Kansas Water Science Center
USGS Water Science Centers are located in each state. |
U.S. Geological Survey
|
Multiply | By | To obtain |
---|---|---|
foot (ft) | 0.3048 | meter |
gram (g) | 0.002205 | pound (lb) |
liter (L) | 33.82 | ounce (oz) |
ounce (oz) | 10.02957 | liter (L) |
kilopascal (kPa) | 0.1450377 | pound per square inch (lb/in²) |
Miscellaneous Abbreviations |
---|
atomic mass units (amu) mass to charge (m/z) meter (m) micrometer (mm) milligram (mg) millimeter (mm) millisecond (ms) minute (min) second (sec) nanogram (ng) |
Abbreviated Water-Quality Units |
liter (L) microgram per liter (mg/L) microliter (mL) milligram per milliliter (mg/mL) mililiter (mL) nanogram per liter (ng/L) nanogram per microliter (ng/mL) |
The use of firm, trade, and brand names in this report is for identification purposes only and does not constitude endorsement by the U.S. Geological Survey.
A method of analysis and quality-assurance practices were developed for the determination of four mosquito insecticides (malathion, metho-prene, phenothrin, and resmethrin) and one synergist (piperonyl butoxide) in water. The analytical method uses liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). Good precision and accuracy were demonstrated in reagent water, urban surface water, and ground water. The mean accuracies as percentages of the true compound concentrations from water samples spiked at 10 and 50 nanograms per liter ranged from 68 to 171 percent, with standard deviations in concentrations of 27 nanograms per liter or less. The method detection limit for all compounds was 5.9 nanograms per liter or less for 247-milliliter samples. This method is valuable for acquiring information about the fate and transport of these mosquito insecticides and one synergist in water.
The persistence of organic pesticides in water is of great importance because of concerns over water quality. Pesticides that find their way into lakes, streams, or drinking-water supplies may pose a potential health threat to wildlife and humans. The U.S. Geological Survey (USGS), as part of the Toxic Substances Hydrology Program, has been studying the fate and transport of four mosquito insecticides and a synergist in the New York City metropolitan area.
Recently, there has been concern in the Northeastern United States about the appearance of the West Nile virus. The West Nile virus was first identified in Africa (Center for Disease Control, 2001a) and has since spread to temperate regions of Europe and North America. It is generally not dangerous to healthy humans but can develop into a deadly form of encephalitis (inflammation of the brain) in the elderly, children, and people with compromised immune systems. In the United States, West Nile virus is transmitted by infected mosquitoes, primarily members of the culex species (Center for Disease Control, 2001b).
A direct way to combat the spread or prevent a recurrent outbreak of West Nile virus is to control the mosquito population. One of the methodologies for control is the use of insecticides, either larvicides or adulticides.
Larvicides for mosquito control include metho-prene, an insect growth regulator. Methoprene controls mosquito larva populations by mimicking the natural juvenile growth hormone, JHIII. This hormone inhibits developing mosquito pupae from molting and passing into the adult stage where they could reproduce. Methoprene is available in suspension, emulsifiable, and soluble concentrate formulations, as well as in briquette, aerosol, and bait form. Methoprene was introduced in the late 1970s as a means of flea and mosquito control.
Adulticides, which may be used in the chemical control of mosquitos, include the organophosphate malathion and the pyrethroids phenothrin, also called sumithrin, and resmethrin. In addition, a synergist compound commonly is applied with pyrethroids to overcome resistance that pests develop with use of insecticides.
Malathion is a nonsystemic, wide-spectrum organophosphate insecticide. It was one of the earliest organophosphate insecticides developed (introduced in 1950). Malathion is used for the control of mosquitoes, flies, household insects, animal parasites (ectoparasites), and head and body lice.
