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Monitoring of pesticides in river water based on samples previously stored in polymeric cartridges followed by on-line solid-phase extraction-liquid chromatography–diode array detection and confirmation by atmospheric pressure chemical ionization mass spectrometry
Analytica Chimica Acta 386 (1999) 237±248
Monitoring of pesticides in river water based on samples previously stored in polymeric cartridges followed by on-line solid-phase extraction-liquid chromatography±diode array detection and con®rmation by atmospheric pressure chemical ionization mass spectrometry C. Aguilara, I. Ferrerb, F. Borrulla, R.M. MarceÂa,*, D. BarceloÂb a
Department of Chemistry, Universitat Rovira i Virgili, Imperial Tarraco 1, 43005, Tarragona, Spain Department of Environmental Chemistry, CID-CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain
b
Received 21 September 1998; received in revised form 7 December 1998; accepted 30 December 1998
Abstract Solid-phase extraction coupled on-line with liquid chromatography±diode array detection was applied to the monitoring study of a group of pesticides and metabolites of different chemical groups in water samples from the Ebro delta area (Tarragona, Spain). Liquid chromatography±atmospheric pressure chemical ionisation-mass spectrometry was used for con®rmatory purposes. The developed method involves the preconcentration of 50 ml of water samples through styrene±divinylbenzene precolumns. The most frequently detected pesticides in the analysed samples were bentazone, molinate, metolachlor and the triazine herbicides simazine, atrazine and its dealkylated metabolites, deisopropilatrazine and deethylatrazine and, at concentration levels ranging from 0.03 to 2.4 mg lÿ1. The stability of several pesticides stored on styrene±divinylbenzene cartridges was also evaluated. The effect of different storage temperatures (room temperature, 48C and ÿ208C) and two storage periods (one week and three months) on the recovery of the pesticides were considered. In general, the recoveries were greater than 90% after three months of storage at ÿ208C on the polymeric cartridges. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Polymeric cartridges; Ebro delta; Water; Pesticides; APCI
1. Introduction Pesticides can be applied directly or indirectly to increase crop yields and their residues may accumu-
*Corresponding author. Tel.: +34-977-55-81-70; fax: +34-97755-95-63; e-mail: [email protected]
late not only on crops, but also in surface waters [1]. Pesticides can also be transformed by physical, chemical and biological processes into one or more transformation products and some of them can be more toxic or persistent than the parent compound [2±5]. Hence, there is a need for analytical procedures for monitoring pesticides and their transformation products in environmental matrices [6,7].
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00033-1
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One of the main aspects to be considered when analysing pesticides is that sometimes samples must be stored for variable periods of time before analysis, so a storage space is needed. It is also essential to know how long the content of a sample may remain stable. In this respect, various studies have been carried out to analyse pesticides after their storage [8±10]. To maintain the integrity of samples containing pesticides until their analysis, different methods of storage have been developed [5,11±15] one of them is to preserve in solid-phase extraction (SPE) materials such as discs or cartridges [11,15±20]. This extraction technique has advantages such as the reduction in the storage space required compared to bottles of water and the fact that the analysis need not be performed immediately after sampling. The type of SPE material is one factor to take into account and although most stability studies have been carried out using C18 sorbent phases [17±20], the stability of pesticides in other kinds of phase such as polymers [21] and carbon [13] has also been tested. Some studies have shown that the stability of organic pollutants stored in SPE materials is, in general, better than when they are stored in a water matrix [18,20,22]. The pesticide extract is mainly analysed by means of gas (GC) or liquid chromatography (LC) coupled to different detectors, depending on the characteristics of the analytes. In the case of LC, diode array detectors (DAD) are extensively used for determining different kinds of pesticide [21,23,24], but in the last few years, the coupling of LC to mass spectrometry (MS) has become a good alternative to classical LC detectors for the unambiguous identi®cation of analytes [21,23,25]. This has been widely used to study the degradation pathways of pesticides [14,21,26]. The development of different interfaces, especially ones with atmospheric pressure ionisation (API) offers new opportunities for the determination of a wide range of pollutants [22,25,27]. The aims of this work were as follows: ®rstly, to develop a method for determining pesticides in water samples, secondly, to monitor the presence of pesticides in an area from the Ebro delta, one of the main agricultural areas in Spain, in order to evaluate the variation in the concentration levels of these contaminants for a four months period and to detect the presence of possible transformation products formed,
and, thirdly, to examine the stability of various pesticides at three different storage temperatures (ÿ208C, 48C and room temperature) for periods of one week or three months in a polymeric cartridge material. By studying stability, it will be possible to determine whether water samples can be stored for up to three months and then analysed all together. 2. Experimental 2.1. Chemicals The pesticides used were 98±99% pure. They were purchased from Riedel-de HaÈen (Seelze, Germany). Stock solutions of 1000 mg lÿ1 were prepared by weighing each of the solutes and dissolving them in HPLC-grade methanol (Scharlau, Barcelona, Spain). They were then stored in the refrigerator at 48C. A stock solution of 5 mg lÿ1 was used to spike water samples at mg lÿ1 level for the determination of the calibration graphs. Ultra-pure water was prepared by ultra®ltration with a Milli-Q water puri®cation system (Millipore, Bedford, MA, USA). Acetic and hydrochloric acid were from Probus (Badalona, Barcelona, Spain). Sodium chloride was also from Probus. Helium for degassing the LC solvents was 99.995% pure and supplied by Carburos MetaÂlicos (Tarragona, Spain). Nitrogen for the APCI interface was 99.998% pure and it was supplied by Air Liquide (Barcelona, Spain). 2.2. Apparatus 2.2.1. LC±DAD LC analyses were performed with an HP 1090 Series II liquid chromatograph (Hewlett-Packard, Palo Alto, CA, USA), equipped with a ternary solventdelivery system, an injection valve with a 25 ml loop and an HP 1040M diode array detector. The chromatographic separation was performed with a Spherisorb ODS-2 column (200.46 cm i.d., 5 mm particle size) (Teknokroma, Barcelona, Spain). The gradient elution was carried out with a binary gradient composed of LC solvent A (methanol) and LC solvent B (water acidi®ed to pH 4.5 by adding acetic acid) according to the following programme: 10% A to 50% A in 20 min,
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then to 70% A in 24 min and to 100% A in 1 min. After 1 min at 100% methanol, the mobile phase was returned to the initial conditions and, after 10 min of stabilisation, the next sample was injected. The ¯ow rate was 1 ml minÿ1. For multi-wavelength monitoring, the DAD was set at 240, 254 and 280 nm with a bandwidth of 4 nm. Absorbance spectra were recorded from 200 to 400 nm. Chromatographic data were collected and recorded using the HP 79994A Workstation and quanti®cation was carried out at the maximum absorbance value for each compound. 2.2.2. LC±APCI±MS The eluent was delivered by a gradient system from Waters 616 pumps controlled by a Waters 600S (Waters, Mildford, MA, USA). A VG Platform from Fisons Instruments (Manchester, UK), equipped with an APCI interface was used. The APCI interface consists of a heater nebulizer probe and the standard atmospheric pressure source was equipped with a corona discharge pin. The operating parameters were a drying gas ¯ow rate of 250±300 l hÿ1 and a nebulizing gas ¯ow rate of 10 l hÿ1. The cone voltage was set at 20 and 40 V and the corona voltage at 3.5 kV. The ion source and probe temperatures were set at 1508C and 4008C, respectively. LC±APCI±MS was used under full-scan mode and the scan range was from 70 to 400 m/z in the positive ion (PI) mode. The instrument control and data processing utilities included MegaLinx software. The chromatographic conditions, ¯ow rate and gradient, were the same as those reported above for LC±DAD. 2.3. Procedures Trace enrichment was performed on a 102.0 mm cartridge packed with styrene±divinylbenzene copolymer (Spark Holland, Emmen, Netherlands). An Applied Biosystems (Ramsey, USA) pump was used to deliver the sample and to condition the precolumn. For the stability study in the SPE cartridges, 50 ml of Milli-Q water (adjusted at pH 2 and with 5 g lÿ1 of NaCl added) spiked to 5 mg lÿ1 with a solution which contains each pesticide was preconcentrated on the precolumns of styrene±divinylbenzene and stored at ÿ208C, 48C and at a room temperature (RT) for a period of one week and three months. For each of the
