Combined High Speed Liquid Chromatography and Bioassay for the Evaluation and Analysis of an Organophosphorus Larvacide R. A. Henry, J. A. Schmit, and J. F. Dieckman Instrument Products Division, E. I . du Pont de Nemours and Co., Wilmington, Dei. 19898
F. J. Murphey Department of Entomology, University of Delaware, Newark, Del.
High speed liquid chromatography has been used to detect and isolate impurities in the pesticide, Abate (o,O,O',O'-tetramethyl-0,O'-thiodi-p-phenylene phosphorothioate). The toxicity of the parent compound and each major impurity to mosquito larvae was determined in a bioassay experiment. The same chromatographic system was also used for the rapid quantitative determination of Abate in water. Samples were extracted with chloroform or n-heptane, concentrated by evaporation, and chromatographed without further cleanup. The ultraviolet photometer detector used in the study was sensitive to about 1 nanogram of Abate. The persistence of Abate was investigated In salt marsh ponds and also under simulated pond conditions in the laboratory. Abate was strongly associated with pond organic matter and dropped rapidly to a steady state concentration in the pond water. The level of Abate on mosquito larvae killed by the pesticide was investigated by an extraction technique and also by direct introduction of larvae into the inlet of the chromatographic column. Results indicated that Abate-killed larvae were concentrating the pesticide 100 times over the bulk pond concentration at normal field dosage levels.
FOR MANY YEARS, biological research to find nonchemical methods for insect control was minimal and the use of synthetic pesticides was indiscriminate and widespread. Recently, however, scientists have found that not only are insects building up tolerances to certain common pesticides, but also that pesticide residues are accumulating in the environment at an alarming rate. The Federal Governmnet has now restricted the use of DDT, aldrin, dieldrin, endrin, heptachlor and several other pesticides. These pesticides are being replaced by Sevin (Union Carbide), malathion, parathion, methoxychlor, Abate (American Cyanamid) and others which are less persistent in the environment. This paper will show the use of a powerful new analytical tool, high speed liquid chromatography, to investigate one of these new pesticides. Abate (0,0,0',0'-tetramethyl-O,O'-thiodi-p-phenylene phosphorothioate) is an organophosphorus insecticide that has found increasing use for control of mosquito larvae in natural waters. The structure of Abate is shown in Figure 1. Abate has several advantages over other pesticides used for mosquito control, perhaps the most important of which is that it is nonpersistent in the environment relative to the chlorinated hydrocarbons. In addition, Abate is an extremely effective control agent, being lethal to Aedes aegypti larvae in concentrations below 0.005 ppm. Also, Abate is not harmful to other inhabitants of the water and has a low mammalian toxicity ( I ) . In fact, its toxicity is so low that (1) T. B. Gaines, Renate Kimbrough, and E. R. Laws, Jr., Arch. Environ. Health, 14,283 (1967).