Pyrethrins are natural insecticides in the flowers of certain species of the chrysanthemum plant. Semisynthetic derivatives of the chrysanthemumic acids have been developed as insecticides. These are called pyrethroids and tend to be more effective than natural pyrethrins, and they are less toxic to mammals. The most frequently used pyrethroids for adult mosquito control are phenothrin, also called sumithrin, and resmethrin.
Insects possess an enzyme system called the mixed-function oxidases (MFOs) that give them the ability to rapidly detoxify and become resistant to many insecticides, especially pyrethroids. Piperonyl butoxide (PBO) inhibits the action of MFOs, which allows the applicator to use less active ingredient to obtain the mortality rate desired or to prolong the usefulness of insecticides by overcoming MFO resistance. As is common with pyrethroid insecticides, the synergist compound piperonyl butoxide (PBO) is applied with phenothrin and resmethrin.
An analytical method and quality-assurance practices were developed for the determination of four mosquito insecticides and one synergist at nanogram-per-liter levels in water samples. The method involves using liquid-liquid extraction (LLE) to isolate the compounds from water samples and gas chromatography/mass spectrometry (GC/MS) to identify and quantify these compounds. Quality-control practices include evaluation of laboratory blank and spiked samples, instrument performance, and corrective actions. Method detection limits (MDLs) are calculated on the basis of procedures recognized by the U.S. Environmental Protection Agency (USEPA) (1992). Mean recoveries of the targeted insecticides and synergist from reagent, surface, and ground water also are presented.
The LLE-GC/MS method of analysis described in this report and used at the USGS Organic Geochemistry Research Laboratory in Lawrence, Kansas, has been assigned the method number "O-2134-01" by the USGS Office of Water Quality in Reston, Virginia. At the Organic Geochemistry Research Laboratory, the method of analysis described herein has been given the analysis code "GCM." This unique analysis code can be used to identify the method.
The method described in this report and used by the USGS Organic Geochemistry Research Laboratory is suitable for the determination of nanogram-per-liter concentrations of four mosquito insecticides and a synergist in filtered, natural water samples. Registry numbers and molecular weights are shown in table 1 for each compound determined by the method. This method is applicable to compounds that are (1) efficiently partitioned from the water phase by hexane liquid extraction and (2) sufficiently volatile and thermally stable for gas chromatography. Suspended particulate matter is removed from the samples by filtration, so this method is suitable only for dissolved-phase compounds.
[water-solubility data from Kidd and James (1991) except where noted; amu, atomic mass units;
mg/L, milligrams per liter; °C, degrees Celsius; CAS, Chemical Abstract Service;
<, less than]
Compound | Class | Molecular weight (amu) |
Weight solubility [mg/L (°C)] |
CAS registry number |
---|---|---|---|---|
Malathion | organophosphate | 330 | ¹145 (25) | 121-75-5 |
Methoprene | insect growth regulator | 310 | 1.4 (25) | 40596-69-8 |
Phenothrin | pyrethroid | 350 | <1 (30) | 26002-80-2 |
Piperonyl butoxide (PBO) | synergist | 338 | ²<.001 | 51-03-6 |
Resmethrin | pyrethroid | 338 | <1 (30) | 10453-86-8 |
¹ Tomlin (1997).
² Nature Conservation Council of New South Wales (2001).
Compounds were selected because of their potential use in controlling mosquitoes in the New York City metropolitan area. The calibration range for the method is equivalent to concentrations from 5 to 100 ng/L without dilution.
Water samples are filtered at the collection site using glass-fiber filters with 0.7-mm nominal pore diameter to remove suspended particulate matter. In the laboratory, a surrogate compound is added, and a small volume of sample is removed from the bottle. Then hexane is added directly to the remaining sample in the bottle and mixed. The hexane extract is removed, spiked with an internal standard, and evaporated under nitrogen. The sample components are separated, identified, and measured by injecting an aliquot of the concentrated extract into a high-resolution, fused-silica capillary column of a GC/MS system under selected-ion mode (SIM). Compounds eluting from the GC column are identified by comparing their measured ions and retention times to reference ions and retention times obtained by the measurement of control samples under the same conditions used for the water samples. The concentration of each identified compound is measured by relating the MS response of the quantitation ion produced by that compound to the MS response of the quantitation ion produced by the surrogate standard.
