Effect of Storage on the Recovery of Different Types of Pesticides Using a Solid-phase Extraction Method C. de la Colina, F. S�anchez-Rasero, G. Dios, E. Romero and A. Pe�na* Estaci�on Experimental del Zaid�ýn (CSIC), Profesor Albareda, 1, E-18008 Granada, Spain Recoveries of different pesticide groups after storage either on C18 cartridges or as dried residues from organic solutions, and their analysis by gas chromatography with electron capture and flame photometric detection, were studied.Two storage temperatures, 4 and 218 °C, and three storage periods, 3, 7 and 30 d, were considered. The effect of storage temperature and storage time on the recovery of 27 pesticides in water was investigated. In general, the pesticide recoveries were !70% after 30 d of storage at 218 °C on C18 cartridges. Exceptions included captan and folpet. The storage of the dried residues generally did not affect the pesticide recovery when kept at 218 °C for up to 30 d. Keywords: Sample handling; sample storage; pesticide stability; water; solid-phase extraction; gas chromatography For analytical data to be valid, they should reflect the concentration of pesticides at the time of sampling, but often in a laboratory, owing to temporary shortages of personnel, problems with or breakdown of analytical equipment or sudden unexpected requirements for equipment to be used for other work, samples must be stored for variable periods before their analysis.For this purpose, and also when samples must be transported to other national or foreign laboratories, it is essential to know how long the content of a sample may remain unchanged.Therefore, stability studies of pesticides and organic contaminants in water and other matrices are important. In a recent report from the US Environmental Protection Agency (EPA),1 in which 147 pesticides in water samples were checked for stability for at least 14 d at 4 °C, 26 pesticides were removed from the list because of a 100% loss, even after biological inhibition of the water microorganisms. Research on the storage of pesticides in different water samples has been undertaken,2–5 that confirmed the instability of many pesticides in natural waters, depending on microbial degradation, hydrolysis and photolysis.To compensate for this instability, various stabilizers or preservatives have been added to aqueous samples, e.g., methanol,6 dichloromethane,7 dilute acid solutions1,8 or HgCl2,1,9 or the sample has been freezedried10 to retard the decomposition of the constituent chemicals.An alternative could be the preservation of the pesticides retained in solid-phase extraction (SPE) cartridges or discs, which are being increasingly used in environmental laboratories, 11 a possibility already used for hydrocarbon samples and some pesticides.9–17 The compounds of interest have been effectively preserved from microbial degradation for up to 54 d on these adsorbents12 and photolysis is avoided because the discs and cartridges are usually stored in the dark.It has been speculated that this preservative effect is a result of the trapping of the organics within the lattice structure of the adsorbent, although protection against hydrolysis is still in question.15,17 In addition, the processed samples may be preserved as organic extracts.1 It was reported that analytes generally remained stable in stored sample extracts, although no information was given about how long and for which analytes this kind of storage was effective.In this work, 27 pesticides, including nine organophosphorus, six organochlorine, a carbamate, a pyrethroid and another seven pesticides of miscellaneous groups, were studied at concentrations between 20 and 100 ng l21. Two ways of storing the different pesticides present in water samples were considered: on C18 SPE cartridges and as organic extracts concentrated to dryness.Experimental Reagents All the pesticides were of > 98.5% purity and all the solvents were of pesticide residue analysis grade. C18 extraction cartridges (J. T. Baker, Phillipsburg, NJ, USA) with 500 mg of packing material were used. Cartridge Storage Water samples of 1 l, obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA), were fortified with 20–100 ng of the chemicals (see Table 1). The cartridge was conditioned by rinsing it with the eluents in reverse order of elution, then with methanol and water.20 After the passage of the 1 l fortified water sample, the cartridge was air dried for 30 min.Solid-phase cartridges on which pesticides had been retained were wrapped in Parafilm and stored, at 4 or 218 °C, for 3, 7 and 30 d. At the end of the storage period they were eluted with ethyl acetate and isooctane and evaporated to dryness under a gentle stream of nitrogen. The final residue was taken up with 1 ml of hexane and the internal standard, bromophos, was added before injection into the gas chromatographs.21 Concentrated Extract Storage A mixture of the pesticides in the amounts shown in Table 1 was added to 4 ml of a mixture of the eluents [ethyl acetate– isooctane (1 : 1)] and then concentrated to dryness under a gentle stream of nitrogen. The glass tubes containing the dried eluates were fitted with a glass stopper, covered with Parafilm and stored at the same temperatures and for the same periods of time as used for the cartridges.The dried residue was treated as for the cartridge. For both storage treatments, cartridges and concentrated extracts, the remainder of the analytical process was finished rapidly with injection into the chromatograph on the same day. Statistical Analysis A completely randomized design with three replications of the whole analytical procedure, with three injections per replicate, of two storage temperatures and three storage times was Analyst, January 1997, Vol. 122 (7–11) 7employed for each storage treatment. Mean percentage recoveries were calculated and separated by Fisher’s least significant difference (LSD) at the 0.05 level of significance. Analytical Methodology All the samples were quantified by GC.20,21 Organophosphorus pesticides were determined using a Hewlett-Packard (Avondale, PA, USA) gas chromatograph with a flame photometric