it has been proposed as a mosquito control agent for drinking water stored in cisterns and reservoirs (2-4). Abate is usually determined in water by an extraction procedure followed by thin-layer chromatography (5) or gas chromatography (6, 7). Thin-layer chromatography is slow and difficult to quantitate and gas chromatography is not generally reliable because of thermal instability of organophosphorus compounds. Also, both thin-layer and gas chromatography frequently exhibit high backgrounds when the insecticide must be extracted from an organic-rich matrix such as pond water. High speed liquid chromatography overcomes these problems and is an ideal method for the analysis of Abate and other thermally unstable compounds. The purity of Abate was investigated by liquid chromatography and the major impurities were isolated by collecting the peaks as they emerged from the detector. The parent compound and each impurity were then investigated in a bioassay experiment to determine toxicity to mosquito larvae. Also, a method was developed for the quantitative determination of Abate in water and a recovery experiment was undertaken to test the procedure. The analytical method was then used to investigate the persistence of Abate in pond water. Finally, the presence of Abate on killed larvae was investigated by an extraction procedure and by an in situ extraction in which larvae were placed directly into the injection port ofthe liquid chromatograph. EXPERIMENTAL,
Apparatus. A Du Pont Model 820 Liquid Chromatograph, which has been described in detail in a previous publication (8), was used throughout the study. The chromatograph was equipped with a high pressure, pulse-free liquid pump which was capable of supplying very constant liquid flow at inlet pressures up to 3000 psig. Samples were introduced to the chromatographic column by a 5-pl high pressure syringe (Model 305N, Hamilton Co., Whittier, Calif.) and a lowdead-volume injection port. Analytical columns were 1 m X 2.1 mm i.d. Trubore stainless steel and were packed with Zipax @u Pont) chromatographic support which had been coated 1.0 wt with @,@'-oxydipropionitrile(BOP). A precolumn to prevent stripping of the analytical column by (2) G. D. Brooks and H. F. Schoof, Proceedings of the Fifty-
second Annual Meeting of the New Jersey Mosquito Exterminating Assoc., Atlantic City, N. J., March 24-26, 1965. (3) W. L. Jakob, Mosquito News, 25, 316 (1965). (4) E. R. Laws, V. A. Sedlak, J. W. Miles, C. R. Joseph, J. R. Lacomba, and A. D. Rivera, Bull. WHO, 38, 439 (1968). ( 5 ) R. C. Blinn, J. Agr. Food Chem., 16, 441 (1968). (6) F. C. Wright, B. N. Gilbert, and J. C. Riner, ibid., 15, 1038 (1967). (7) W. E. Dale and J. W.Miles, ibid., 17,60 (1969). (8) H. R. Felton, J. Chromatogr. Sci., 7,13 (1969).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
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Figure 2. Impuritiw in Abete by liquid chromatography the mobile phase was packed with Chromosorb WAW (80-100 mesh) which had been coated 30 wt % with BOP. An ultraviolet photometer with a 254-nm low pressure mercury source and a low-dead-volume Row cell was used as the detector throughout the study (9). A manuallyoperated fraction collection valve was located a t the detector outlet to collect the peaks as they emerged. Reagents and Materials. Analytical columns packed with BOP-coated Zipax and precolumns packed with BOP-coated Chromosorb WAW are commercially available (E. I. du Pont de Nemours & Co., Instrument Products Div., Wilmington, Del.). Heptane, which was used as the mobile phase in the chromatographic experiment, was Eastman reagent grade (Fisher Chemical Co.). Abate was technical grade (American Cyanamid Co.) and was supplied as a 39 wt. solution in an aromatic petroleum solvent. Larvae for the bioassay experiments were supplied by the Entomology Department of the University of Delaware. RESULTS AM) DISCUSSION
Bioactivity of Abate and Major Impurities. Several impurities in technical grade Abate were separated from the parent compound by high speed liquid chromatography. The peaks were collected and introduced to mosquito larvae (9) J. J. Kirkland, ANAL.CHDM., 4@, 391 (1968).