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storage conditions four cartridges were stored. After this period of time, the retained analytes were desorbed by placing the precolumns in a holder and starting the mobile phase gradient in the back¯ush mode. Before analysis, the cartridges were thawed for a period which depends on the storage conditions in order to remove the frozen water matrix involving the stationary phase. Afterwards, these samples were analysed using the protocol described above and for each of the storage conditions two replicate analyses were performed for DAD. For the monitoring study, several aliquots were collected from ®ve different sampling sites in the Ebro delta area once a month from April to July, when pesticide application to crops was greatest, and four cartridges of each sample was stored each month. The sample sites were selected in order to give overall information about the quality of the water. Hydrochloric acid was added to the samples immediately after collection in order to minimise possible biological degradation and they were also ®ltered through a 0.45 mm PTFE ®lter (Millipore) to remove particulate matter. Then, a 50 ml water sample was preconcentrated at a ¯ow rate of 4 ml minÿ1 and the cartridges were stored at ÿ208C until their analysis. For these samples the analytes trapped on the cartridges were eluted in the same way as for the stability study. 3. Results and discussion 3.1. General remarks The analytes monitored in this study included 10 pesticides (aldicarb, atrazine, bentazone, carbaryl, carbofuran, cyanazine, malathion, metolachlor, molinate and simazine) and three metabolites (deethylatrazine (DEA), deisopropilatrazine (DIA) and malaoxon). They were chosen to cover a broad range of chemical classes and because they are commonly detected in surface waters of the studied area and also because they are on the priority lists of pesticides of the European Community [2]. The use of a DAD detector made it possible for all analytes to be determined at the wavelengths of maximum absorbance in order to obtain an improvement in sensitivity and selectivity. The analytes were iden-
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Table 1 Wavelengths used to measure absorbance, molecular weight, main ions obtained under APCI±MS under PI mode of operation at a cone voltage of 20 and 40 V, linearity range, correlation coefficients, r2, and limits of detection (LODs) obtained by on-line SPE±LC±DAD for all target compounds Pesticide
Deisopropilatrazine (DEA) Bentazone Deethylatrazine (DIA) Aldicarb Cyanazine Carbofuran Simazine Carbaryl Malaoxon Atrazine Malathion Molinate Metolachlor
(nm)
240 240 240 254 240 280 240 280 280 240 280 240 254
MW
187 240 173 190 241 221 202 201 314 215 330 187 284
m/z (V) 20
40
174 120 188 116 241 222 202 145 127 216 285 188 284
96 120 146 89 216 165 132 145 127 174 127 126 252
ti®ed by comparing the absorption spectra and retention times of each compound with those of standard solutions. Table 1 shows the wavelengths used for the quanti®cation of each analyte. In order to con®rm the data obtained from DAD detection, samples were also analysed by means of LC±APCI±MS. The operational conditions were optimised previously [25]. Table 1 shows the molecular weight of each analyte and the major ions obtained, under full-scan conditions using the positive ion (PI) mode of acquisition at cone voltages of 20 and 40 V, by ¯ow-injection analysis of the pesticides at a concentration of 50 mg lÿ1 into a carrier stream of methanol/water (with the addition of acetic acid). The corresponding fragments to the ions shown in this table are widely explained in a previous paper [25]. 3.2. On-line SPE Preconcentration was performed by on-line SPE on styrene±divinylbenzene copolymer. This stationary phase was selected taking into account previous results obtained using this phase and C18. In general, retention was higher when the polymeric sorbent was used, particularly for medium and highly polar pesticides [20,21,23±25]. The different parameters which can affect the response for the on-line SPE procedure were opti-
Linearity range (mg lÿ1)
r2
LOD (mg lÿ1)
0.1±10 0.1±10 0.05±10 0.1±10 0.025±10 0.1±10 0.025±10 0.1±10 0.2±10 0.025±10 0.2±10 0.1±10 0.1±10
0.996 0.995 0.999 0.997 0.999 0.998 0.996 0.996 0.996 0.997 0.999 0.999 0.998
0.04 0.04 0.02 0.04 0.01 0.04 0.01 0.04 0.10 0.01 0.10 0.04 0.04
mised, so the effect of the sample pH and the addition of sodium chloride on the recoveries of the analytes were evaluated. Initially, the in¯uence of sample pH was studied by analysing samples at different pH values. For the most polar compounds, such as bentazone, DEA and DIA, and under acidic conditions, recoveries were greater than those for neutral samples and consequently for further studies the samples were acidi®ed to pH 2 by adding hydrochloric acid before being preconcentrated. The effect of adding salt was also tested by adding different amounts of NaCl to the sample. For some compounds, particularly the organophosphorous pesticides, the addition of 5 g lÿ1 of sodium chloride enhanced the recovery as had previously been reported [24]. The breakthrough volumes of the pesticides were obtained by percolating different sample volumes, between 25 and 100 ml, of Milli-Q water acidi®ed at pH 2 and with the addition of NaCl through the styrene±divinylbenzene precolumns. The recoveries obtained for each pesticide after the preconcentration of 25, 50 and 100 ml of Milli-Q water sample spiked at the 5 mg lÿ1 level are shown in Table 2. Sample volumes higher than 50 ml gave losses for the two dealkylated metabolites of atrazine, DEA and DIA, so all pesticides were determined and quanti®ed by preconcentrating a volume of 50 ml for which the recov-
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Table 2 Mean recoveries and relative standard deviation (RSD) (n5) of pesticides at the 5 mg lÿ1 level in Milli-Q water at different sample volumes acidified to pH 2 and with the addition of 5 g lÿ1 of NaCl Pesticide
Deisopropilatrazine(DEA) Bentazone Deethylatrazine (DIA) Aldicarb Cyanazine Carbofuran Simazine Carbaryl Malaoxon Atrazine Malathion Molinate Metolachlor
25 ml
50 ml
100 ml
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
83 90 82 89 92 81 94 99 79 109 82 90 91
8 9 12 6 7 9 11 9 8 7 5 4 9
78 89 75 86 97 78 94 96 80 99 83 89 87
11 8 8 5 7 12 7 4 6 11 8 7 9
53 76 48 74 92 76 97 91 73 92 71 75 77
5 7 9 11 10 9 6 5 9 8 7 9 12
eries for most of the analytes were between 80% and 100%. 3.3. Quality parameters Under optimum conditions of the calibration plots were constructed by percolating 50 ml of Milli-Q water spiked with the pesticides at levels between 0.025 and 10 mg lÿ1 using on-line SPE± LC±DAD and quantifying each compound at its maximum wavelength. The limits of detection (LODs) were calculated by using a signal-to-noise ratio of 3. The results are shown in Table 1. The linearity was also checked for river Ebro water and the corresponding results were similar as reported for Milli-Q water. In order to study the repeatability of the method, river samples spiked at a level of 5 mg lÿ1 were preconcentrated and the relative standard deviation (n5) was below 10% for all the analytes. 3.4. Stability study Pesticides can be degraded during storage by such processes as hydrolysis or microbial decomposition. Storage conditions can affect the stability of pesticides in the precolumns; different temperatures and periods of time were studied in order to evaluate the optimum conditions for storing the pesticides on the styrene± divinylbenzene cartridges.