detector (FPD), provided with an HP-1 capillary column (12 m 3 0.2 mm id, 0.33 mm film thickness) with the following oven temperature programme: 45 °C (1 min), increased at 30 °C min21 to 170 °C (2 min), at 4 °C min21 to 200 °C (2 min) and at 20 °C min21 to 270 °C (2 min).The injector and detector temperatures were 250 and 275 °C, respectively. The other pesticides were determined using a Hewlett- Packard gas chromatograph with an electron capture detector (ECD), in which an Ultra-2 capillary column (25 m 3 0.32 mm id, 0.17 mm film thickness) was installed, with the following oven temperature programme: 160 °C (1 min), increased at 4 °C min21 to 230 °C (2 min) and at 20 °C min21 to 280 °C (6 min).The injector temperature was 250 °C and the detector temperature was 300 °C. Results and Discussion The pesticides included in this study, were tested at concentrations lower than or equal to the maxima allowed by EU legislation. Such concentrations are at least 102–103 times more dilute than the values previously reported in some storage studies using extraction discs15,16 or cartridges.17 Therefore, the amounts used in this study approach legal conditions and avoid higher concentrations which can influence degradation, as has already been reported for some organophosphorus and carbamate pesticides in soil columns.22 Fig. 1 shows the separation of a pesticide standard solution, with the different detection methods, at the concentrations indicated in Table 1. Cartridge Storage The recoveries obtained for the pesticides retained on the C18 cartridges are given in Table 2 for the pesticides in which no interaction between storage time and temperature was encountered and in Table 3 for the chemicals with a significant interaction between the storage factors studied.As can be seen for the effect of temperature (Table 2), only alachlor, capA-DDE and chlorpyrifos-ethyl show significant differences for the two temperatures studied. Nevertheless, for most of these chemicals only small differences in the recoveries for the two storage temperatures are observed (@10%).Captan, and the chemically related folpet, exhibited an important reduction in recovery, higher for the freezer (218 °C) than for the refrigerator (4 °C), for which no explanation has Table 1 Pesticide amounts used for both storage treatments and their physico-chemical properties. Data from ref. 18 Amount Vapour Water added/ pressure/ solubility/ Pesticide ng* mPa Log Kow mg l21 Trifluralin 20 9.5 4.0† 0.221 Lindane 20 5.6 3.7† 7.3 Triallate 40 16 na‡ 4 Alachlor 80 2.9 na 242 Captan 40 1.3 2.5† 3.3 Folpet 40 1.3 3.1 1 o,pA-DDE 20 na na na p,pA-DDE 20 0.025 5.7–7.0† na Oxyfluorfen 20 0.0267 4.5 0.116 o,pA-DDT 20 na 5.8† na p,pA-DDT 20 na 6.2–6.9† na Bromopropylate 20 0.011 5.4 < 0.5 Dicofol 40 0.053 4.3 0.8 Tetradifon 20 3.231025 4.6 0.08 Deltamethrin 40 0.002 4.6 < 0.0002 Dimethoate 100 1.1 0.7 23800 Fonofos 100 28 3.9 13 Diazinon 100 12 3.3 60 Formothion 100 0.113 na 2600 Fenitrothion 100 18 3.4 21 Malathion 100 5.3 2.9† 145 Fenthion 100 0.74 4.8 4.2 Chlorpyrifos 100 2.7 5.3† 1.4 Methidathion 100 0.25 2.2 200 Phosmet 100 0.065 3.0 25 Azinphos-methyl 100 0.18 3.0 28 Phosalone 100 < 0.067 4.3† 1.7 * The same amount was added to 1 l of water or to 4 ml of the desorption solution. † Data from ref. 19. ‡ Not available. Table 2 Effect of storage temperature and storage time on recoveries of pesticides retained on C18 cartridges Storage Storage temperature/°C* time/d† Pesticide 218 4 LSD‡ 3 7 30 LSD‡ Trifluralin 90 92 86 89 101 5.1 Lindane 96 97 95 97 98 Triallate 89 86 96 86 82 6.6 Atachlor 94 98 2.7 96 95 97 Captan 58 73 9.1 74 66 58 11.2 Folpet 38 50 50 41 47 o,pA-DDE 69 64 4.0 63 72 65 4.9 p,pA-DDE 67 66 64 67 68 Oxyfluorfen 85 84 81 84 88 3.5 o,pA-DDT 67 69 63 71 70 4.9 p,pA-DDT 70 69 68 71 70 Bromopropylate 84 83 84 84 84 Dicofol 87 86 85 86 87 Tetradifon 95 98 100 96 94 4.0 Diazinon 87 87 89 89 84 3.8 Fenitrothion 94 95 99 97 88 4.0 Malathion 94 94 98 98 87 3.6 Chlorpyriphos-ethyl 82 79 2.4 85 83 72 2.9 Phosmet 95 96 103 87 97 9.7 * Average recovery for nine observations (three injections per observation).† Average recovery for six observations (three injections per observation). ‡ LSD = least significant difference (P < 0.05). The LSD values are provided for those compounds with a significant difference between the means. Table 3 Effect of temperature and time of storage on recovery of pesticides retained on C18 cartridges Recovery (%)* Refrigerator Freezer (4 °C) (218 °C) Pesticide 3 d 7 d 30 d 3 d 7 d 30 d LSD† Fonofos 75 68 55 79 64 65 7.9 Methidathion 105 98 89 101 103 99 6.9 Azinphos-methyl 121 102 129 119 114 167 17.8 Phosalone 102 96 87 110 93 98 6.9 * Average recovery for three observations (three injections per observation). † See Table 2. 8 Analyst, January 1997, Vol. 122been found. In a previous study, stability problems with captan were reported (54% recovery on a C18 disc at 4 °C and 32% at 218 °C after 30 d of storage) and ascribed to hydrolysis under the C18 packing of the SPE disc15 and, later, to volatilization,17 although the vapour pressure of this fungicide ( < 1.3 mPa, Table 1) is lower than that of some other pesticides included in this study, which do not show a loss.In addition, the captan recovery is also affected by the storage time (Table 2), so this method of preservation is not appropriate for this pesticide.When captan was stored on extraction discs,15 removal of residual water was recommended. The length of the storage affects several compounds, apart from captan, as can be seen in Table 2. In general, for the pesticides affected (trifluralin, triallate, o,pA-DDE, oxyfluorfen, o,pA-DDT, tetradifon, diazinon, fenitrothion, malathion, chlorpyrifos- ethyl and phosmet), the recovery decreases with increasing storage time, but remains within a ±10–15% variation for the whole length of the storage period.This variation range is within that commonly found in other storage studies.15,17 In addition, the final recovery after 30 d remains over 70% (the minimum recovery required by the EPA regulations23), except for DDEs, with recoveries of !65%, and captan and folpet. Low recoveries