Organic compounds having identical mass ions and GC retention times to those of the compounds of interest may interfere.
Following USGS protocol, sampling methods capable of collecting water samples that accurately represent the water-quality characteristics of the surface water or ground water at a given time or location are used. Detailed descriptions of sampling methods used by the USGS to obtain surface-water samples are given in Edwards and Glysson (1988) and Ward and Harr (1990). Similar descriptions of sampling methods for obtaining ground-water samples are given in Hardy and others (1989).
Briefly, sample-collection equipment is free of tubing, gaskets, and other components made of nonfluorinated plastic material that might leach interferences into water samples or sorb organic compounds from the water. The water samples from each site are composited in a single container and filtered through a 0.7-µm glass-fiber filter using a peristaltic pump (Sandstrom, 1995). Filters are leached with about 200 mL of sample prior to filtration of sample. The filtrate for analysis is collected in baked 8-oz amber glass bottles with Teflon-lined lids. Samples are chilled immediately and shipped to the laboratory via an overnight carrier. At the laboratory, samples are logged in, assigned identification numbers, and extracted on the day they arrive.
(1),
RRTc = RTc/RTi,
where RTc = uncorrected retention time of the quantitation ion of the selected compound or surrogate compound, in minutes, and
where RTi = uncorrected retention time of the quantitation ion of the internal standard (phenanthrene-d10), in minutes.
See table 2 for an example of retention times, relative retention times, quantitation ions, and qualification ions.
[min, minute; m/z, mass to charge ratio; --, not applicable]
Compound | Retention time (min) |
Relative retention time (dimen- sionless) |
Quantita- tion ion (m/z) |
Qualification ion(s) (m/z) |
||||
---|---|---|---|---|---|---|---|---|
Insecticides and synergist (in order of increasing retention time) | ||||||||
Malathion | 15.720 | 1.170 | 173 | 127, 93, 158 | ||||
Methoprene | 17.320 | 1.289 | 73 | 111, 153, 191 | ||||
Pieronyl butoxide (PBO) |
21.710 | 1.615 | 176 | 177, 149, 119 | ||||
Resmethrin | 21.800 | 1.622 | 123 | 171, 143, 128 | ||||
Phenothrin I | 23.380 | 1.740 | 123 | 183, 81 | ||||
Phenothrin II | 23.580 | 1.754 | 123 | 183, 81 | ||||
Internal standard | ||||||||
Phenanthrene-d10 | 13.440 | 1.000 | 188 | -- | ||||
Surrogate standard | ||||||||
Terbuthylazine | 13.520 | 1.006 | 214 | 173, 229 |
Mass spectrometer performance is evaluated by assessing isotopic ratios, contamination, electron multiplier sensitivity, and abundance.
(2),
RT = (RRTc)(RTi),
where RT = expected retention time of the selected compound, in minutes
where RRTc = relative retention time of the selected compound, dimensionless; and
where RTi = uncorrected retention time of the internal standard, in minutes.
(3),
DF = (247 / (247 - Va) ,
where DF = dilution factor, and
where Va = volume added = milliliters of distilled water added to a sample that contains less than 247 mL.
The dilution factor is incorporated into the calculation for determining final concentrations of samples.
(4),
C = ((Ac/Ai)²+ (b) (Ac/Ai) + 0) x (DF) x (SC) ,
where C = concentration of the selected insecticide or synergist in the sample, in nanograms per liter;
where A = coefficient of x² in the quadratic curve fit;
where Ac= area of the quantitation ion for the selected insecticide or synergist identified;
where Ai = area of the quantitation ion of the surrogate standard, terbuthylazine;
where b = coefficient of x in the quadratic curve fit;
where DF = dilution factor as calculated in equation 3; and
where CF = slope correction.
