PESTICIDES - Revised methods for analysis of water, suspended sediment, and bottom material July 6, 1973 QUALITY OF WATER BRANCH TECHNICAL MEMORANDUM N0. 74.0l Subject: PESTICIDES - Revised methods for analysis of water, suspended sediment, and bottom material The attached write-ups describe the current methodology for analysis of pesticides in water, suspended sediment, and bottom material. These sections replace the indicated pages in TWRI Book 5 Chapter A3. R. J. Pickering Chief, Quality of Water Branch Attachments (2) WRD Distribution: A, FO-L, PO (HARD COPIES NOT AVAILABLE IN OFFICE OF WATER QUALITY) Replaces pages 30-32 in TWRI Book 5 Chapter A3 INSECTICIDES IN WATER Gas chromatographic method 1. Summary of method Insecticides are extracted from the filtered water sample with n-hexane. After drying and removing the bulk of the solvent, the insecticides are isolated from extraneous material by microcolumn adsorption chromatography on alumina. Semi-microcolumn absorption chromatography is employed for the analysis of insecticides in waters that contain large amounts of organic compounds other than pesticides. Polychlorinated biphenyl and chloronaphthalene compounds, which otherwise interfere with the analysis, are separated on a silica-gel column. Interferring sulfur and organosulfur compounds, if present, may also be removed. The insecticides are determined by gas chromatography. This method is a modification and extension of the procedures developed by Lamar, Goerlitz and Law (1965, 1966). 2. Application This method is usable for the analysis of water only. The insecticides and associated chemicals (aldrin, p,p', DDD, p,p'- DDE, o,p'-DDT, p,p'-DDT, dieldrin, endrin, heptachlor, heptochlor epoxide, isodrin, lindane (BHC), and methoxychlor) may be determined to 0.005 ug/l in l-liter water samples. The insecticides carbphenothion, chlordan, dioxathion, diazinon, ethion, malathion, methyl parathion, Methyl Trithion, parathion, toxaphene, and VC-13 may be determined when present ot higher levels (method for organophosphorus pesticides similar to that of Zweig and Devine, 1969). Also, the chemicals chlordene, hexachlorobicycloheptadiene, and hexachlorocyclopentadiene, which are pesticide manufacturing precursors, may be analyzed by this method. 3. Interferences Any compound or compounds having chemical and physical properties similar to the pesticide of interest may cause interference. The procedure incorporates a column chromatographic technique which eliminates most extraneous material. Polychlorinated biphenyls and polychlorinated naphthalenes, which otherwise interfere with analysis, are separated from most of the pesticides named in this procedure. Special precautions are necessary to avoid contamination during sampling and analysis. 4. Apparatus See step 4, "Gas Chromatographic Analysis of Pesticides." 4. 4.1 Concentrating apparatus: A Kuderna-Danish concentrator, 250-ml capacity with a l-ball Snyder column, is used for the initial concentration step. Final concentration is performed in the receiver using a l-ball Snyder microcolumn. A calibrated 4.00-ml receiver tube is used with the concentration apparatus. 4.2 Chromatography columns: general utility glass columns, 10 mm ID and 300 mm long, having a sealed-in coarse porosity fitted disc. The columns are prepared by filling to a depth of 1 cm with granular-anhydrous sodium sulfate. Add sufficient adsorbent to form an 8-cm layer and top with another l-cm layer of sodium sulfate. 4.3 Cleanup microcolumns: Disposable Pasteur pipets, 14-cm long and 5-mm ID, are used for the chromatographic cleanup columns. The pipets are washed in warm detergent solution, thoroughly rinsed with dilute hydrochloric acid and organic-free distilled water, then heated to 300 C overnight to remove any traces of organic matter. A column is prepared by plugging the pipet with a small amount of specially cleaned glass wool, adding enough deactivated alumina through a microfunnel to fill 3 cm of the column, followed by another 0.5 cm of anhydrous sodium sulfate. 4.4 Filters: Glass fiber, having no organic binder, 142 mm diameter. The filters should remove 99.7 percent of dioctyl phthalate particles larger than 0.3 um. The filters are rinsed in acetone and hexane, the solvent evaporated, and then heated at 300 C overnight before use. 4.5 Pressure filtration apparatus: Stainless steel, Millipore YY2214200 or equivalent, accepting 142-mm diameter filters. A 4-1iter capacity barrel, specially fabricated to fit between the top and bottom filter flanges and hydrostatically tested to 200 psi is required. The modified apparatus may be obtained from Offenhauser Co., Houston, Texas. 