For each storage time and temperature two samples were analysed by the DAD and ®nally the recovery for each pesticide was evaluated by comparing the recoveries obtained after preconcentration of 50 ml of Milli-Q water sample spiked at the 5 mg lÿ1 level. The RSD of one of the storing conditions (48C and three months) was checked and was lower than 9% for all the analytes (n3). 3.4.1. Storage at ÿ208C The precolumns kept at ÿ208C were defrosted for 4±6 h at room temperature before the retained analytes were eluted, because previous studies [20] have demonstrated that if the extraction process is carried out immediately after the cartridge is removed from the freezer, the quanti®cation and identi®cation of the compounds is dif®cult. The recoveries of each pesticide after storing the precolumns for one week and three months at ÿ208C are shown in Table 3. It can be seen that for all compounds the recoveries were between 85% and 100% at this low temperature independently of the storage time. No parent compounds were lost during the period of time studied because the pesticides did not degrade and were completely recovered even after three months of storage. 3.4.2. Storage at 48C The precolumns stored at 48C were kept at room temperature for 1 h before their elution. Table 3 shows
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Table 3 Recovery values (%) of the pesticides after different storage times at ÿ208C, 48C and room temperature (RT) obtained after a 50 ml volume of Milli-Q water spiked to 5 mg lÿ1 level with each pesticide was loaded on the PLRP-S cartridge Pesticide
DEA Bentazone DIA Aldicarb Cyanazine Carbofuran Simazine Carbaryl Malaoxon Atrazine Malathion Molinate Metolachlor
ÿ208C
48C
One week
Three months
98 102 97 94 102 107 96 102 93 109 97 94 107
81 82 94 81 87 93 89 87 90 95 93 90 94
RT
One week 92 89 93 96 97 84 94 98 91 102 92 101 97
the results of studying the stability of pesticides stored at this temperature. These results show that losses were greater (20±30%) after the three month period for carbofuran, as was previously reported by other authors who used C18 disks for the storage [16]. For one week of storage recovery values were higher than 84% for all the compounds studied, so these storage conditions are feasible for the pesticides evaluated. 3.4.3. Room temperature Losses at room temperature (RT) were quite important for some of the target compounds even after short periods of storage. For the ®rst week of storage under this temperature most of the pesticides were stable whereas losses were as high as 30% for aldicarb, carbaryl and bentazone and as high as 40% for carbofuran. Consequently, samples containing those pesticides have to be stored under low temperatures. These results indicated that the N-methylcarbamate pesticides aldicarb, carbaryl and carbofuran were the least stable of the pesticides included in this study since they were seriously affected by the storage conditions. Their recoveries tend to reduce with the storage temperature, as previously reported using C18 as the SPE sorbent [16]. The length of storage time at room temperature also affected several compounds such as malathion or some triazine compounds such as deethylatrazine and atrazine for which the recovery decreases between
Three months
One week
Three months
89 82 88 78 92 72 92 94 86 93 88 89 92
89 78 83 72 93 62 90 74 84 94 87 92 91
77 62 75 63 90 43 88 71 87 85 79 86 87
10% and 15% as storage time increases. These results are in agreement with the data on the literature on malathion [3,19,20] and the triazine herbicides atrazine, deethylatrazine or simazine [14,17,18] although the sorbent used is C18. In the chromatograms obtained after the elution of the cartridges stored at RT, the same peaks obtained for 48C appeared. By comparing all the data of the different storage conditions, it can be concluded that the most reliable method for storing pesticides in SPE sorbents is to keep them at ÿ208C because the integrity of most of the analytes is not affected even after long storage periods; these results are in agreement with previous results [3,15]. However, storage at 48C is also feasible for most of the compounds. Fig. 1 shows the chromatograms corresponding to the preconcentration of 50 ml of Milli-Q water spiked with the pesticides at the 5 mg lÿ1 level and obtained after storage at ÿ208C, 48C and room temperature for three months. 3.5. LC±APCI±MS A peak appears very close to atrazine in all the DAD chromatograms obtained after the elution of the cartridges stored at any conditions. In order to obtain more structural information about the interfering peak, some of the cartridges were analysed by coupling them on-line with the LC±APCI±MS system under
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243
Fig. 1. HPLC±DAD chromatograms at 240 nm obtained after the on-line preconcentration of 50 ml of Milli-Q water after storing the PLRP-S cartridges with the pesticides for a period of three months at: (a) RT, (b) 48C and (c) ÿ208C. Peaks1: Deisopropilatrazine (DEA); 2: Bentazone; 3: Deethylatrazine (DIA); 4: Aldicarb; 5: Cyanazine; 6: Carbofuran; 7: Simazine; 8: Carbaryl; 9: Malaoxon; 10: Atrazine; 11: Malathion; 12: Molinate; 13: Metolachlor.
full-scan conditions and positive acquisition. The unidenti®ed peak presented m/z 177 as its base peak and an ion at m/z 149 under the experimental conditions; in order to obtain more structural information an extraction voltage of 40 V was used to induce greater fragmentation but no additional information was obtained because the only difference between this spectrum and the one obtained for 20 V was the relative abundance of ions, the base peak being the ion at m/z 149. The peak was tentatively identi®ed as a phthalate compound since the ion at m/z 149 is a typical fragment of this kind of compound. Likewise, the ion at m/z 177 is the reported base peak for many phthalate esters and the ion at m/z 149 is also a typical peak for these compounds in their spectra obtained by
electron impact ionisation [28]. With all this information the peak could be tentatively identi®ed as diethyl phthalate or diethyl ter-phthalate since their molecular weight was 222 and in the spectrum obtained at 20 V there was an ion at m/z 223 which can be assigned to [MH] and an ion at m/z 245 corresponding to [MNa]. The presence of a phthalate ester in the samples could be due to the use of plasticware or tubing that often contains phthalate esters as plasticizers [28]. Another small peak at a retention time of about 8.5 min appeared in the chromatograms obtained under 48C and the samples were also analysed by on-line SPE±LC±APCI±MS method under full-scan conditions in the positive ion mode. The correspond-
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ing spectra showed an ion at m/z 132 as the base peak and other ions at m/z 166 and 181. The comparison between its spectrum and the corresponding mass spectra of the analytes initially included in this study, obtained at a cone voltage of 40 V, showed that for the chlorotriazines DIA, simazine and atrazine, the ions at m/z 132 and 166 were also present, so the unknown peak was tentatively assigned to a transformation product of a chlorotriazine herbicide. The ion at m/z 132 is described in literarure [29] as a common peak for chlorotriazines with an ethyl substituent, such as those previously mentioned. 3.6. Monitoring study In order to determine the levels of pesticides in water samples from the Ebro delta, ®ve different sampling sites were established in the area. The samples, obtained from each of the sites, were collected monthly from April to July. The sampling sites were selected on the basis of monitoring zones
exposed to high agricultural activity since the Ebro river delta is a typical cultivation area where some pesticides are applied in large amounts [30]. The selected sampling points are shown in Fig. 2. First, the analyses were carried out by HPLC±DAD which provided a primary screening of the samples collected since the different peaks that appeared in the corresponding chromatograms were compared with the DAD spectra of the target compounds. LC±APCI± MS was used to con®rm the data obtained from DAD detection and also to identify the presence of possible unknowns which were found to be present in the samples. The data obtained in this study have been useful for determining the occurrence and temporal distribution of pesticides and their metabolites in the evaluated area. The results are shown in Table 4 and RSDs (n3) checked for river water were lower than 11 for all compounds. The results show that there are some differences during the sampling period and it can be pointed out that there is a relationship with the
Fig. 2. Map of delta Ebro area showing the location of sampling sites: (1) Ebro river, (2) Carrete channel (irrigation ditch which receives water from surronding fields, (3) Encanyissada lagoon (it receives irrigation channels from the rice fields, (4) a part of the Encanyissada lagoon which is closer to the sea, and (5) Tancada lagoon (it receives irrigation channels from the rice fields).