have been also reported for DDE and DDT retained on Empore filters for up to 4 weeks, from waters spiked at 50 ng l21.14 Fonofos, methidathion, azinphos-methyl and phosalone showed a significant interaction between temperature and length of storage (Table 3) and therefore both factors cannot be considered individually.For fonofos, methidathion and phosalone, a trend of loss on storage in the refrigerator after 30 d was observed. For fonofos, its storage on a cartridge is not recommended, because its recovery is already below 70% after 7 d at the two temperatures investigated. A complete loss of fonofos, which is the most volatile pesticide included in the present study (Table 1), has been reported for storage in similar cartridges at 4 °C after 1.5 months.17 In the contrast, the same group17 reported complete recovery of fonofos when stored at 220 °C for 8 months.Our results show a 14% loss after 1 month of storage in the freezer. For azinphos-methyl, which is not completely separated from phosalone in the chromatographic column, an artifact appears after 30 d of storage and therefore its quantification was inaccurate.Fig. 2(A) shows the separation of the pesticide mixture with electron capture detection (ECD) after storage in the cartridge for 30 d at 218 °C. The recoveries of the fungicides captan and folpet are clearly affected. The additional peaks correspond to the organophosphorus pesticides that can be detected by ECD. As indicated previously, several organophosphorus pesticides included in the present study (azinphos-methyl, fenitrothion, malathion, diazinon and phosmet) have been eliminated from the US EPA survey list owing to their instability;1 the first three are nevertheless included in the 76/464/EEC Council Directive List of Pesticides to be monitored in the aquatic environment.23 In addition, phosmet has been repeatedly reported to be unstable on storage in water.2,5 These pesticides could be stabilized by storing them on the cartridge packing at 4 or 218 °C for at least 30 d.Nevertheless, certain losses have been observed for some of the pesticides, especially those with low partition coefficients, which confirms the results of Lacorte et al.,17 who pointed to hydrolysis and microbial degradation as the main factors causing instability with this kind of storage. Concentrated Extract Storage The recoveries obtained for the pesticides stored in the dry extract are given in Table 4 for the pesticides in which no interaction between storage time and temperature was encountered and in Table 5 for the chemicals with a statistically significant interaction.For this storage treatment, three additional organophosphorus and one pyrethroid pesticide not considered in the cartridge storage were included: dimethoate, formothion, fenthion and deltamethrin. These compounds could only be recovered between 12 and 59% from water samples after the whole SPE Fig. 1 Gas chromatogram of a standard pesticide solution, at the concentrations shown in Table 1, with electron capture (ECD) and flame photometric detection (FPD).TR, trifluralin; L, lindane; TL, triallate; A, alachlor; IS, bromophos (internal standard); CP, captan; FP, folpet; OAE, o,pA-DDE; PAE, p,pADDE; OX, oxyfluorfen; OAT, o,pA-DDT; PAT, p,pA-DDT; BP, bromopropylate; DF, dicofol; TF, tetradifon; DT, deltamethrin; D, dimethoate; FO, fonofos; DZ, diazinon; FR, formothion; FN, fenitrothion; M, malathion; FT, fenthion; CL, chlorpyriphos; MT, methidathion; PM, phosmet; AZ, azinphos-methyl; and PS, phosalone.Analyst, January 1997, Vol. 122 9process,20 which did not allow an appropriate study of their stability when retained on the cartridge packing. Pesticides stored after evaporation to dryness in general give good recoveries. In this experiment, no real extraction in water was carried out and only the effect of storage on the simulated eluate was studied. Table 4 indicates that for this storage factor, higher recoveries were obtained on storage at 218 °C.Seven out of the 20 compounds listed are affected by storage temperature. Nevertheless, the difference in the recovery for the two temperatures is !10% only for captan, folpet, dimethoate, formothion and fenthion. In Fig. 2(B), decrease in recovery of the last three pesticides after storage for 30 d at 4 °C is shown. Captan and folpet, which already showed instability during cartridge storage, together with the last three organophosphorus pesticides, especially fenthion, which were not included previously, appear to be difficult to stabilize also in this dried form.For captan, the reason cannot be hydrolysis, because no water was present. Volatilization is also possible, but again captan, folpet and fenthion, with differences in recoveries > 25% for the two temperatures considered, have low vapour pressures (Table 1). Molecular structure may be responsible for the instability, as has already been reported for fenthion.5 The effect of storage time for the same chemicals is also shown in Table 4.Many pesticides show a statistically significant difference among the storage periods investigated but, for most of them, the recoveries fall within a 10% variation. Only for dimethoate, formothion and fenthion was the loss Table 4 Effect of storage temperature and storage time on recoveries of pesticides stored after their concentration to dryness Recovery (%) Storage Storage temperature/°C* time/d† Pesticide 218 4 LSD‡ 3 7 30 LSD‡ Alachlor 97 95 101 88 100 3.1 Captan 99 76 5.6 91 90 82 6.8 Folpet 101 74 7.3 93 87 83 o,pA-DDE 97 94 106 89 91 4.0 p,pA-DDE 98 96 1.8 100 89 102 2.2 Oxyfluorfen 96 94 96 96 93 2.7 o,pA-DDT 100 99 98 93 107 2.7 p,pA-DDT 101 98 99 92 108 3.9 Bromopropylate 104 101 101 97 111 6.8 Dicofol 105 98 5.3 97 99 108 6.5 Tetradifon 103 100 100 98 107 5.3 Deltamethrin 102 97 98 96 106 5.5 Dimethoate 93 80 9.9 86 98 75 12.1 Formothion 121 102 7.2 140 117 76 8.8 Malathion 105 105 103 100 111 3.9 Fenthion 74 33 8.0 64 53 43 9.9 Methidathion 101 99 106 99 96 5.0 Phosmet 107 108 109 104 111 Azinphos-methyl 102 103 104 103 101 Phosalone 106 107 105 101 114 5.4 * Average recovery for nine observations (three injections per observation).