4.6 Sandbath, fluidized, Tecam, or equivalent. 4.7 Separatory funnels, Squibb form, 1- or 2-1iter capacity. No lubricant is used on the stopcocks. 5. Reagents 5.1 Alumina, neutral aluminum oxide, activity grade I, Woelm. Weigh 45.0g activated alumina into a dry 250-ml glass-stoppered erlenmeyer flask and quickly add 5.0 ml distilled water. Stopper the flask and mix the contents thoroughly by tumbling. Allow 2 hr.before use. Keep tightly stoppered and store in a desiccator. 5.2 Benzene, distilled in glass, pesticide-analysis quality. 5.3 n-Hexane, distilled in glass, pesticide-analysis quality. 5.4 Silica ~, activity grade I, Woelm. Weigh 48.5 g activated silica gel into a dry 25b-ml glass-stoppered erlenmeyer flask and quickly add 1.5 ml distilled water. Stopper the flask and mix the contents thoroughly by tumbling. Allow 2 hr before use. Keep tightly stoppered and store in a desiccator. 5.5 Sodium sulfate, anhydrous, granular. Prepare by heating at 300 C overnight and store at 130!C. 5.6 Mercury, metallic, reagent gradeO 5.7 Water, distilled, obtained from a high-purity tin-lined still. The feed water is passed through an activated carbon filter. The distillate is collected in a tin-sliver-lined storage tank, and the water is constantly irradiated with ultraviolet light during storage. A gravity delivery system is used, and no plastic material other than teflon is allowed to contact the distilled water. 6. Procedure Samples should be collected according to the recommended practice for the collection of samples for organic analysis. Owing to the hydrophobic nature of most chlorinated hydrocarbon insecticides, consideration of sorption on particulate matter is very important. Suspended sediment sampling techniques should be employed for sampling when necessary. The sample and the bottles used for collecting and/or compositing thesample should be taken or promptly shipped directly to the laboratory. No preservative is used and unless analyzed without delay, the sample should be protected from light and refrigerated. 6.1 Water is separated from particulate matter by filtration. Weigh the sample and container (s) and pour into the pressure filtration apparatus. Filter the water through a glass-fiber filter, using minimum pressure. Collect the fillrate in a tared beaker. See also step 6.1 "Chlorinated Hydrocarbon Insecticides in Suspended Sediment and Bottom Material." 6.2 Determine the weight of the filtered water sample to three significant figures and pour the water into a 1 or 2-1iter separatory funnel. 6.3 Add 25 ml n-hexane to the sample in the separatory funnelO Stopper and shake the separatory funnel vigorously for 1 full min, venting the pressure often. Allow the contents to separate for 10 min and draw off the aqueous layer into the original beaker. If the hexane layer emulsifies, separate as much water as possible, then shake the contents of the funnel very vigorously so that the liquids contact the entire inside surface of the vessel. (CAUTION: Vent often!) Allow the layers to separate and add approximately 5 ml distilled water to aid the separation, if necessary. Remove the water and pour the extract from the top of the separatory funnel into a 125-ml erlenmeyer flask containing about 005 g anhydrous sodium sulfate. 6.4 Repeat a second and thirdextraction of the water sample in the same manner using 25 ml n-hexane each time, and collect the extracts in the 125-ml erlenmeyer flask containing the drying agent. Cover the flask containing the extract with foil and set aside for 30 min. 6.5 Filter the dried extract through glass wool into the Kuderna- Danish apparatus. Add a sand-sized boiling stone and remove most of the hexane by heating on a fluidized sandbath at 100 C in a hood. When the ball in the Snyder column just stops bouncing, remove the apparatus from the heat and allow to cool. Reduce the volume to between 0.4 and 0.5 ml by directing a stream of dry nitrogen on the surface of the liquid while warming the receiver with a heat lamp. 6.6 Purification 6 6.1 Microcolumn cleanup: Quantitatively transfer the contents of the Kuderna-Danish receiver (0.4-0.5 ml) to the top of a deactivated alumina cleanup microcolumn. Use a disposable pipet to transfer. Not more than 0.1-0.2 ml hexane should be needed for washing. Using hexane, elute the extract from the column to a volume of 8.5 ml in a calibrated lO.OO-ml receiver. Add only enough hexane so that the solvent