to the parent compound, showed larvacidal activity. Fivemicroliter samples of 0.05% (w/v) and 0.5% (w/v) Abate in chloroform were chromatographed to simulate field dosage level and ten times field dosage level in the beakers. Typical field dosage level for Abate in salt marsh ponds is estimated to be 0.02 ppm. Figure 2 shows the chromatogram of technical grade Abate with the peaks numbered in the order in which they were collected. Peaks were collected in small vials, evaporated to near-dryness, and sealed under nitrogen. An unchromatographed sample of Abate was also injected into a vial and sealed to verify the larvacidal activity of technical grade Abate. A mobile phase b1ar.k was collected before the Abate sample was chromatographed to show that the small amount of BOP stationary phase that was dissolved in the mobile phase was not harmful to the larvae and also after the Abate sample was chromatographed to verify that the chromatographic system had not become contaminated by Abate during the experiment. The contents of each of the vials were washed with a minimum quantity of acetone into a beaker containing 200 ml of water and 25 mosquito larvae. Blank experiments were run to establish that the quantity of acetone used to rinse the vials was not harmful to the larvae. The beakers were allowed to stand for 24 hours and the dead larvae were counted. The data are presented in duplicate in Table I with the peaks identified by the numbers assigned in Figure 2. The results of the bioassay experiment indicate that the parent compound (peak 4 in Figure 2) is the only fraction that exhibits definite larvacidal activity. In every other case where fractions showed some larvacidal activity, the dead larvae were small ones (first instar) which were probably killed by residual BOP from the chromatographic fraction, or by the acetone that was used to rinse the fraction into the beaker. In support of this conclusion, some mortality of small larvae was observed in the blank collected after chromatographing the 0.5 Abate sample. Quantitative Determination of Abate in Water. The same liquid chromatographic system used in the bioassay experiment was also used as the basis for a quantitative procedure to determine Abate in water. Initial experiments were carried out in micro-marsh ponds which had been constructed a t the University of Delaware for graduate research in toxicity of pesticides (10). Each pond could be prepared to simulate the salt marsh ponds of Delaware where mosquitos breed and mosquito control agents can be effectively em-
(10) F. J. Murphey, “Toxicity of Mosquito Control Chemicals to Salt Marsh Wildlife,” Proc. Twelfth Ann. Meeting Northeastern Mosquito Control Assoc., 1966, pp 1-14.
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Table I. Mortality of Mosquito Larvae as an Indication of Toxicity of Chromatographic Fractions Mortality 24 hr Mortality 24 hr Fraction A B Concn Fraction A B 0 0.05 Blank 0 Blank 0 0 16 0 Peak 1 0 0 Peak 1 0 0 Peak 2 26 18 Peak 2 24 0 Peak 3 Peak 3 0 8 100 100 Peak 4 100 Peak 4 (Abate) 100 0 0 Peak 5 Peak 5 0 0 0 0 Blank Blank 6 0 100 100 Unchromatographed Unchromatographed 100 100 sample sample ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
Conlrol Pond 0 ppm 0 hrs.
Figure 3. Determination of Abate in salt marsh pond extrectr
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Heptane was the preferred extraction solvent in this investigation because heptane was also used as the mobile phase in the chromatographic experiment; therefore heptane extracts gave smaller chromatographic backgrounds. A low background is particularly important when operating at high detector sensitivities. In the quantitative extraction procedure, a 100-ml sample of pond water along with suspended organic matter was acidified and placed in a separatory funnel with 50 ml of heptane. Abate was extracted into the heptane layer by shaking vigorously for three one-minute periods, allowing the two layers to completely separate between shakings. The bottom aqueous layer was drawn off and discarded. When chloroform was used as the extraction solvent, the bottom layer contained Abate and was retained. The top heptane layer was then transferred to a beaker, and the separatory funnel was rinsed twice with several milliliters of acetone which were then added to the heptane extract to be certain that all of the Abate had been transferred from the separatory funnel to the beaker. No attempt was made to filter solids from extracts because they were not present in large amounts and also because loss of some Abate in the additional filtering step might occur. The extract was reduced in volume under a stream of nitrogen and transferred to a vial. The evaporation procedure can be accelerated, with little danger of losing Abate, by warming the extract in a water bath. The beaker was rinsed thoroughly with several milliliters of heptane, which were also transferred to the vial. The contents of the vial were reduced to dryness
pkjyed. Into three ponds was pipetted 0.026, 0.078, and 0.131 ppm Abate to simulate lX, 3X, and 5X field dosage. A fourth pond was left free of Abate for control purposes. Ponds were stirred gently to distribute the Abate, and 500-ml aliquots were removed from each pond and taken to the laboratory for extraction. A 100-ml portion of each pond was extracted vigorously with a single 50-ml portion of chloroform. The pond water was acidified with 5 ml of 1N HC1 before extraction to accelerate the formation of a clean interface between the solvents and improve the efficiency of the extraction procedure. The chloroform layer was removed and evaporated to 1 ml for chromatographic analysis. A 5-pl sample of each evaporated extract was chromatographed and the chromatograms are shown in Figure 3. No Abate was found in the control pond extract while Abate was detected in the extracts of the treated ponds in quantities proportional to the amount of Abate added originally to the pond. Proportionately smaller amounts of Abate were detected in 24-hour pond extracts. It was concluded then that the extraction procedure, coupled with high speed liquid chromatography, could be refined into an analytical method for the determination of Abate in water. Also, these preliminary results showed that Abate concentration in the pond water had dropped rapidly in 24 hours. Both hydrocarbons and chloroform can be used for the quantitative removal of Abate from water (5-7). A single 2xtractio:i procedure can be used because of the favorable distribution of Abate between these solvents and water.