† Average recovery for six observations (three injections per observation). ‡ See Table 2. Fig. 2 Gas chromatograms of the pesticides stored A, in the cartridge for 30 d at 218 °C, with ECD, and B, in the concentrated extract at 4 °C for 30 d, with FPD.Abbreviations as in Fig. 1. Table 5 Effect of temperature and length of storage on recovery of pesticides stored after their concentration to dryness Recovery (%)* Refrigerator Freezer (4 °C) (218 °C) Pesticide 3 d 7 d 30 d 3 d 7 d 30 d LSD† Trifluralin 84 62 44 96 86 109 6.5 Lindane 78 58 47 93 84 97 5.9 Triallate 87 68 63 96 87 94 4.7 Fonofos 82 65 44 97 87 97 6.9 Diazinon 91 76 49 100 93 97 7.0 Fenitrothion 100 97 88 103 97 100 3.5 Chlorpyriphos-ethyl 101 95 86 98 94 107 6.3 * Average recovery for three observations (three injections per observation).† See Table 2. 10 Analyst, January 1997, Vol. 122> 10%. Nevertheless, except for fenthion, their recoveries were > 70%, which complies with EPA regulations.23 Table 5 shows the trends of a reduction in recovery with time and storage temperature which apply to all of the compounds. For trifluralin, lindane, triallate, fonofos and diazinon, the loss is already clear after 7 d of storage at 4 °C, and is more evident after 30 d with recoveries, in many cases, around 50%. If their vapour pressures are considered, a clear relationship between this property (Table 1) and pesticide loss can be observed.A high degree of correlation was found between vapour pressure and pesticide loss after 30 d at 4 °C for fonofos, triallate and chlorpyrifos. The losses of diazinon, trifluralin and lindane are higher than expected and those of fenitrothion are lower, if the only reason considered to be involved in the process was volatility.For diazinon, an imidoyl phosphate, it has been reported3 that the oxygen–aromatic moiety linkage may be activated and cleaved under both acidic and alkaline conditions to form the corresponding carbonyl compounds with a doublebond shift. This functional group is also present in the structure of chlorpyrifos, so the losses must be due to a mixture of both effects.In contrast, all the pesticides present in Table 5 show recoveries between 94 and 117% at 218 °C after 30 d of storage. It can be concluded from these results that the storage of dried eluates in the refrigerator may be problematic for volatile compounds, as already reported for chlormephos and dichlorvos (7600 and 1600 mPa, respectively) when stored in water samples5 or in disposable SPE cartridges.17 Nevertheless, each case should be studied carefully to verify the observed tendencies.The comparison with physico-chemical data provided in Table 1 indicates that, in general, the most volatile compounds are affected by storage in dried form in the refrigerator, although at 218 °C the decrease in recovery is almost negligible for all of them except for fenthion, which showed great instability, independently of the storage conditions. The storage of the dried eluates in the freezer at 218 °C seems to be a good alternative for these compounds, except for fenthion. Stability problems with triallate, tetradifon, diazinon, fenitrothion, malathion, chlorpyrifos-ethyl and fonofos, which could not be properly controlled when retained on the C18 cartridges, could be solved by storing them as dried eluates for up to 30 d at 218 °C.Conclusions Although immediate extraction and analysis are the best way of obtaining the most accurate residue data, this is not always possible. Therefore, two different storage conditions, corresponding to different stages in the over-all SPE process, were investigated. The results indicated that storage of different pesticide classes on the C18: silica gel surface of solid-phase cartridges for 30 d at 4 or 218 °C is effective for 17 out of the 23 pesticides studied.Triallate, captan, fenitrothion, malathion, chlorpyriphos and fonofos are affected. These pesticides, except for captan and fonofos, can be safely stored in the cartridges for up to 3 or 7 d, and their recoveries after 30 d are still > 70%.All of them, including captan and fonofos, could alternatively be stored for up to 30 d at 218 °C in the dried extract. For this kind of storage, only formothion and fenthion showed considerable losses along the storage period studied. Formerly, only a limited number of 1 or 2 l bottles could be stored in the laboratory. For both treatments considered here, owing to the small size of the cartridges and glass tubes, the space required for their storage has been reduced and a large number of samples can be preserved in a conventional freezer.The authors thank the Comisi�on Interministerial de Ciencia y Tecnolog�ýa (CICYT) for financial support (Project No. NAT91- 0407). Ma. D. Maroto is acknowledged for technical assistance and Ma. D. Mingorance is thanked for her assistance with the statistical analysis. References 1 Munch, D. J., and Frebis, C. P., Environ. Sci. Technol., 1992, 26, 921. 2 Ripley, B. D., Wilkinson, R.J., and Chau, A. S. Y., J. Assoc. Off. Anal. Chem., 1974, 57, 1033. 3 Chau, A. S. Y., Ripley, B. D., and Kawahara, F., in Analysis of Pesticides in Water, ed. Chau, A. S. Y., and Afghan, B. K., CRC Press, Boca Raton, FL, 1982, vol. II, pp. 61–154. 4 Barcel�o, D., Chiron, S., Lacorte, S., Mart�ýnez, E., Salau, J. S., and Hennion, M. C., TrAC, Trends Anal. Chem., (Pers. Ed.), 1994, 13, 352. 5 Lartiges, S. B., and Garrigues, P. P., Environ. Sci. Technol., 1995, 29, 1246. 6 Liska, I., Brower, E. R., Ostheimer, A. G. L., Lingeman, H., and Brinkman, U. A. Th.. Environ. Anal. Chem., 1992, 47, 267. 7 Bourne, S., J. Environ. Sci. Health, 1978, B13, 75. 8 Chau, A. S. Y., and Thomson, K., J. Assoc. Off. Anal. Chem., 1978, 61, 1481. 9 Lopez-Avila, V., Wesselman, R., and Edgell, K., J. Assoc. Off. Anal. Chem., 1990, 73, 276. 10 Barcel�o, D., House, W. A., Maier, E. A., and Griepink, B., Int. J. Environ. Anal. Chem., 1994, 57, 237. 11 Font, G., Ma�nes, J., Molt�o, J.C., and Pic�o, Y., J. Chromatogr., 1993, 642, 135. 12 Green, D. R., and Le Pape, D., Anal. Chem., 1987, 59, 699. 13 Berkane, K., Caissie, G. E., and Mallet, V. N., J. Chromatogr., 1977, 139, 386. 