level enters the column packing just as the 8.5-ml elution level is reached. Change receivers and continue the elution using 1:1 benzene-hexane solvent. Collect 8.5 ml of eluate in a second receiver. The first fraction of elute should contain all the chlorinated hydrocarbon insecticides, and carbophenthion, Methyl Trithion, and VC-13. The remaining phosphoruscontaining pesticides are eluted in the benzene-hexane fraction. Reduce the volume of each eluate to 1.00 ml by directing a stream of dry nitrogen on the surface of the liquid while warming the receiver with a heat lamp. 6.6.2 Column chromatographic separation and purification, for samples containing large amounts of extraneous organic matter, sulfur, polychlorinated biphenyls and polychlorinated naphthalenes. 6.6.2.1 Using a dropping pipet, quantitatively transfer the contents of the receiver to the top of an alumina chromatographic column as prepared in step 4.2. 6.6.2.2 Elute the extract from the column using 40 ml of hexane (see figure 1). Collect the first 20-ml fraction (A-l) and the second fraction, 15 ml (A-2) in graduated centrifuge tubes. The column holdup Figure l.--Purification Scheme Hexane Extract (0.5-ml) Alumina - 0-20 ml fraction A-l 20-35 ml fraction A-2 35-50 ml fraction A-3 (hexane) (hexane (benzene) Aldrin Dieldrin Carbophenothion Chlordan Endrin Dioxathion DDD Heptachlor epoxide Ethion DDE VC-13 Malathion DDT Methyl parathion Heptachlor Parathion Lindane Methyl trithion Polychlorinated biphenyls Polychlorinated naphthalenes Toxaphene Mercury (0.5 ml) Silica gel I 0-25 ml fraction S-l 25-45 ml fraction S-2 (hexane) (benzene) Aldrin Chlordan Polychlorinated biphenyls DDD Polychlorinated naphthalenes DDE DDT Heptachlor Lindane Toxaphene is approximately 5 ml. Add 15-ml of benzene to the top of the column just as the last of the hexane enters the sodium sulfate layer and collect the 30 to 50-ml fraction (A-3), in a graduated centrifuge tube. Reduce the volume of each fraction to 1.0 ml by directing a gentle stream of dry nitrogen on the surface of the liquid whiie heating the tube with a lamp. Use a dropping pipet as a nozzle. Analyze by gas chromatography. 6.6.2.3 To remove sulfur, fraction A-l is treated by thoroughly mixing with 0.1 to 0.2 ml of mercury on a mechanical test tube shaking device for one minute. If the mercury darkens or if a black precipitate forms, transfer the solution to a clean centrifuge tube, filtering through glass wool, and repeat the mercury treatment. Reduce the volume to 1.0 ml for gas chromatography. 6.602.4 A second column chromatographic separation of fraction A-l is required if polychlorinated biphenyl or polychlorinated naphthalene are present. Reduce the volume of fraction A-l to 0.5 ml and transfer to the silica gel column. Add 30 ml hexane and collect 25 ml of eluate in a graduated centrifuge tube (S-l, see figure 1). Add 20 ml of benzene to the top of the column just as the last of the hexane enters the sodium sulfate layer and collect the 25 to 45 ml fraction (S-2) in a graduated centrifuge tube. Reduce the volume of each fraction to 1.0 ml for gas chromatographic analysis. 6.7 Analyze the eluates by gas chromatography under conditions optimized for the particular gas chromatographic system being used. Run the first analysis on the electron-capture gas chromatograph using the DC-200 column. For components in concentrations ranging from 0.01 ug/l to 1.0 ug/l, a second analysis by electron capture on the QF-l column is required. Pesticides in concentrations greater than l~O ug/l must either be analyzed by microcoulometric or flame-photometric gas chromatography on both the DC-200 and the QF-l columns or identified by mass spectrometry. 7 Calculations See step 7, "Gas Chromatographic Analysis." 8. Report The pesticide concentrations in water samples are reported as follows: Less than 1.0 ug/l, two decimals and report less than 0.005 ug/l as 0.00 ug/l; 1.0 ug/l and above, to two significant figures. If more than one column or gas chromatographic system is used, report the lowest value. 