C. Aguilar et al. / Analytica Chimica Acta 386 (1999) 237±248 Table 4 Mean value for the pesticide concentration (mg lÿ1) over the period of study at each sampling point Compound
Months
Sampling point 1
2
3
4
5
Atrazine
April May June July
0.18 0.24 0.19 0.06
0.06 0.03 0.05 n.d.
n.d. 0.03 0.04 n.d.
n.d. n.d. 0.03 n.d.
n.d. n.d. n.d. n.d.
DIA
April May June July
0.15 0.23 0.14 0.10
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
DEA
April May June July
0.11 0.12 0.10 n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
Simazine
April May June July
0.14 1.06 0.13 0.05
0.07 0.07 0.04 n.d.
0.04 0.05 n.d. n.d.
n.d. 0.04 n.d. n.d.
n.d. 0.03 0.05 n.d.
Molinate
April May June July
n.d. 0.29 0.11 0.08
0.92 1.30 2.39 1.25
0.22 0.41 0.64 0.39
0.28 0.46 0.51 0.33
0.12 0.10 0.11 0.11
Bentazone
April May June July
n.d. n.d. n.d. n.d.
n.d. 0.32 0.20 0.11
n.d. 0.24 0.19 0.10
n.d. 0.10 0.10 n.d.
n.d. 0.22 n.d. n.d.
Metolachlor
April May June July
0.20 0.12 n.d. n.d.
n.d. n.d. 0.10 n.d.
0.22 0.12 n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d.: not detected.
increase of the agricultural activities in the area evaluated. The various pesticides which were found in the analysed samples belong to different chemical groups as a consequence of the variety of crops of the sampled area. The triazine herbicides, atrazine and simazine, and the thiocarbamate molinate were found in the greatest number of samples. Other pesticides such as metolachlor, bentazone and the dealkylated metabolites of atrazine also appeared in some samples. Pesticides were not distributed uniformly throughout the different sampling sites; whereas the concentration of the triazine herbicides was, in general,
245
higher in the river Ebro (sampling point 1), the concentration of molinate, bentazone or propanil was higher at the sampling points which were inside the delta. The presence of these pesticides in samples from the delta river water is not surprising because they are used in large amounts in this zone [30]. Atrazine is applied to various crops along the river and its presence in the river Ebro samples can be attributed to its transport from its application site as was previously found [12,25]. The presence of deethylatrazine and deisopropilatrazine in some of the samples can be attributed to the degradation of the triazine herbicides since in the Ebro river basin important amounts of some triazine herbicides are used. The other compounds found are typical rice pesticides and, of these, molinate was found to be the most used, being encountered at the highest concentration with respect the other pesticides. The maximum concentration levels for the typical rice herbicides were observed at sampling point 2. This is logical since this point receives waters from rice ®elds, where pesticides are being applied, while the lower concentrations at points 3, 4 and 5 are probably due to the dilution effect. As an example, Fig. 3 shows the chromatograms obtained for the period studied, from April to July, corresponding to the sampling point 2. Numerous unknowns were found to be present in the analysed samples and some peaks were tentatively identi®ed as the transformation products of pesticides. In recent years, considerable interest has been shown in the detection of pesticide metabolites, which are potentially toxic compounds and in this sense the use of mass spectrometry allows the presence of some compounds to be con®rmed. In this study, the presence of some degradation products of molinate was con®rmed by the corresponding mass spectra obtained at 20 and 40 V. They were assigned as photoproducts of this thiocarbamate pesticide since, as was previously reported, photodegradation is the major route of dissipation of this compound in water [30,31]. As an example, Fig. 4 shows the ion chromatogram corresponding to m/z 204 and the spectra tentatively assigned to 3-ketomolinate at 20 and 40 V, but, as the standard was not available, the retention time could not be con®rmed. As was previously mentioned, a peak appeared in the chromatograms of the stability that was assigned to
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Fig. 3. On-line SPE±LC±DAD chromatograms at 240 nm obtained for the sampling point 2 over the period of the monitoring study: (a) April, (b) May, (c) June and (d) July. Peak numbering is reported in Fig. 1.
phthalate compound and now the same peak in the chromatograms of the monitoring study, thus reinforces the assumption that the presence of this compound is due to the plasticware or tubing. 4. Conclusions The analysis of water samples from the Ebro delta area by HPLC±DAD and HPLC±APCI±MS enabled the organic micropollutants to be characterised and the
variation in the concentration of the different pesticides at different points in the area during a four month period to be investigated. The results indicate that it is recommended to store all the pesticides studied on the styrene±divinylbenzene cartridges at ÿ208C for a period of up to three months. Storage at 48C is also feasible for most of compounds. The storage at room temperature is not recommended because the recovery of such pesticides as carbamates is considerably lower.
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247
Fig. 4. Chromatogram obtained by on-line SPE±LC±APCI±MS in full-scan and positive ion mode for a sample from the Ebro delta area corresponding to sampling site 2 and took in June in which molinate and a degradation product (*) of this thiocarbamate pesticide appears (a) and extracted ion chromatograms corresponding to m/z 126 (b), and 204 (c).
One of the biggest advantages of stabilising pesticides in the polymeric cartridges is that they require less storage space and a large number of samples can be preserved in a conventional freezer for periods up to three months before their analysis, since this need not to be made immediately after sampling. Acknowledgements This work has been supported by the Commission of the European Communities, Environ-
ment and Climate Program 1994±98 (ENV4-CT960333). References [1] C. Wesseling, R. McConnell, T. Partanen, C. Hogstedt, Int. J. Health Serv. 27 (1997) 273. [2] M. Fielding, D. BarceloÂ, A. Helweg, S. Galassi, L. Torstensson, P. Van Zoonen, R. Wolter, G. Angeletti, Pesticides in Ground and Drinking Water, Water Pollution Research Report 27, Comission of the European Communities, Brussels, Belgium, 1992, p. 1.
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[3] D. BarceloÂ, M.F. Alpendurada, Chromatographia 42 (1996) 704. [4] A. OubinÄa, I. Ferrer, J. GascoÂn, D. BarceloÂ, Environ. Sci. Technol. 30 (1996) 3551. [5] B. Berger, J. Chromatogr. A 769 (1997) 338. [6] M.C. Hennion, V. Pichon, D. BarceloÂ, Trends Anal. Chem. 13 (1994) 361. [7] S. Papilloud, W. Haerdi, S. Chiron, D. BarceloÂ, Environ. Sci. Technol. 30 (1996) 1822. [8] G. Mascolo, A. LoÂpez, R. Passino, G. Ricco, G. Tiravanti, Water Res. 28 (1994) 2499. [9] S. Lacorte, N. Ehresmann, D. BarceloÂ, Environ. Sci. Technol. 30 (1996) 917. [10] S. Lacorte, D. BarceloÂ, J. Chromatogr. A 712 (1995) 103. [11] B.A. Tomkins, R. Merriweather, R.A. Jenkins, Ch.K. Bayne, J. AOAC Int. 75 (1992) 1091. [12] D. BarceloÂ, J. Chromatogr. 643 (1993) 117. [13] C. Crescenzi, A. Di Corcia, M. Diab Madbouly, R. Sampieri, Environ. Sci. Technol. 29 (1995) 2185. [14] E. MartõÂnez, D. BarceloÂ, Chromatographia 42 (1996) 72. [15] D. BarceloÂ, S. Chiron, S. Lacorte, E. MartõÂnez, J.S. Salau, M.C. Hennion, Trends Anal. Chem. 13 (1994) 352. [16] W.G. Johnson, T.L. Lavy, S.A. Senseman, J. Environ. Qual. 23 (1994) 1027. [17] S.A. Senseman, T.L. Lavy, J.D. Mattice, B.M. Myers, B.W. Skulman, Environ. Sci. Technol. 27 (1993) 516. [18] S.A. Senseman, T.L. Lavy, J.D. Mattice, Anal. Chem. 67 (1995) 3064.