14 Tomkins, B. A., Merriweather, R., Jenkins, R. A., and Bayne, C. K., J. Assoc. Off. Anal. Chem. Int., 1992, 75, 1091. 15 Senseman, S. A., Lavy, T. L., Mattice, J. D., Myers, B. M., and Skulman, B. W., Environ. Sci. Technol., 1993, 27, 516. 16 Johnson, W. J., Lavy, T.L., and Senseman, S. A., J. Environ. Qual., 1994, 23, 1027. 17 Lacorte, S., Ehresmann, N., and Barcel�o, D., Environ. Sci. Technol., 1995, 29, 2834. 18 The Pesticide Manual. A World Compendium, British Crop Protection Council, Thornton Heath, UK, 8th edn., 1987. 19 Noble, A., J. Chromatogr., 1993, 642, 3. 20 de la Colina, C., S�anchez-Rasero, F., Dios, G., Romero, E., and Pe�na, A., Analyst, 1995, 120, 1723. 21 de la Colina, C., Pe�na, A., Mingorance, M. D., and S�anchez- Rasero, F., J.Chromatogr. A, 1996, 733, 275. 22 Belisle, A. A., and Swineford, D. M., Environ. Toxicol. Chem., 1988, 7, 749. 23 Barcel�o, D., J. Chromatogr., 1993, 643, 117. Paper 6/05275D Received July 29, 1996 Accepted September 30, 1996 Analyst, January 1997, Vol. 122 11 Effect of Storage on the Recovery of Different Types of Pesticides Using a Solid-phase Extraction Method C. de la Colina, F. S�anchez-Rasero, G. Dios, E. Romero and A. Pe�na* Estaci�on Experimental del Zaid�ýn (CSIC), Profesor Albareda, 1, E-18008 Granada, Spain Recoveries of different pesticide groups after storage either on C18 cartridges or as dried residues from organic solutions, and their analysis by gas chromatography with electron capture and flame photometric detection, were studied.Two storage temperatures, 4 and 218 °C, and three storage periods, 3, 7 and 30 d, were considered. The effect of storage temperature and storage time on the recovery of 27 pesticides in water was investigated.In general, the pesticide recoveries were !70% after 30 d of storage at 218 °C on C18 cartridges. Exceptions included captan and folpet. The storage of the dried residues generally did not affect the pesticide recovery when kept at 218 °C for up to 30 d. Keywords: Sample handling; sample storage; pesticide stability; water; solid-phase extraction; gas chromatography For analytical data to be valid, they should reflect the concentration of pesticides at the time of sampling, but often in a laboratory, owing to temporary shortages of personnel, problems with or breakdown of analytical equipment or sudden unexpected requirements for equipment to be used for other work, samples must be stored for variable periods before their analysis.For this purpose, and also when samples must be transported to other national or foreign laboratories, it is essential to know how long the content of a sample may remain unchanged. Therefore, stability studies of pesticides and organic contaminants in water and other matrices are important.In a recent report from the US Environmental Protection Agency (EPA),1 in which 147 pesticides in water samples were checked for stability for at least 14 d at 4 °C, 26 pesticides were removed from the list because of a 100% loss, even after biological inhibition of the water microorganisms. Research on the storage of pesticides in different water samples has been undertaken,2–5 that confirmed the instability of many pesticides in natural waters, depending on microbial degradation, hydrolysis and photolysis.To compensate for this instability, various stabilizers or preservatives have been added to aqueous samples, e.g., methanol,6 dichloromethane,7 dilute acid solutions1,8 or HgCl2,1,9 or the sample has been freezedried10 to retard the decomposition of the constituent chemicals. An alternative could be the preservation of the pesticides retained in solid-phase extraction (SPE) cartridges or discs, which are being increasingly used in environmental laboratories, 11 a possibility already used for hydrocarbon samples and some pesticides.9–17 The compounds of interest have been effectively preserved from microbial degradation for up to 54 d on these adsorbents12 and photolysis is avoided because the discs and cartridges are usually stored in the dark.It has been speculated that this preservative effect is a result of the trapping of the organics within the lattice structure of the adsorbent, although protection against hydrolysis is still in question.15,17 In addition, the processed samples may be preserved as organic extracts.1 It was reported that analytes generally remained stable in stored sample extracts, although no information was given about how long and for which analytes this kind of storage was effective.In this work, 27 pesticides, including nine organophosphorus, six organochlorine, a carbamate, a pyrethroid and another seven pesticides of miscellaneous groups, were studied at concentrations between 20 and 100 ng l21.Two ways of storing the different pesticides present in water samples were considered: on C18 SPE cartridges and as organic extracts concentrated to dryness. Experimental Reagents All the pesticides were of > 98.5% purity and all the solvents were of pesticide residue analysis grade. C18 extraction cartridges (J. T. Baker, Phillipsburg, NJ, USA) with 500 mg of packing material were used.Cartridge Storage Water samples of 1 l, obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA), were fortified with 20–100 ng of the chemicals (see Table 1). The cartridge was conditioned by rinsing it with the eluents in reverse order of elution, then with methanol and water.20 After the passage of the 1 l fortified water sample, the cartridge was air dried for 30 min. Solid-phase cartridges on which pesticides had been retained were wrapped in Parafilm and stored, at 4 or 218 °C, for 3, 7 and 30 d.At the end of the storage period they were eluted with ethyl acetate and isooctane and evaporated to dryness under a gentle stream of nitrogen. The final residue was taken up with 1 ml of hexane and the internal standard, bromophos, was added before injection into the gas chromatographs.21 Concentrated Extract Storage A mixture of the pesticides in the amounts shown in Table 1 was added to 4 ml of a mixture of the eluents [ethyl acetate– isooctane (1 : 1)] and then concentrated to dryness under a gentle stream of nitrogen. The glass tubes containing the dried eluates were fitted with a glass