part of the molecule is formed, while in the presence of UV radiation, a degradation product forms similar to the M3 identified for AMIDO and deriving from the breaking of the S–N bond, the main difference being the insertion of –OR groups, detected only in our study. This different behaviour could be ascribed to the different polarities of the solvents, acting on radical formation, since the analyte was dissolved in acetonitrile/water (80:20) in the work from Braschi et al. (2000), while water was used in our case.

Table 1 Molecular structures and their photodegradation products of amidosulfuron, tribenuron-methyl and nicosulfuron CH3O

R2

O

N

C N

N

H

O

R1

O

CH3

S N H

S O

N

CH3O

R2

O

N

C

O

CH3

N

CH3O

O

CH3

C

N

R1

O

CH3

CH3O

O

N

N

S

H

H

O

N

N

NICO

N

N

S

H

H

O

OCH3

N NH2

NH2

NH2

N

N

N

N

N1

CH3O

A1

N

O

C

N

N

N

S

H

H

O

O NH2 NH

C

-

OCH3

N2

CH3 O

A2

T1

CH3O

O

CH3 O

Loss of R1 group R2 C-N bond breaking

R1

CH3O N

N

Loss of R1 group S-N bond breaking Insertion of -OCH3

C O

TRIBE CH3O

CH3O

C

CH3O

CH3O

Hydrolysis

O

N N

CH3O

AMIDO

R2

O

CH3

C

N

CH3

O

C N

N

S

H

H

O

N3

N

A3 O

Dealkylation on R1 and R2 Ureic N-C bond break Loss of R2

O

HO N

-

N

N

N

S

NH2

H

H

O

N

CH3O

-

O

CH3

C

N

N4 O C

CH3

NH2

OCH3

O

O S

NH2 N

O

Ureic N-C bond break Loss of R2 Cyclization

C O

C

S

N5

T2

O O

-

-

C

HN O

S O

T3

Author's personal copy Environ Sci Pollut Res

For class b, Bhattacherjee and Dureja (1999a) found a strong fragmentation that led to the formation of many degradation products that only in part correspond to those found in our studies; this kind of strong fragmentation is reported also in other works characterised by the application of solvent extraction procedures on the photoirradiated solutions before the injection in the chromatographic system and/or by the use of organic solvents to dissolve the analytes (Bhattacherjee and Dureja 1999b, 2002; Bossi et al. 1999; Perreau et al. 2007). The experimental conditions were proven to play an important role on the fragmentation pattern observed, probably due to the effect played by the solvent on radical formation during photolysis or to its effect on the stability of the R1 and R2 groups or even to its effect on the mechanisms of fragment formation. For class c, Sabadie (2002) considers nicosulfuron deposited on minerals that have undergone hydrolysis and alcoholysis; the hydrolysis products correspond to those obtained by our group and obviously does not produce fragments with the insertion of –OR groups; alcoholysis instead corresponds to the formation of fragments characterised by the insertion of the alcoholic –OR group. Scrano et al. (1999) consider the different degradation pathways of rimsulfuron; they found two major and three minor photodegradation and hydrolysis degradation products: surprisingly, only the structures of the minor degradation products correspond to those identified for NICO in our work (Benzi et al. 2011), while no evidence of the formation of the two major degradation products was found. Also, in this case, we think that this difference could be attributed to different solvents used, since in the study of Scrano et al. (1999), the analyte stock solution was prepared in acetonitrile and not in water. Moreover, we noted that, when acetonitrile is used to dissolve the analyte, –OR groups are not found in the fragments, while water seems to have an opposite behaviour. In particular, the insertion of –OCH3 groups was detected in our studies, while –OR groups are inserted in the study of Sabadie (2002), according to the alcohol used during alcoholysis. All the experimental evidence discussed show clearly that the experimental conditions used in hydrolysis and photodegradation studies deeply influence the obtained fragmentation. In particular, the effect of the solvent used for dissolving the analyte seems relevant; however, it is important to point out that the amount of solvent used for this purpose has to be considered: effects due to solvent can probably be neglected if its concentration in the final samples analysed is low. In spite of the difficulties encountered in the attempt to draw general consideration on the natural photodegradation of sulfonylurea herbicides, it is important to point out that all the studies present in literature report that the complete mineralisation of the sample is never reached: the parent ions are only partially

degraded or, even when totally degraded, they are transformed into other organic compounds; in any case, if subjected to ecotoxicological tests, the degradation products have at least the same toxicity as the precursor herbicides.

Conclusions In this work, the photodegradation of amidosulfuron in aqueous solutions was studied by an HPLC-DAD-MS/MS method that allowed the identification of three possible degradation products. Then, the results obtained from the photodegradation of three sulfonylurea herbicides, obtained in identical experimental conditions in our laboratories, were also used. This is the first time, to our knowledge, that the behaviour of different classes of sulfonylurea herbicides exposed to photodegradation processes can be compared in the same experimental conditions. The results obtained by our laboratory were also compared to those reported in the literature even if the quite different experimental conditions adopted during the degradation processes make the direct comparison of the results quite difficult. We can confirm here the hypotheses drawn by Morrica et al. (2004): the breaking of the sulfonylureic bridge is always detected, while fragments involving different bonds depend on the structures of the R1 and R2 groups. Above all, it can be pointed out that the choice of the experimental conditions applied during degradation deeply affect the degradation products: the use of organic solvents to obtain complete solubilization of the sample plays a fundamental role and deeply influences the degradation processes that, therefore, not always fully adhere to actual natural photodegradation pathways. Moreover, considerations about toxicity were driven since the complete mineralisation of the sample is not reached: even when the parent pesticides are totally degraded, they are, however, transformed into other organic compounds showing, if subject to ecotoxicological tests, at least the same toxicity of the precursor herbicides. The evidence here presented suggests that, at least for the class of sulfonylurea pesticides, their professed low persistence actually does not produce any real advantage. Acknowledgements The authors gratefully acknowledge the financial support from AATF (Associazione Ambiente-Territorio e Formazione, Alessandria, Italy), from Regione Piemonte, Direzione Igiene e Sanità Pubblica (Turin, Italy) and from MIUR (Ministero Italiano Università e Ricerca, Rome, Italy).

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