[19] C. de la Colina, F. SaÂnchez-Rasero, G. Dios, E. Romero, A. PenÄa, Analyst 122 (1997) 7. [20] S. Lacorte, N. Ehresmann, D. BarceloÂ, Environ. Sci. Technol. 29 (1995) 2834. [21] C. Molina, P. Grasso, E. Benfenati, D. BarceloÂ, J. Chromatogr. A 737 (1996) 47. [22] I. Ferrer, D. BarceloÂ, J. Chromatogr. A 778 (1997) 161. [23] C. Aguilar, F. Borrull, R.M. MarceÂ, Chromatographia 43 (1996) 592. [24] C. Aguilar, F. Borrull, R.M. MarceÂ, J. Chromatogr. A 754 (1996) 77. [25] C. Aguilar, I. Ferrer, F. Borrull, R.M. MarceÂ, D. BarceloÂ, J. Chromatogr. A 794 (1998) 147. [26] S. Lacorte, S.B. Lartigues, Ph. Garrigues, D. BarceloÂ, Environ. Sci. Technol. 29 (1995) 431. [27] C. Crescenzi, A. Di Corcia, E. Guerriero, R. Sampieri, Environ. Sci. Technol. 31 (1997) 479. [28] S.C. Leung, B.Y. Giang, Bull. Environ. Contam. Toxicol. 50 (1993) 528. [29] J. AbiaÂn, G. Durand, D. BarceloÂ, J. Agric. Food Chem. 41 (1993) 1264. [30] D. BarceloÂ, S. Chiron, A. FernaÂndez-Alba, A. Valverde, M.F. Alpendurada, Herbicide Metabolites in Surface Water and Groundwater, ACS Symposium Series, vol. 630, 1996. [31] C.J. Soderquist, J.B. Bowers, D.G. Crosby, J. Agric. Food Chem. 25 (1977) 940.
Monitoring of pesticides in river water based on samples previously stored in polymeric cartridges followed by on-line solid-phase extraction-liquid chromatography±diode array detection and con®rmation by atmospheric pressure chemical ionization mass spectrometry C. Aguilara, I. Ferrerb, F. Borrulla, R.M. MarceÂa,*, D. BarceloÂb a
Department of Chemistry, Universitat Rovira i Virgili, Imperial Tarraco 1, 43005, Tarragona, Spain Department of Environmental Chemistry, CID-CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain
b
Received 21 September 1998; received in revised form 7 December 1998; accepted 30 December 1998
Abstract Solid-phase extraction coupled on-line with liquid chromatography±diode array detection was applied to the monitoring study of a group of pesticides and metabolites of different chemical groups in water samples from the Ebro delta area (Tarragona, Spain). Liquid chromatography±atmospheric pressure chemical ionisation-mass spectrometry was used for con®rmatory purposes. The developed method involves the preconcentration of 50 ml of water samples through styrene±divinylbenzene precolumns. The most frequently detected pesticides in the analysed samples were bentazone, molinate, metolachlor and the triazine herbicides simazine, atrazine and its dealkylated metabolites, deisopropilatrazine and deethylatrazine and, at concentration levels ranging from 0.03 to 2.4 mg lÿ1. The stability of several pesticides stored on styrene±divinylbenzene cartridges was also evaluated. The effect of different storage temperatures (room temperature, 48C and ÿ208C) and two storage periods (one week and three months) on the recovery of the pesticides were considered. In general, the recoveries were greater than 90% after three months of storage at ÿ208C on the polymeric cartridges. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Polymeric cartridges; Ebro delta; Water; Pesticides; APCI
1. Introduction Pesticides can be applied directly or indirectly to increase crop yields and their residues may accumu-
*Corresponding author. Tel.: +34-977-55-81-70; fax: +34-97755-95-63; e-mail: [email protected]
late not only on crops, but also in surface waters [1]. Pesticides can also be transformed by physical, chemical and biological processes into one or more transformation products and some of them can be more toxic or persistent than the parent compound [2±5]. Hence, there is a need for analytical procedures for monitoring pesticides and their transformation products in environmental matrices [6,7].
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00033-1
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One of the main aspects to be considered when analysing pesticides is that sometimes samples must be stored for variable periods of time before analysis, so a storage space is needed. It is also essential to know how long the content of a sample may remain stable. In this respect, various studies have been carried out to analyse pesticides after their storage [8±10]. To maintain the integrity of samples containing pesticides until their analysis, different methods of storage have been developed [5,11±15] one of them is to preserve in solid-phase extraction (SPE) materials such as discs or cartridges [11,15±20]. This extraction technique has advantages such as the reduction in the storage space required compared to bottles of water and the fact that the analysis need not be performed immediately after sampling. The type of SPE material is one factor to take into account and although most stability studies have been carried out using C18 sorbent phases [17±20], the stability of pesticides in other kinds of phase such as polymers [21] and carbon [13] has also been tested. Some studies have shown that the stability of organic pollutants stored in SPE materials is, in general, better than when they are stored in a water matrix [18,20,22]. The pesticide extract is mainly analysed by means of gas (GC) or liquid chromatography (LC) coupled to different detectors, depending on the characteristics of the analytes. In the case of LC, diode array detectors (DAD) are extensively used for determining different kinds of pesticide [21,23,24], but in the last few years, the coupling of LC to mass spectrometry (MS) has become a good alternative to classical LC detectors for the unambiguous identi®cation of analytes [21,23,25]. This has been widely used to study the degradation pathways of pesticides [14,21,26]. The development of different interfaces, especially ones with atmospheric pressure ionisation (API) offers new opportunities for the determination of a wide range of pollutants [22,25,27]. The aims of this work were as follows: ®rstly, to develop a method for determining pesticides in water samples, secondly, to monitor the presence of pesticides in an area from the Ebro delta, one of the main agricultural areas in Spain, in order to evaluate the variation in the concentration levels of these contaminants for a four months period and to detect the presence of possible transformation products formed,
and, thirdly, to examine the stability of various pesticides at three different storage temperatures (ÿ208C, 48C and room temperature) for periods of one week or three months in a polymeric cartridge material. By studying stability, it will be possible to determine whether water samples can be stored for up to three months and then analysed all together. 2. Experimental 2.1. Chemicals The pesticides used were 98±99% pure. They were purchased from Riedel-de HaÈen (Seelze, Germany). Stock solutions of 1000 mg lÿ1 were prepared by weighing each of the solutes and dissolving them in HPLC-grade methanol (Scharlau, Barcelona, Spain). They were then stored in the refrigerator at 48C. A stock solution of 5 mg lÿ1 was used to spike water samples at mg lÿ1 level for the determination of the calibration graphs. Ultra-pure water was prepared by ultra®ltration with a Milli-Q water puri®cation system (Millipore, Bedford, MA, USA). Acetic and hydrochloric acid were from Probus (Badalona, Barcelona, Spain). Sodium chloride was also from Probus. Helium for degassing the LC solvents was 99.995% pure and supplied by Carburos MetaÂlicos (Tarragona, Spain). Nitrogen for the APCI interface was 99.998% pure and it was supplied by Air Liquide (Barcelona, Spain). 2.2. Apparatus 2.2.1. LC±DAD LC analyses were performed with an HP 1090 Series II liquid chromatograph (Hewlett-Packard, Palo Alto, CA, USA), equipped with a ternary solventdelivery system, an injection valve with a 25 ml loop and an HP 1040M diode array detector. The chromatographic separation was performed with a Spherisorb ODS-2 column (200.46 cm i.d., 5 mm particle size) (Teknokroma, Barcelona, Spain). The gradient elution was carried out with a binary gradient composed of LC solvent A (methanol) and LC solvent B (water acidi®ed to pH 4.5 by adding acetic acid) according to the following programme: 10% A to 50% A in 20 min,