stopper, covered with Parafilm and stored at the same temperatures and for the same periods of time as used for the cartridges.The dried residue was treated as for the cartridge. For both storage treatments, cartridges and concentrated extracts, the remainder of the analytical process was finished rapidly with injection into the chromatograph on the same day.Statistical Analysis A completely randomized design with three replications of the whole analytical procedure, with three injections per replicate, of two storage temperatures and three storage times was Analyst, January 1997, Vol. 122 (7–11) 7employed for each storage treatment. Mean percentage recoveries were calculated and separated by Fisher’s least significant difference (LSD) at the 0.05 level of significance. Analytical Methodology All the samples were quantified by GC.20,21 Organophosphorus pesticides were determined using a Hewlett-Packard (Avondale, PA, USA) gas chromatograph with a flame photometric detector (FPD), provided with an HP-1 capillary column (12 m 3 0.2 mm id, 0.33 mm film thickness) with the following oven temperature programme: 45 °C (1 min), increased at 30 °C min21 to 170 °C (2 min), at 4 °C min21 to 200 °C (2 min) and at 20 °C min21 to ctor and detector temperatures were 250 and 275 °C, respectively. The other pesticides were determined using a Hewlett- Packard gas chromatograph with an electron capture detector (ECD), in which an Ultra-2 capillary column (25 m 3 0.32 mm id, 0.17 mm film thickness) was installed, with the following oven temperature programme: 160 °C (1 min), increased at 4 °C min21 to 230 °C (2 min) and at 20 °C min21 to 280 °C (6 min).The injector temperature was 250 °C and the detector temperature was 300 °C.Results and Discussion The pesticides included in this study, were tested at concentrations lower than or equal to the maxima allowed by EU legislation. Such concentrations are at least 102–103 times more dilute than the values previously reported in some storage studies using extraction discs15,16 or cartridges.17 Therefore, the amounts used in this study approach legal conditions and avoid higher concentrations which can influence degradation, as has already been reported for some organophosphorus and carbamate pesticides in soil columns.22 Fig. 1 shows the separation of a pesticide standard solution, with the different detection methods, at the concentrations indicated in Table 1. Cartridge Storage The recoveries obtained for the pesticides retained on the C18 cartridges are given in Table 2 for the pesticides in which no interaction between storage time and temperature was encountered and in Table 3 for the chemicals with a significant interaction between the storage factors studied.As can be seen for the effect of temperature (Table 2), only alachlor, captan, o,pA-DDE and chlorpyrifos-ethyl show significant differences for the two temperatures studied. Nevertheless, for most of these chemicals only small differences in the recoveries for the two storage temperatures are observed (@10%). Captan, and the chemically related folpet, exhibited an important reduction in recovery, higher for the freezer (218 °C) than for the refrigerator (4 °C), for which no explanation has Table 1 Pesticide amounts used for both storage treatments and their physico-chemical properties. Data from ref. 18 Amount Vapour Water added/ pressure/ solubility/ Pesticide ng* mPa Log Kow mg l21 Trifluralin 20 9.5 4.0† 0.221 Lindane 20 5.6 3.7† 7.3 Triallate 40 16 na‡ 4 Alachlor 80 2.9 na 242 Captan 40 1.3 2.5† 3.3 Folpet 40 1.3 3.1 1 o,pA-DDE 20 na na na p,pA-DDE 20 0.025 5.7–7.0† na Oxyfluorfen 20 0.0267 4.5 0.116 o,pA-DDT 20 na 5.8† na p,pA-DDT 20 na 6.2–6.9† na Bromopropylate 20 0.011 5.4 < 0.5 Dicofol 40 0.053 4.3 0.8 Tetradifon 20 3.231025 4.6 0.08 Deltamethrin 40 0.002 4.6 < 0.0002 Dimethoate 100 1.1 0.7 23800 Fonofos 100 28 3.9 13 Diazinon 100 12 3.3 60 Formothion 100 0.113 na 2600 Fenitrothion 100 18 3.4 21 Malathion 100 5.3 2.9† 145 Fenthion 100 0.74 4.8 4.2 Chlorpyrifos 100 2.7 5.3† 1.4 Methidathion 100 0.25 2.2 200 Phosmet 100 0.065 3.0 25 Azinphos-methyl 100 0.18 3.0 28 Phosalone 100 < 0.067 4.3† 1.7 * The same amount was added to 1 l of water or to 4 ml of the desorption solution.† Data from ref. 19. ‡ Not available. Table 2 Effect of storage temperature and storage time on recoveries of pesticides retained on C18 cartridges Storage Storage temperature/°C* time/d† Pesticide 218 4 LSD‡ 3 7 30 LSD‡ Trifluralin 90 92 86 89 101 5.1 Lindane 96 97 95 97 98 Triallate 89 86 96 86 82 6.6 Atachlor 94 98 2.7 96 95 97 Captan 58 73 9.1 74 66 58 11.2 Folpet 38 50 50 41 47 o,pA-DDE 69 64 4.0 63 72 65 4.9 p,pA-DDE 67 66 64 67 68 Oxyfluorfen 85 84 81 84 88 3.5 o,pA-DDT 67 69 63 71 70 4.9 p,pA-DDT 70 69 68 71 70 Bromopropylate 84 83 84 84 84 Dicofol 87 86 85 86 87 Tetradifon 95 98 100 96 94 4.0 Diazinon 87 87 89 89 84 3.8 Fenitrothion 94 95 99 97 88 4.0 Malathion 94 94 98 98 87 3.6 Chlorpyriphos-ethyl 82 79 2.4 85 83 72 2.9 Phosmet 95 96 103 87 97 9.7 * Average recovery for nine observations (three injections per observation).† Average recovery for six observations (three injections per observation). ‡ LSD = least significant difference (P < 0.05). The LSD values are provided for those compounds with a significant difference between the means. Table 3 Effect of temperature and time of storage on recovery of pesticides retained on C18 cartridges Recovery (%)* Refrigerator Freezer (4 °C) (218 °C) Pesticide 3 d 7 d 30 d 3 d 7 d 30 d LSD† Fonofos 75 68 55 79 64 65 7.9 Methidathion 105 98 89 101 103 99 6.9 Azinphos-methyl 121 102 129 119 114 167 17.8 Phosalone 102 96 87 110 93 98 6.9 * Average recovery for three observations (three injections per observation). † See Table 2. 