9. Precision The results may vary as much as ~ 15 percent for compounds in the 0.01 to O.lO-ug/l concentration range. Recovery and precision data are given in table 4. REFERENCES Lamar, W. L., Goerlitz, D. F., and Law, L. M., 1965, Identification and measurement of chlorinated organic pesticides in water by electroncapture gas chromatography: U.S. Geol; Survey Water-Supply Paper 1817-B, 12 p. Lamar, W. L., Goerlitz, D. F., and Law, L. M., 1966, Determination of organic insecticides in water by electron-capture gas chromatography, in Organic pesticides in the environment: Am. Chem. Soc., Advances in Chemistry, ser. 60, p. 187-199. Law, L. M., and Goerlitz, D. F., 1970, Microcolumn chromatographic cleanup for the analysis of pesticides in water: Assoc. Official Anal. Chemists Jour., v. 53, no. 6, p. 1276-1286. [U.S. ] Federal Water Pollution Control Administration, 1969, FWPCA method for chlorinated hydrocarbon pesticides in water and wastewater: Cincinnati, Federal Water Pollution Control Adm., 29 p. Zweig, Gunter, and Devine, J. M., 1969, Determination of organophosphorus pesticides in water: Residue Rev., v. 26, p. 17-36. Replaces pages 33-35 in TWRI Book 5 Chapter A3 CHLORINATED HYDROCARBON INSECTICIDES IN SUSPENDED SEDIMENT AND BOTTOM MATERIAL Gas chromatographic method 1. Summary of method The insecticides are extracted from the sediment or bottom material using acetone and n-hexane. The solid is dispersed first in acetone, and then hexane is added to recover the acetone together with the desorbed insecticides. The extract is washed with distilled water and dried over sodium sulfate. A preliminary gas chromatographic analysis is performed before concentration and cleanup. Following this, the volume is reduced and extraneous material is removed by adsorption chromatography. The insecticides are determined by gas chromatography. 2. Application Sediment and bed material may be analyzed by this methodO Water samples containing suspended sediment may also be analyzed by this technique. The insecticides aldrin, p,p',-DDD, p,p'-DDE, o,p'-DDT, p,p'-DDT, dieldrin, endrin, heptachlor, heptachlor epoxide, isodrin, lindane, and methoxychlor, may be determined down to 0.20 ~g/kg for a SO.O-gsample, on a dry-weight basisO The pesticide chemicals chlordan, chlordene, hexachlorobicycloheptadiene, hexachlorocyclopentadiene, and toxaphene may also be determined by this method. 3. Interferences As in the analysis of water for pesticides, chlorinated by hydrocarbon compounds similar to pesticide chemicals give the most interference. As such, polychlorinated biphenyls and polychlorinated naphthalenes are separated 5.3 Sodium sulfate, anhydrous, granular. Prepare by heating at 300!C overnight and store at 130 CO 5.4 Water, distilled water obtained from a high-purity tin-lined still. The feed water is passed through an activated carbon filter. The distillate is collected in a tin-silver-lined storage tank, and the water is constantly irradiated with ultraviolet light during storage. A gravity system is used, and no plastic material other than teflon is allowed to contact the distilled water. 6. Procedure Samples should be collected according to the recommended practice for suspended sediment and bed materials. Special care should be taken to avoid contaminating the sample with oil from the sampling device. Rubber gaskets should be replaced with teflon. Two suspended-sediment samples should be taken, one for insecticide analysis and the other for determining the sediment concentration and/or particle-size distribution. A suspended sediment sample of at least l-liter is needed for insecticide determination. analysis. The bed material Collect 150 g of solids, if possible, for bed material/should be wetsieved through a screen having 2 mm openings, as soon as possible. Any particle-size separations should be performed without delay. No preservative is added. Unless analyzed within a few days of collection, the samples should be refrigerated and protected from light. 6.1 Filtration and extraction of suspended sediment. A reagent blank must accompany the analysis. 6.1.1 Weigh the sample and containers and pour the contents into the prepared pressure filtration apparatus (See step 4 "Insecticides in Water"). Filter the water through a glass-fiber filter. Collect the filtrate in a beaker and use it to wash all the sediment from the sample containers into the filtration apparatus and also to rinse any sediment from the sides of the filter barrel. 6.1.2 Filter the sample to completion using minimum pressure. Open the apparatus, and using forceps, carefully fold or roll the filter over the filter cake and transfer to a 250 ml glass- stoppered erlenmeyer flask. 6.1.3 Saturate the filter in the erlenmeyer flask by dropwise addition of distilled water. Measure 40 ml of acetone into the flask, clamp the stopper in place and mix the contents vigorously for 20 min on a wristaction shaker. Add 80 ml of hexane and shake again for 10 min. Decant the extract into a l-liter separatory funnel Extract the sample again by adding 20 ml of acetone to the contents of the erlenmeyer flask and mix for 20 min. Add 80 ml of hexane and shake for 10 minutes. Decant the extract into a l-liter separatory funnel. Repeat as in the second extraction one more time. Proceed with the analysis at step 6.3. 