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Figure 5. Abate concentration in pond water as a function of time
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steady state concentration of 0.02-0.04ppm in a few hours, No significant differences were observed in Abate persistence at the three temperatures. In the kinetic study, occasional high concentrations of Abate were found in the aliquots at all temperatures. It was discovered that these spurious points were most prevalent near the end of the kinetic experiment when only a few hundred milliliters were left in the beakers. It appeared that the high Abate concentrations in certain aliquots could be correlated with organic matter which had been inadvertently drawn into the pipet, especially near the end of the experiment when the water remaining in the beaker was heavy with sediment. In order to verify the observation, the water remaining in the bottom of a beaker after a kinetic experiment was filtered, and both the filtrate and organic residue were extracted with chloroform. Figure 6 shows that the organic residue still contained a large percentage of Abate 96 hours after addition to the pond water. It can be concluded that, while some hydrolytic and oxidative degradation undoubtedly takes place, the majority of Abate added to mosquito ponds is adsorbed by organic matter, and then slowly released into the pond over a longer period of time. The apparent affinity of Abate for organic matter might also be helpful in explaining its toxicity to mosquito larvae at very low concentrations. If Abate concentrates on mosquito larvae or on microorganisms which are fed upon by the larvae, then the effective concentration in the vicinity of a larva could be very high. Detection of Abate on Dead Larvae, The high sensitivity of the chromatographic system was used to detect Abate on large larvae (fourth instar) which had been killed by the
at ambient temperature with a stream of nitrogen, and an appropriate aliquot of heptane was pipetted accurately into the vial. The vial was tightly stoppered and vigorously agitated for a few minutes to dissolve the Abate prior to injecting into the liquid chromatograph. The efficiency of the above extraction procedure using chloroform and heptane extriction solvents is shown in Figure 4. Aliquots of aqueous Abate stock solutions were pipetted into stirring beakers containing water. Identical aliquots were pipetted into vials, evaporated to dryness at 50 "C with a stream of nitrogen and dissolved in 1 ml of chloroform and heptane, respectively, to serve as reference standards for the extraction procedures. After stirring vigorously for a few minutes, the contents of the beakers were transferred to separatory funnels and extracted. The samples from the extractions were then compared with the reference samples. If an emulsion forms during the extraction procedure so that a sharp interface between the two extraction layers is not achieved, a second extraction may be required. Kinetic Study. Abate was added to pond water in glass beakers and samples were removed at regular intervals to determine the persistence of Abate under simulated pond conditions. Abate was determined in the pond samples by the extraction method described above. Diuron was used as an internal standard in the liquid chromatographic experiment. Concentration of Abate us. time is shown in Figure 5 for three different temperatures covering the extremes normally encountered in Delaware ponds during the mosquito breeding season. The curves in Figure 5 show that the Abate concentration in the pond water drops quickly from an initial level of 0.1 ppm immediately after application, to a