8 Analyst, January 1997, Vol. 122been found. In a previous study, stability problems with captan were reported (54% recovery on a C18 disc at 4 °C and 32% at 218 °C after 30 d of storage) and ascribed to hydrolysis under the C18 packing of the SPE disc15 and, later, to volatilization,17 although the vapour pressure of this fungicide ( < 1.3 mPa, Table 1) is lower than that of some other pesticides included in this study, which do not show a loss.In addition, the captan recovery is also affected by the storage time (Table 2), so this method of preservation is not appropriate for this pesticide. When captan was stored on extraction discs,15 removal of residual water was recommended. The length of the storage affects several compounds, apart from captan, as can be seen in Table 2.In general, for the pesticides affected (trifluralin, triallate, o,pA-DDE, oxyfluorfen, o,pA-DDT, tetradifon, diazinon, fenitrothion, malathion, chlorpyrifos- ethyl and phosmet), the recovery decreases with increasing storage time, but remains within a ±10–15% variation for the whole length of the storage period. This variation range is within that commonly found in other storage studies.15,17 In addition, the final recovery after 30 d remains over 70% (the minimum recovery required by the EPA regulations23), except for DDEs, with recoveries of !65%, and captan and folpet.Low recoveries have been also reported for DDE and DDT retained on Empore filters for up to 4 weeks, from waters spiked at 50 ng l21.14 Fonofos, methidathion, azinphos-methyl and phosalone showed a significant interaction between temperature and length of storage (Table 3) and therefore both factors cannot be considered individually.For fonofos, methidathion and phosalone, a trend of loss on storage in the refrigerator after 30 d was observed. For fonofos, its storage on a cartridge is not recommended, because its recovery is already below 70% after 7 d at the two temperatures investigated. A complete loss of fonofos, which is the most volatile pesticide included in the present study (Table 1), has been reported for storage in similar cartridges at 4 °C after 1.5 months.17 In the contrast, the same group17 reported complete recovery of fonofos when stored at 220 °C for 8 months.Our results show a 14% loss after 1 month of storage in the freezer. For azinphos-methyl, which is not completely separated from phosalone in the chromatographic column, an artifact appears after 30 d of storage and therefore its quantification was inaccurate. Fig. 2(A) shows the separation of the pesticide mixture with electron capture detection (ECD) after storage in the cartridge for 30 d at 218 °C.The recoveries of the fungicides captan and folpet are clearly affected. The additional peaks correspond to the organophosphorus pesticides that can be detected by ECD. As indicated previously, several organophosphorus pesticides included in the present study (azinphos-methyl, fenitrothion, malathion, diazinon and phosmet) have been eliminated from the US EPA survey list owing to their instability;1 the first three are nevertheless included in the 76/464/EEC Council Directive List of Pesticides to be monitored in the aquatic environment.23 In addition, phosmet has been repeatedly reported to be unstable on storage in water.2,5 These pesticides could be stabilized by storing them on the cartridge packing at 4 or 218 °C for at least 30 d.Nevertheless, certain losses have been observed for some of the pesticides, especially those with low partition coefficients, which confirms the results of Lacorte et al.,17 who pointed to hydrolysis and microbial degradation as the main factors causing instability with this kind of storage.Concentrated Extract Storage The recoveries obtained for the pesticides stored in the dry extract are given in Table 4 for the pesticides in which no interaction between storage time and temperature was encountered and in Table 5 for the chemicals with a statistically significant interaction. For this storage treatment, three additional organophosphorus and one pyrethroid pesticide not considered in the cartridge storage were included: dimethoate, formothion, fenthion and deltamethrin.These compounds could only be recovered between 12 and 59% from water samples after the whole SPE Fig. 1 Gas chromatogram of a standard pesticide solution, at the concentrations shown in Table 1, with electron capture (ECD) and flame photometric detection (FPD). TR, trifluralin; L, lindane; TL, triallate; A, alachlor; IS, bromophos (internal standard); CP, captan; FP, folpet; OAE, o,pA-DDE; PAE, p,pADDE; OX, oxyfluorfen; OAT, o,pA-DDT; PAT, p,pA-DDT; BP, bromopropylate; DF, dicofol; TF, tetradifon; DT, deltamethrin; D, dimethoate; FO, fonofos; DZ, diazinon; FR, formothion; FN, fenitrothion; M, malathion; FT, fenthion; CL, chlorpyriphos; MT, methidathion; PM, phosmet; AZ, azinphos-methyl; and PS, phosalone.Analyst, January 1997, Vol. 122 9process,20 which did not allow an appropriate study of their stability when retained on the cartridge packing.Pesticides stored after evaporation to dryness in general give good recoveries. In this experiment, no real extraction in water was carried out and only the effect of storage on the simulated eluate was studied. Table 4 indicates that for this storage factor, higher recoveries were obtained on storage at 218 °C. Seven out of the 20 compounds listed are affected by storage temperature. Nevertheless, the difference in the recovery for the two temperatures is !10% only for captan, folpet, dimethoate, formothion and fenthion. In Fig. 2(B), decrease in recovery of the last three pesticides after storage for 30 d at 4 °C is shown. Captan and folpet, which already showed instability during cartridge storage, together with the last three organophosphorus pesticides, especially fenthion, which were not included previously, appear to be difficult to stabilize also in this dried form. For captan, the reason cannot be hydrolysis, because no water was present.Volatilization is also possible, but again captan, folpet and fenthion, with differences in recoveries > 25% for the two temperatures considered, have low vapour pressures (Table 1). Molecular structure may be responsible for the instability, as has already been reported for fenthion.5 The effect of storage time for the same chemicals is also shown in Table 4. Many pesticides show a statistically significant difference among the storage periods investigated but, for most of them, the recoveries fall within a 10% variation.Only for dimethoate, formothion and fenthion was the loss