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then to 70% A in 24 min and to 100% A in 1 min. After 1 min at 100% methanol, the mobile phase was returned to the initial conditions and, after 10 min of stabilisation, the next sample was injected. The ¯ow rate was 1 ml minÿ1. For multi-wavelength monitoring, the DAD was set at 240, 254 and 280 nm with a bandwidth of 4 nm. Absorbance spectra were recorded from 200 to 400 nm. Chromatographic data were collected and recorded using the HP 79994A Workstation and quanti®cation was carried out at the maximum absorbance value for each compound. 2.2.2. LC±APCI±MS The eluent was delivered by a gradient system from Waters 616 pumps controlled by a Waters 600S (Waters, Mildford, MA, USA). A VG Platform from Fisons Instruments (Manchester, UK), equipped with an APCI interface was used. The APCI interface consists of a heater nebulizer probe and the standard atmospheric pressure source was equipped with a corona discharge pin. The operating parameters were a drying gas ¯ow rate of 250±300 l hÿ1 and a nebulizing gas ¯ow rate of 10 l hÿ1. The cone voltage was set at 20 and 40 V and the corona voltage at 3.5 kV. The ion source and probe temperatures were set at 1508C and 4008C, respectively. LC±APCI±MS was used under full-scan mode and the scan range was from 70 to 400 m/z in the positive ion (PI) mode. The instrument control and data processing utilities included MegaLinx software. The chromatographic conditions, ¯ow rate and gradient, were the same as those reported above for LC±DAD. 2.3. Procedures Trace enrichment was performed on a 102.0 mm cartridge packed with styrene±divinylbenzene copolymer (Spark Holland, Emmen, Netherlands). An Applied Biosystems (Ramsey, USA) pump was used to deliver the sample and to condition the precolumn. For the stability study in the SPE cartridges, 50 ml of Milli-Q water (adjusted at pH 2 and with 5 g lÿ1 of NaCl added) spiked to 5 mg lÿ1 with a solution which contains each pesticide was preconcentrated on the precolumns of styrene±divinylbenzene and stored at ÿ208C, 48C and at a room temperature (RT) for a period of one week and three months. For each of the
239
storage conditions four cartridges were stored. After this period of time, the retained analytes were desorbed by placing the precolumns in a holder and starting the mobile phase gradient in the back¯ush mode. Before analysis, the cartridges were thawed for a period which depends on the storage conditions in order to remove the frozen water matrix involving the stationary phase. Afterwards, these samples were analysed using the protocol described above and for each of the storage conditions two replicate analyses were performed for DAD. For the monitoring study, several aliquots were collected from ®ve different sampling sites in the Ebro delta area once a month from April to July, when pesticide application to crops was greatest, and four cartridges of each sample was stored each month. The sample sites were selected in order to give overall information about the quality of the water. Hydrochloric acid was added to the samples immediately after collection in order to minimise possible biological degradation and they were also ®ltered through a 0.45 mm PTFE ®lter (Millipore) to remove particulate matter. Then, a 50 ml water sample was preconcentrated at a ¯ow rate of 4 ml minÿ1 and the cartridges were stored at ÿ208C until their analysis. For these samples the analytes trapped on the cartridges were eluted in the same way as for the stability study. 3. Results and discussion 3.1. General remarks The analytes monitored in this study included 10 pesticides (aldicarb, atrazine, bentazone, carbaryl, carbofuran, cyanazine, malathion, metolachlor, molinate and simazine) and three metabolites (deethylatrazine (DEA), deisopropilatrazine (DIA) and malaoxon). They were chosen to cover a broad range of chemical classes and because they are commonly detected in surface waters of the studied area and also because they are on the priority lists of pesticides of the European Community [2]. The use of a DAD detector made it possible for all analytes to be determined at the wavelengths of maximum absorbance in order to obtain an improvement in sensitivity and selectivity. The analytes were iden-
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Table 1 Wavelengths used to measure absorbance, molecular weight, main ions obtained under APCI±MS under PI mode of operation at a cone voltage of 20 and 40 V, linearity range, correlation coefficients, r2, and limits of detection (LODs) obtained by on-line SPE±LC±DAD for all target compounds Pesticide
Deisopropilatrazine (DEA) Bentazone Deethylatrazine (DIA) Aldicarb Cyanazine Carbofuran Simazine Carbaryl Malaoxon Atrazine Malathion Molinate Metolachlor
(nm)
240 240 240 254 240 280 240 280 280 240 280 240 254
MW
187 240 173 190 241 221 202 201 314 215 330 187 284
m/z (V) 20
40
174 120 188 116 241 222 202 145 127 216 285 188 284
96 120 146 89 216 165 132 145 127 174 127 126 252
ti®ed by comparing the absorption spectra and retention times of each compound with those of standard solutions. Table 1 shows the wavelengths used for the quanti®cation of each analyte. In order to con®rm the data obtained from DAD detection, samples were also analysed by means of LC±APCI±MS. The operational conditions were optimised previously [25]. Table 1 shows the molecular weight of each analyte and the major ions obtained, under full-scan conditions using the positive ion (PI) mode of acquisition at cone voltages of 20 and 40 V, by ¯ow-injection analysis of the pesticides at a concentration of 50 mg lÿ1 into a carrier stream of methanol/water (with the addition of acetic acid). The corresponding fragments to the ions shown in this table are widely explained in a previous paper [25]. 3.2. On-line SPE Preconcentration was performed by on-line SPE on styrene±divinylbenzene copolymer. This stationary phase was selected taking into account previous results obtained using this phase and C18. In general, retention was higher when the polymeric sorbent was used, particularly for medium and highly polar pesticides [20,21,23±25]. The different parameters which can affect the response for the on-line SPE procedure were opti-
Linearity range (mg lÿ1)
r2
LOD (mg lÿ1)
0.1±10 0.1±10 0.05±10 0.1±10 0.025±10 0.1±10 0.025±10 0.1±10 0.2±10 0.025±10 0.2±10 0.1±10 0.1±10
0.996 0.995 0.999 0.997 0.999 0.998 0.996 0.996 0.996 0.997 0.999 0.999 0.998
0.04 0.04 0.02 0.04 0.01 0.04 0.01 0.04 0.10 0.01 0.10 0.04 0.04
mised, so the effect of the sample pH and the addition of sodium chloride on the recoveries of the analytes were evaluated. Initially, the in¯uence of sample pH was studied by analysing samples at different pH values. For the most polar compounds, such as bentazone, DEA and DIA, and under acidic conditions, recoveries were greater than those for neutral samples and consequently for further studies the samples were acidi®ed to pH 2 by adding hydrochloric acid before being preconcentrated. The effect of adding salt was also tested by adding different amounts of NaCl to the sample. For some compounds, particularly the organophosphorous pesticides, the addition of 5 g lÿ1 of sodium chloride enhanced the recovery as had previously been reported [24]. The breakthrough volumes of the pesticides were obtained by percolating different sample volumes, between 25 and 100 ml, of Milli-Q water acidi®ed at pH 2 and with the addition of NaCl through the styrene±divinylbenzene precolumns. The recoveries obtained for each pesticide after the preconcentration of 25, 50 and 100 ml of Milli-Q water sample spiked at the 5 mg lÿ1 level are shown in Table 2. Sample volumes higher than 50 ml gave losses for the two dealkylated metabolites of atrazine, DEA and DIA, so all pesticides were determined and quanti®ed by preconcentrating a volume of 50 ml for which the recov-
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241
Table 2 Mean recoveries and relative standard deviation (RSD) (n5) of pesticides at the 5 mg lÿ1 level in Milli-Q water at different sample volumes acidified to pH 2 and with the addition of 5 g lÿ1 of NaCl Pesticide
Deisopropilatrazine(DEA) Bentazone Deethylatrazine (DIA) Aldicarb Cyanazine Carbofuran Simazine Carbaryl Malaoxon Atrazine Malathion Molinate Metolachlor
25 ml
50 ml
100 ml
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
83 90 82 89 92 81 94 99 79 109 82 90 91
8 9 12 6 7 9 11 9 8 7 5 4 9
78 89 75 86 97 78 94 96 80 99 83 89 87
11 8 8 5 7 12 7 4 6 11 8 7 9
53 76 48 74 92 76 97 91 73 92 71 75 77
5 7 9 11 10 9 6 5 9 8 7 9 12
eries for most of the analytes were between 80% and 100%. 3.3. Quality parameters Under optimum conditions of the calibration plots were constructed by percolating 50 ml of Milli-Q water spiked with the pesticides at levels between 0.025 and 10 mg lÿ1 using on-line SPE± LC±DAD and quantifying each compound at its maximum wavelength. The limits of detection (LODs) were calculated by using a signal-to-noise ratio of 3. The results are shown in Table 1. The linearity was also checked for river Ebro water and the corresponding results were similar as reported for Milli-Q water. In order to study the repeatability of the method, river samples spiked at a level of 5 mg lÿ1 were preconcentrated and the relative standard deviation (n5) was below 10% for all the analytes. 3.4. Stability study Pesticides can be degraded during storage by such processes as hydrolysis or microbial decomposition. Storage conditions can affect the stability of pesticides in the precolumns; different temperatures and periods of time were studied in order to evaluate the optimum conditions for storing the pesticides on the styrene± divinylbenzene cartridges.