The four insecticides and the synergist are reported in concentrations ranging from 5 to 100 ng/L. If the concentration is greater than 100 ng/L, the sample is reextracted with a 1:10 dilution (sample:distilled water) and reanalyzed for those compounds that were greater than 100 ng/L.
A reagent-water sample, a surface-water sample collected from the Kisco River below Mt. Kisco, New York, and a ground-water sample collected from a 27-ft deep well near Halstead, Kansas, were used to test the method performance. The surface- and ground-water samples were collected in 45-L carboys. Aliquots of each sample were fortified with either 10 or 50 ng/L of primary fortification standard. Then they were split into eight 247-mL samples at each concentration (10 and 50 ng/L). In addition, unfortified samples of reagent, surface, and ground water were extracted and analyzed to determine background concentrations of the pesticides. All samples were analyzed in one laboratory (the USGS Organic Geochemistry Research Laboratory in Lawrence, Kansas) using one GC/MS system. Each sample set was extracted and analyzed on different days from April through May 2001, so comparison of different matrices and concentrations included bias from day-to-day variation. Accuracy and precision data from the analyses are listed in tables 3, 4, and 5.
[ng/L, nanogram per liter]
Samples spiked at 10 ng/L | Samples spiked at 50 ng/L | |||||||
---|---|---|---|---|---|---|---|---|
Compound | Mean observed compound (ng/L) |
Standard deviation (ng/L) |
Relative standard deviation (percent) |
Mean accuracy (percentage of true con- centration) |
Mean observed compound (ng/L) |
Standard deviation (ng/L) |
Relative standard deviation (percent) |
Mean accuracy (percentage of true con- centration) |
Malathion | 11.4 | 1 | 12 | 114 | 52.1 | 9 | 17 | 104 |
Methoprene | 17.1 | 3 | 20 | 171 | 65.5 | 13 | 21 | 131 |
Phenothrin, total | 15.6 | 3 | 18 | 156 | 54.7 | 27 | 49 | 109 |
Piperonyl butoxide (PBO) | 12.2 | 1 | 10 | 122 | 59.6 | 10 | 17 | 119 |
Resmethrin | 13.4 | 4 | 26 | 134 | 48.3 | 18 | 38 | 97 |
[ng/L, nanogram per liter]
Samples spiked at 10 ng/L | Samples spiked at 50 ng/L | |||||||
---|---|---|---|---|---|---|---|---|
Compound | Mean observed compound (ng/L) |
Standard deviation (ng/L) |
Relative standard deviation (percent) |
Mean accuracy (percentage of true con- centration) |
Mean observed compound (ng/L) |
Standard deviation (ng/L) |
Relative standard deviation (percent) |
Mean accuracy (percentage of true con- centration) |
Malathion | 10.2 | 1 | 14 | 102 | 47.8 | 9 | 18 | 96 |
Methoprene | 10.4 | 3 | 28 | 104 | 47.0 | 16 | 33 | 94 |
Phenothrin, total | 12.9 | 2 | 13 | 129 | 42.9 | 9 | 21 | 86 |
Piperonyl butoxide (PBO) | 11.4 | 1 | 6 | 114 | 55.5 | 8 | 14 | 111 |
Resmethrin | 11.8 | 3 | 25 | 118 | 41.3 | 14 | 33 | 83 |
[ng/L, nanogram per liter]
Samples spiked at 10 ng/L | Samples spiked at 50 ng/L | |||||||
---|---|---|---|---|---|---|---|---|
Compound | Mean observed compound (ng/L) |
Standard deviation (ng/L) |
Relative standard deviation (percent) |
Mean accuracy (percentage of true con- centration) |
Mean observed compound (ng/L) |
Standard deviation (ng/L) |
Relative standard deviation (percent) |
Mean accuracy (percentage of true con- centration) |
Malathion | 10.8 | 2 | 15 | 108 | 51.5 | 10 | 19 | 103 |
Methoprene | 10.2 | 3 | 34 | 102 | 50.4 | 22 | 43 | 101 |
Phenothrin, total | 11.9 | 4 | 33 | 119 | 33.8 | 10 | 30 | 68 |
Piperonyl butoxide (PBO) | 11.2 | 2 | 16 | 112 | 54.9 | 8 | 15 | 110 |
Resmethrin | 10.8 | 3 | 29 | 108 | 40.0 | 19 | 47 | 80 |
Corrections for background concentrations-Neither the surface- nor ground-water sample required correction for background concentrations of insecticides or synergist. The reagent-water sample also had no detections of insecticides or synergist.