Table 4 Effect of storage temperature and storage time on recoveries of pesticides stored after their concentration to dryness Recovery (%) Storage Storage temperature/°C* time/d† Pesticide 218 4 LSD‡ 3 7 30 LSD‡ Alachlor 97 95 101 88 100 3.1 Captan 99 76 5.6 91 90 82 6.8 Folpet 101 74 7.3 93 87 83 o,pA-DDE 97 94 106 89 91 4.0 p,pA-DDE 98 96 1.8 100 89 102 2.2 Oxyfluorfen 96 94 96 96 93 2.7 o,pA-DDT 100 99 98 93 107 2.7 p,pA-DDT 101 98 99 92 108 3.9 Bromopropylate 104 101 101 97 111 6.8 Dicofol 105 98 5.3 97 99 108 6.5 Tetradifon 103 100 100 98 107 5.3 Deltamethrin 102 97 98 96 106 5.5 Dimethoate 93 80 9.9 86 98 75 12.1 Formothion 121 102 7.2 140 117 76 8.8 Malathion 105 105 103 100 111 3.9 Fenthion 74 33 8.0 64 53 43 9.9 Methidathion 101 99 106 99 96 5.0 Phosmet 107 108 109 104 111 Azinphos-methyl 102 103 104 103 101 Phosalone 106 107 105 101 114 5.4 * Average recovery for nine observations (three injections per observation).† Average recovery for six observations (three injections per observation). ‡ See Table 2. Fig. 2 Gas chromatograms of the pesticides stored A, in the cartridge for 30 d at 218 °C, with ECD, and B, in the concentrated extract at 4 °C for 30 d, with FPD. Abbreviations as in Fig. 1. Table 5 Effect of temperature and length of storage on recovery of pesticides stored after their concentration to dryness Recovery (%)* Refrigerator Freezer (4 °C) (218 °C) Pesticide 3 d 7 d 30 d 3 d 7 d 30 d LSD† Trifluralin 84 62 44 96 86 109 6.5 Lindane 78 58 47 93 84 97 5.9 Triallate 87 68 63 96 87 94 4.7 Fonofos 82 65 44 97 87 97 6.9 Diazinon 91 76 49 100 93 97 7.0 Fenitrothion 100 97 88 103 97 100 3.5 Chlorpyriphos-ethyl 101 95 86 98 94 107 6.3 * Average recovery for three observations (three injections per observation).† See Table 2. 10 Analyst, January 1997, Vol. 122> 10%. Nevertheless, except for fenthion, their recoveries were > 70%, which complies with EPA regulations.23 Table 5 shows the trends of a reduction in recovery with time and storage temperature which apply to all of the compounds. For trifluralin, lindane, triallate, fonofos and diazinon, the loss is already clear after 7 d of storage at 4 °C, and is more evident after 30 d with recoveries, in many cases, around 50%. If their vapour pressures are considered, a clear relationship between this property (Table 1) and pesticide loss can be observed.A high degree of correlation was found between vapour pressure and pesticide loss after 30 d at 4 °C for fonofos, triallate and chlorpyrifos. The losses of diazinon, trifluralin and lindane are higher than expected and those of fenitrothion are lower, if the only reason considered to be involved in the process was volatility. For diazinon, an imidoyl phosphate, it has been reported3 that the oxygen–aromatic moiety linkage may be activated and cleaved under both acidic and alkaline conditions to form the corresponding carbonyl compounds with a doublebond shift.This functional group is also present in the structure of chlorpyrifos, so the losses must be due to a mixture of both effects. In contrast, all the pesticides present in Table 5 show recoveries between 94 and 117% at 218 °C after 30 d of storage. It can be concluded from these results that the storage of dried eluates in the refrigerator may be problematic for volatile compounds, as already reported for chlormephos and dichlorvos (7600 and 1600 mPa, respectively) when stored in water samples5 or in disposable SPE cartridges.17 Nevertheless, each case should be studied carefully to verify the observed tendencies.The comparison with physico-chemical data provided in Table 1 indicates that, in general, the most volatile compounds are affected by storage in dried form in the refrigerator, although at 218 °C the decrease in recovery is almost negligible for all of them except for fenthion, which showed great instability, independently of the storage conditions.The storage of the dried eluates in the freezer at 218 °C seems to be a good alternative for these compounds, except for fenthion. Stability problems with triallate, tetradifon, diazinon, fenitrothion, malathion, chlorpyrifos-ethyl and fonofos, which could not be properly controlled when retained on the C18 cartridges, could be solved by storing them as dried eluates for up to 30 d at 218 °C.Conclusions Although immediate extraction and analysis are the best way of obtaining the most accurate residue data, this is not always possible. Therefore, two different storage conditions, corresponding to different stages in the over-all SPE process, were investigated. The results indicated that storage of different pesticide classes on the C18: silica gel surface of solid-phase cartridges for 30 d at 4 or 218 °C is effective for 17 out of the 23 pesticides studied.Triallate, captan, fenitrothion, malathion, chlorpyriphos and fonofos are affected. These pesticides, except for captan and fonofos, can be safely stored in the cartridges for up to 3 or 7 d, and their recoveries after 30 d are still > 70%. All of them, including captan and fonofos, could alternatively be stored for up to 30 d at 218 °C in the dried extract. For this kind of storage, only formothion and fenthion showed considerable losses along the storage period studied.Formerly, only a limited number of 1 or 2 l bottles could be stored in the laboratory. For both treatments considered here, owing to the small size of the cartridges and glass tubes, the space required for their storage has been reduced and a large number of samples can be preserved in a conventional freezer. The authors thank the Comisi�on Interministerial de Ciencia y Tecnolog�ýa (CICYT) for financial support (Project No. NAT91- 0407). Ma. D. Maroto is acknowledged for technical assistance and Ma. D. Mingorance is thanked for her assistance with the statistical analysis. References 1 Munch, D. J., and Frebis, C. P., Environ. Sci. Technol., 1992, 26, 921. 2 Ripley, B. D., Wilkinson, R. J., and Chau, A. S. Y., J. Assoc. Off. Anal. 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Chem., 1988, 7, 749. 23 Barcel�o, D., J. Chromatogr., 1993, 643, 117. Paper 6/05275D Received July 29, 1996 Accepted September 30, 1996 Analys