For each storage time and temperature two samples were analysed by the DAD and ®nally the recovery for each pesticide was evaluated by comparing the recoveries obtained after preconcentration of 50 ml of Milli-Q water sample spiked at the 5 mg lÿ1 level. The RSD of one of the storing conditions (48C and three months) was checked and was lower than 9% for all the analytes (n3). 3.4.1. Storage at ÿ208C The precolumns kept at ÿ208C were defrosted for 4±6 h at room temperature before the retained analytes were eluted, because previous studies [20] have demonstrated that if the extraction process is carried out immediately after the cartridge is removed from the freezer, the quanti®cation and identi®cation of the compounds is dif®cult. The recoveries of each pesticide after storing the precolumns for one week and three months at ÿ208C are shown in Table 3. It can be seen that for all compounds the recoveries were between 85% and 100% at this low temperature independently of the storage time. No parent compounds were lost during the period of time studied because the pesticides did not degrade and were completely recovered even after three months of storage. 3.4.2. Storage at 48C The precolumns stored at 48C were kept at room temperature for 1 h before their elution. Table 3 shows
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Table 3 Recovery values (%) of the pesticides after different storage times at ÿ208C, 48C and room temperature (RT) obtained after a 50 ml volume of Milli-Q water spiked to 5 mg lÿ1 level with each pesticide was loaded on the PLRP-S cartridge Pesticide
DEA Bentazone DIA Aldicarb Cyanazine Carbofuran Simazine Carbaryl Malaoxon Atrazine Malathion Molinate Metolachlor
ÿ208C
48C
One week
Three months
98 102 97 94 102 107 96 102 93 109 97 94 107
81 82 94 81 87 93 89 87 90 95 93 90 94
RT
One week 92 89 93 96 97 84 94 98 91 102 92 101 97
the results of studying the stability of pesticides stored at this temperature. These results show that losses were greater (20±30%) after the three month period for carbofuran, as was previously reported by other authors who used C18 disks for the storage [16]. For one week of storage recovery values were higher than 84% for all the compounds studied, so these storage conditions are feasible for the pesticides evaluated. 3.4.3. Room temperature Losses at room temperature (RT) were quite important for some of the target compounds even after short periods of storage. For the ®rst week of storage under this temperature most of the pesticides were stable whereas losses were as high as 30% for aldicarb, carbaryl and bentazone and as high as 40% for carbofuran. Consequently, samples containing those pesticides have to be stored under low temperatures. These results indicated that the N-methylcarbamate pesticides aldicarb, carbaryl and carbofuran were the least stable of the pesticides included in this study since they were seriously affected by the storage conditions. Their recoveries tend to reduce with the storage temperature, as previously reported using C18 as the SPE sorbent [16]. The length of storage time at room temperature also affected several compounds such as malathion or some triazine compounds such as deethylatrazine and atrazine for which the recovery decreases between
Three months
One week
Three months
89 82 88 78 92 72 92 94 86 93 88 89 92
89 78 83 72 93 62 90 74 84 94 87 92 91
77 62 75 63 90 43 88 71 87 85 79 86 87
10% and 15% as storage time increases. These results are in agreement with the data on the literature on malathion [3,19,20] and the triazine herbicides atrazine, deethylatrazine or simazine [14,17,18] although the sorbent used is C18. In the chromatograms obtained after the elution of the cartridges stored at RT, the same peaks obtained for 48C appeared. By comparing all the data of the different storage conditions, it can be concluded that the most reliable method for storing pesticides in SPE sorbents is to keep them at ÿ208C because the integrity of most of the analytes is not affected even after long storage periods; these results are in agreement with previous results [3,15]. However, storage at 48C is also feasible for most of the compounds. Fig. 1 shows the chromatograms corresponding to the preconcentration of 50 ml of Milli-Q water spiked with the pesticides at the 5 mg lÿ1 level and obtained after storage at ÿ208C, 48C and room temperature for three months. 3.5. LC±APCI±MS A peak appears very close to atrazine in all the DAD chromatograms obtained after the elution of the cartridges stored at any conditions. In order to obtain more structural information about the interfering peak, some of the cartridges were analysed by coupling them on-line with the LC±APCI±MS system under
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243
Fig. 1. HPLC±DAD chromatograms at 240 nm obtained after the on-line preconcentration of 50 ml of Milli-Q water after storing the PLRP-S cartridges with the pesticides for a period of three months at: (a) RT, (b) 48C and (c) ÿ208C. Peaks1: Deisopropilatrazine (DEA); 2: Bentazone; 3: Deethylatrazine (DIA); 4: Aldicarb; 5: Cyanazine; 6: Carbofuran; 7: Simazine; 8: Carbaryl; 9: Malaoxon; 10: Atrazine; 11: Malathion; 12: Molinate; 13: Metolachlor.