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Figure 7. Detection of Abate in extract of whole larvae pesticide. Five Abate-killed larvae and five control larvae were extracted with a minimum quantity of chloroform, which was then reduced to 10 microliters and injected into the liquid chromatograph. The chromatograms in Figure 7 indicate about 5 nanograms of Abate in the extract of the pesticide-killed larvae. Assuming an average larva weight of 1 rng, the concentration of Abate on each of the five larva was approximately 1 pprn. This was about 100 times the concentration of Abate in the water which was used to kill the larvae. Similar results were obtained by placing freshly killed and control larva directly in the inlet of the chromatographic column, and pressurizing the system to effect an instantaneous extraction by the heptane mobile phase. Figure 8 shows the chromatograms obtained by this in situ extraction method. The quality of the chromatograms is not as good as in Figure 7 because the base line requires several minutes to stabilize after starting the flow at maximum sensitivity (0.005 absorbance unit full
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Figure 8. Detection of Abate on larvae by an in situ extraction in the inlet of a chromatographiccolumn scale). Also contributing to base-line disturbance were air bubbles, which were introduced while positioning larvae in the injection port. Results obtained by the in situ method were in qualitative agreement with those found by the extraction method. From these data, it appears that the larva are concentrating Abate about 100 times the bulk pond concentration. Further work would have to be done to determine whether the pesticide is concentrated on the surface of, or inside, the larva. ACKNOWLEDGMENT
The authors thank Robert W. Lake and Kenneth M. Lornax of the Department of Entomology, University of Delaware, for their assistance. RECEIVED for review February 12, 1971. Accepted April 22, 1971.
Laser Pyrolysis Gas Chromatography-Application to Polymers 0. F. Folmer, Jr. Continental Oil Company, Ponca City, Okla. 74601
I n a continuation of previous work, the effects of different operating conditions and methods of sample preparation on fragmentation patterns have been studied. Clear or translucent samples give reproducible results if mixed with carbon. The concentration of carbon is critical: it has a reat effect on the fragmentation pattern. Some poyymers were run whose pyrolysis chromatograms show great difference in pattern. Others had patterns which were quite similar. To better compare these similar patterns and the patterns arising from different operating conditions, a statistical method was devised. An attempt was made to correlate these comparisons of patterns with some of the known characteristics of the polymers.
PREVIOUS PUBLISHED WORK (1-4) on laser pyrolysis has pointed out some of the advantages of laser pyrolysis (rapid heating (1) 0. F. Folmer, Jr., and Leo V. Azarraga, J. Chrornatogr. Sci., 7. 665 11969).
(2)'Bohdan T:-Guran, Robert J. O'Brien, and Don H. Anderson, ANAL. CHEM., 42, 115 (1970). (3) Tsugio Kojima and Fujio Morishita, J. Chromatogr. Sci., 8, 4710 (1970). --(4) William T. Ristau and Nicholas E. Vanderborgh, ANAL. CHEM., 42, 1848 (1970). -I.
and cooling of the sample, and relatively simple fragmentation patterns) and some of the disadvantages (dependence on sample color). That work was largely qualitative in nature depending upon correlation of data by inspection of pyrolysis chromatograms. The work reported here is a continuation of that of Folmer and Azarraga (I). It is an attempt to describe results in numerical terms ; to quantitatively correlate the fragmentation patterns with operating conditions and with sample structure. The effects of various operating parameters and methods of sample preparation on fragmentation patterns were investigated. The knowledge gained was used in applying laser pyrolysis to some polymer samples. EXPERIMENTAL
A Gen-a-lite Model 3R laser (General Laser Corp., Natick, Mass. 01760) and a Focuscope (General Laser Corp.) focusing device were used. The laser uses a 31/&1, ruby rod with a maximum energy outut of 2 joules with a pulse length of 600 microseconds. The instruments were anounied on an optical rail so that the focused beam entered a box containing a ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
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