Method detection limits (MDLs)-An MDL is defined as the minimum concentration of a substance that can be identified, measured, and reported with 99-percent confidence that the compound concentration is greater than zero. MDLs were determined according to procedures outlined by the U.S. Environmental Protection Agency (1992) using fortified reagent water. Two liters of reagent water were fortified with 5.0 ng/L of primary fortification standard and split into eight 247-mL samples. These were extracted and analyzed to determine MDLs (table 6). Each sample was analyzed on different days during April through May 2001, so day-to-day variation is included in the results.
The MDL was calculated using the following equation:
(5),
MDL = (S)t(n-1,1-a)=0.99) ,
where S = standard deviation of replicate analysis, in nanograms per liter, at the fortified concentration;
where t(n-1, 1-a= 0.99) = Student's t-value for the 99-percent confidence level with n-1 degrees of freedom (U.S. Environmental Protection Agency, 1992); and
where n = number of replicate analyses.
The estimated mean MDLs ranged from 1.7 to 5.9 ng/L (table 6). According to the U.S. Environmental Protection Agency (1992) procedure, the fortified concentrations should be no more than five times the estimated MDL. The fortified concentrations were within five times the MDL.
[ng/L, nanograms per liter; MDL, method detection limit]
Compound | Mean observed concentration (ng/L) |
Mean standard deviation (ng/L) |
MDL (ng/L) |
---|---|---|---|
Malathion | 5.5 | 1.2 | 3.7 |
Methoprene | 8.6 | 1.6 | 4.8 |
Phenothrin, total | 8.6 | 2.0 | 5.9 |
Piperonyl butoxide (PBO) | 5.8 | .6 | 1.7 |
Resmethrin | 7.1 | 1.8 | 5.3 |
Mean accuracy-Mean accuracy in reagent-, surface-, and ground-water samples was determined by comparing the mean observed concentration (see "Quantitation" section) from eight replicate samples to the spiked concentration. Mean accuracy as a percentage of the true concentration was best in surface water fortified at 50 ng/L (table 4). The mean accuracy of all compounds spiked at the concentrations in tables 3, 4, and 5 were averaged to calculate the mean recovery for the three matrixes. Mean recoveries in reagent-water samples were farther from 100 percent than the mean recoveries in surface- and ground-water samples. The mean recovery in reagent water was 139 and 112 percent at 10 and 50 ng/L, respectively. The mean recovery in surface water was 113 and 94 percent at 10 and 50 ng/L, respectively. The mean recovery in ground water was 110 and 92 percent at 10 and 50 ng/L, respectively.
Extraction absolute recovery-Absolute recovery of each insecticide and synergist was determined by comparing standard curves (0 to 50 ng/L) prepared internally and externally to the extraction procedure. The same mass of compound from the primary fortification standard was added either to a reagent-water sample or directly to a test tube spiked with internal standard (phenanthrene-d10). The internal standard curve samples were processed using the aforementioned extraction procedure. Then both standard curves were injected on the GC/MS. For each compound in each standard curve, a graph was made with the ratio of the area of the compounds'quantitation ion divided by the area of the quantitation ion of the internal standard. A linear best-fit trend line was calculated for each graph. Finally, the slope of the internal standard curve was divided by the slope of the external curve for each compound to determine the absolute recovery for that compound. Absolute recoveries are listed in table 7. Absolute recovery is different than mean accuracies listed in tables 3, 4, and 5 in that mean accuracies are calculated from an initial calibration curve that is processed in the same manner as the samples, thus correcting for routine analyte losses.