full-scan conditions and positive acquisition. The unidenti®ed peak presented m/z 177 as its base peak and an ion at m/z 149 under the experimental conditions; in order to obtain more structural information an extraction voltage of 40 V was used to induce greater fragmentation but no additional information was obtained because the only difference between this spectrum and the one obtained for 20 V was the relative abundance of ions, the base peak being the ion at m/z 149. The peak was tentatively identi®ed as a phthalate compound since the ion at m/z 149 is a typical fragment of this kind of compound. Likewise, the ion at m/z 177 is the reported base peak for many phthalate esters and the ion at m/z 149 is also a typical peak for these compounds in their spectra obtained by
electron impact ionisation [28]. With all this information the peak could be tentatively identi®ed as diethyl phthalate or diethyl ter-phthalate since their molecular weight was 222 and in the spectrum obtained at 20 V there was an ion at m/z 223 which can be assigned to [MH] and an ion at m/z 245 corresponding to [MNa]. The presence of a phthalate ester in the samples could be due to the use of plasticware or tubing that often contains phthalate esters as plasticizers [28]. Another small peak at a retention time of about 8.5 min appeared in the chromatograms obtained under 48C and the samples were also analysed by on-line SPE±LC±APCI±MS method under full-scan conditions in the positive ion mode. The correspond-
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ing spectra showed an ion at m/z 132 as the base peak and other ions at m/z 166 and 181. The comparison between its spectrum and the corresponding mass spectra of the analytes initially included in this study, obtained at a cone voltage of 40 V, showed that for the chlorotriazines DIA, simazine and atrazine, the ions at m/z 132 and 166 were also present, so the unknown peak was tentatively assigned to a transformation product of a chlorotriazine herbicide. The ion at m/z 132 is described in literarure [29] as a common peak for chlorotriazines with an ethyl substituent, such as those previously mentioned. 3.6. Monitoring study In order to determine the levels of pesticides in water samples from the Ebro delta, ®ve different sampling sites were established in the area. The samples, obtained from each of the sites, were collected monthly from April to July. The sampling sites were selected on the basis of monitoring zones
exposed to high agricultural activity since the Ebro river delta is a typical cultivation area where some pesticides are applied in large amounts [30]. The selected sampling points are shown in Fig. 2. First, the analyses were carried out by HPLC±DAD which provided a primary screening of the samples collected since the different peaks that appeared in the corresponding chromatograms were compared with the DAD spectra of the target compounds. LC±APCI± MS was used to con®rm the data obtained from DAD detection and also to identify the presence of possible unknowns which were found to be present in the samples. The data obtained in this study have been useful for determining the occurrence and temporal distribution of pesticides and their metabolites in the evaluated area. The results are shown in Table 4 and RSDs (n3) checked for river water were lower than 11 for all compounds. The results show that there are some differences during the sampling period and it can be pointed out that there is a relationship with the
Fig. 2. Map of delta Ebro area showing the location of sampling sites: (1) Ebro river, (2) Carrete channel (irrigation ditch which receives water from surronding fields, (3) Encanyissada lagoon (it receives irrigation channels from the rice fields, (4) a part of the Encanyissada lagoon which is closer to the sea, and (5) Tancada lagoon (it receives irrigation channels from the rice fields).
C. Aguilar et al. / Analytica Chimica Acta 386 (1999) 237±248 Table 4 Mean value for the pesticide concentration (mg lÿ1) over the period of study at each sampling point Compound
Months
Sampling point 1
2
3
4
5
Atrazine
April May June July
0.18 0.24 0.19 0.06
0.06 0.03 0.05 n.d.
n.d. 0.03 0.04 n.d.
n.d. n.d. 0.03 n.d.
n.d. n.d. n.d. n.d.
DIA
April May June July
0.15 0.23 0.14 0.10
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
DEA
April May June July
0.11 0.12 0.10 n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
Simazine
April May June July
0.14 1.06 0.13 0.05
0.07 0.07 0.04 n.d.
0.04 0.05 n.d. n.d.
n.d. 0.04 n.d. n.d.
n.d. 0.03 0.05 n.d.
Molinate
April May June July
n.d. 0.29 0.11 0.08
0.92 1.30 2.39 1.25
0.22 0.41 0.64 0.39
0.28 0.46 0.51 0.33
0.12 0.10 0.11 0.11
Bentazone
April May June July
n.d. n.d. n.d. n.d.
n.d. 0.32 0.20 0.11
n.d. 0.24 0.19 0.10
n.d. 0.10 0.10 n.d.
n.d. 0.22 n.d. n.d.
Metolachlor
April May June July
0.20 0.12 n.d. n.d.
n.d. n.d. 0.10 n.d.
0.22 0.12 n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d.: not detected.
increase of the agricultural activities in the area evaluated. The various pesticides which were found in the analysed samples belong to different chemical groups as a consequence of the variety of crops of the sampled area. The triazine herbicides, atrazine and simazine, and the thiocarbamate molinate were found in the greatest number of samples. Other pesticides such as metolachlor, bentazone and the dealkylated metabolites of atrazine also appeared in some samples. Pesticides were not distributed uniformly throughout the different sampling sites; whereas the concentration of the triazine herbicides was, in general,
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higher in the river Ebro (sampling point 1), the concentration of molinate, bentazone or propanil was higher at the sampling points which were inside the delta. The presence of these pesticides in samples from the delta river water is not surprising because they are used in large amounts in this zone [30]. Atrazine is applied to various crops along the river and its presence in the river Ebro samples can be attributed to its transport from its application site as was previously found [12,25]. The presence of deethylatrazine and deisopropilatrazine in some of the samples can be attributed to the degradation of the triazine herbicides since in the Ebro river basin important amounts of some triazine herbicides are used. The other compounds found are typical rice pesticides and, of these, molinate was found to be the most used, being encountered at the highest concentration with respect the other pesticides. The maximum concentration levels for the typical rice herbicides were observed at sampling point 2. This is logical since this point receives waters from rice ®elds, where pesticides are being applied, while the lower concentrations at points 3, 4 and 5 are probably due to the dilution effect. As an example, Fig. 3 shows the chromatograms obtained for the period studied, from April to July, corresponding to the sampling point 2. Numerous unknowns were found to be present in the analysed samples and some peaks were tentatively identi®ed as the transformation products of pesticides. In recent years, considerable interest has been shown in the detection of pesticide metabolites, which are potentially toxic compounds and in this sense the use of mass spectrometry allows the presence of some compounds to be con®rmed. In this study, the presence of some degradation products of molinate was con®rmed by the corresponding mass spectra obtained at 20 and 40 V. They were assigned as photoproducts of this thiocarbamate pesticide since, as was previously reported, photodegradation is the major route of dissipation of this compound in water [30,31]. As an example, Fig. 4 shows the ion chromatogram corresponding to m/z 204 and the spectra tentatively assigned to 3-ketomolinate at 20 and 40 V, but, as the standard was not available, the retention time could not be con®rmed. As was previously mentioned, a peak appeared in the chromatograms of the stability that was assigned to
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Fig. 3. On-line SPE±LC±DAD chromatograms at 240 nm obtained for the sampling point 2 over the period of the monitoring study: (a) April, (b) May, (c) June and (d) July. Peak numbering is reported in Fig. 1.
phthalate compound and now the same peak in the chromatograms of the monitoring study, thus reinforces the assumption that the presence of this compound is due to the plasticware or tubing. 4. Conclusions The analysis of water samples from the Ebro delta area by HPLC±DAD and HPLC±APCI±MS enabled the organic micropollutants to be characterised and the
variation in the concentration of the different pesticides at different points in the area during a four month period to be investigated. The results indicate that it is recommended to store all the pesticides studied on the styrene±divinylbenzene cartridges at ÿ208C for a period of up to three months. Storage at 48C is also feasible for most of compounds. The storage at room temperature is not recommended because the recovery of such pesticides as carbamates is considerably lower.
C. Aguilar et al. / Analytica Chimica Acta 386 (1999) 237±248
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Fig. 4. Chromatogram obtained by on-line SPE±LC±APCI±MS in full-scan and positive ion mode for a sample from the Ebro delta area corresponding to sampling site 2 and took in June in which molinate and a degradation product (*) of this thiocarbamate pesticide appears (a) and extracted ion chromatograms corresponding to m/z 126 (b), and 204 (c).
One of the biggest advantages of stabilising pesticides in the polymeric cartridges is that they require less storage space and a large number of samples can be preserved in a conventional freezer for periods up to three months before their analysis, since this need not to be made immediately after sampling. Acknowledgements This work has been supported by the Commission of the European Communities, Environ-
ment and Climate Program 1994±98 (ENV4-CT960333). References [1] C. Wesseling, R. McConnell, T. Partanen, C. Hogstedt, Int. J. Health Serv. 27 (1997) 273. [2] M. Fielding, D. BarceloÂ, A. Helweg, S. Galassi, L. Torstensson, P. Van Zoonen, R. Wolter, G. Angeletti, Pesticides in Ground and Drinking Water, Water Pollution Research Report 27, Comission of the European Communities, Brussels, Belgium, 1992, p. 1.
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