Compound | Absolute recovery (percent) |
---|---|
Malathion | 77 |
Methoprene | 64 |
Phenothrin, total | 84 |
Piperonyl butoxide (PBO) | 98 |
Resmethrin | 86 |
Quality-control data are produced to quantitatively check the measurement process for environmental samples. The types of quality-control data collected include results of the analysis of duplicate samples, matrix-spiked samples, laboratory blank samples, and controls of differing concentrations.
Each extraction set of as many as six samples contains a minimum of one duplicate sample. The duplicate samples are analyzed concurrently and reanalyzed if agreement of the calculated concentration for any detected insecticide or synergist is not within 40 percent, as determined by the relative percentage difference.
(6),
RPD = | X1 - X 2 / X | x 100 ,
where RPD = relative percentage difference;
where |X1 - X 2| = absolute value of the difference between the two values; and
where X = mean of the two values.
Recovery of all target compounds is determined for each matrix-spiked sample. After the water sample is received in the laboratory, 12.35 µL of the primary fortification standard are added prior to extraction. Any compounds present in the unspiked sample are subtracted from the matrix-spiked sample's values. These final concentration values are reported.
Laboratory blank samples are used to demonstrate that laboratory equipment or instruments are cleaned adequately and that no contamination is contributed by the laboratory procedures. A laboratory blank consists of reagent water that is processed exactly like samples. If any insecticide or synergist is detected at any concentration greater than the MDL in the laboratory blank control, the source of the problem is determined and corrected. Samples analyzed in that extraction set then are reevaluated for contamination.
Low and high concentration controls are used to verify the calibration curve being used for quantification. The recoveries for each insecticide and synergist are determined. A new calibration curve is prepared if the recovery is outside the control limits in two consecutive runs. Control limits are initially set at ±20 percent until an adequate number of controls have been analyzed to calculate a relevant standard deviation. Control warning limits are set at ±1.5 standard deviations from the mean and the control limits at ±2 standard deviations from the mean.
Recovery of the surrogate, terbuthylazine, is determined for each sample, including all control samples. Control charts for the terbuthylazine recovery are constructed using the mean; the warning limits are set at 1.5 standard deviations from the mean and the control limits at ±2 standard deviations from the mean. The control charts are constructed using all previous sample terbuthylazine recoveries. A sample is reextracted and reanalyzed on the GC/MS if the recovery is outside the control limits.
This report presents a method of analysis and quality-assurance practices for the determination of four mosquito insecticides and one synergist in natural water samples. From the data presented in this report, liquid-liquid extraction with gas chromatography/ mass spectrometry detection are shown to be a sensitive and reliable method for the determination of nanogram-per-liter concentrations. Good precision and accuracy were demonstrated. Method detection limits ranged from 1.7 to 5.9 ng/L. The mean accuracies of the mosquito insecticides and synergist from water samples spiked at 10 and 50 ng/L ranged from 68 to 171 percent, with relative standard deviations of 6 to 49 percent. Information about the fate and transport of the four mosquito insecticides and one synergist in water can be acquired from the analysis of surface- and ground-water samples. These methods also can be useful for water-quality determinations and analytical verification in toxicological studies.
For additional information contact:
Betty Scribner
U.S. Geological Survey
4821 Quail Crest Place
Lawrence, KS 66049-3839
Telephone: (785) 832-3564
Fax: (785) 832-3500
Email: scribner@usgs.gov