6.2 Bottom material extraction, A reagent blank must accompany the analysis. 6.2.1 Desiccated samples, such as bed material from dry streams, should be moistened with distilled water (to about 15 percent by weight). Samples to which water is added are first pulverized, then mixed with the water, and an then kept in /air tight glass container. A minimum of 2 hr should be allowed for equilibration. Start the analysis of homogeneous samples at step 6.2.3, below. 6.2.2 Excessive water in sediment and bottom-mud samples must be separated from the solids in order to obtain a homogeneous fraction of the sample. A proportionate amount of this water is used later so that any suspended material is included in the analysis. This technique may also be used whenever water and solids are to be analyzed separately. Weight the container and contents and transfer the sample to centrifuge bottles. Spin the solids at a relative centrifuge force of soo-i,ooo times gravity. Use the supernatant water to complcte the transfer and repeat the centrifugation as necessary. Decant the separated water into the empty tared sample container. Calculate the weight of the solid by difference. Discard the water. 6.2.3 Thoroughly mix the moist solid until homogeneous and then weigh 50.0 g into a 250-ml erlenmeyer flask having a ground-glass stopper. Also at this time, weigh an additional 10.00 g of the solid into a tared 50-ml beaker to be heated at 130!C overnight for moisture determination. 6.2.4 Measure 40 ml acetone into the erlenmeyer flask containing the sample and clamp the stopper in place. (If the sediment is sandy, use 20 ml acetone instead.) Mix the contents of the flask for 20 min using a wristaction shaker. Add 80 ml hexane and shake again for 10 min. Decant the extract into a separatory funnel containing 500 ml distilled water. Add 20 ml acetone to the erlenmeyer flask and shake 20 min. Again add 80 ml hexane, shake 10 min, and decant the extract into the separatory funnel. Repeat as in the second extraction one more time. NOTE.--If the sediment is not wet enough to agglomerate when the hexane is added, add water, by drops, while swirling the flask and observe if this helps. Very sandy material may remain dispersed. Extremely wet, mucky sediments may be better handled by the additon of anhydrous sodium sulfate. Add sodium sulfate? in small quantities, until the desired consistency is attained or until the amount added approximates the weight of the sediment. The extract volume recovered should be measured at each extraction to insure that 75 percent or more is regained. If not, additiGnal extractions are necessary to obtain quantitative removal of the insecticides. Proceed with the analysis at step 6.3. 6.3 Concentration and purification 6.3.1 Add 500 ml of distilled water to the combined extracts in the l-liter separatory funnel. Gently mix the contents for about one minute and allow the layers to separate. Collect the water in a beaker and decant the extract into a 500-ml erlenmeyer flask. Back-extract the water wash with 25 ml of hexane. Discard the water. Combine both extracts in the l-liter separatory funnel and wash with fresh 500-ml quantities of distilled water two more times. Discard the water layers and collect the washed extract in 500-ml erlenmeyer flask to which has been added about 0.5-g anhydrous sodium sulfateO 6.302 Filter the dried extract through glass wool into a Kuderna- Danish evaporative concentrator. Add a sand-sized boiling stone and remove most of the hexane by heating on a 100!C fluidized sand bath in a hood. As the ball of the Snyder column just stops bouncing, remove the apparatus from heat and allow to stand undisturbed until coolO Reduce the volume of extract to 0.5 ml by directing a stream of dry nitrogen on the surface of the liquid while warming the receiver with a heat lamp. Proceed with the analysis at step 6.6.2 "Insecticides in Water". 7. Calculations See step 7, "Gas Chromatographic Analysis of Pesticides." the water and on the suspended sediment in The pesticide concentrations in~water-sediment mixtures should be reported as in step 8, "Insecticides in Water," and the sediment concentration should accompany the report. The bed material is reported on a dryweight basis as follows: Less than 1.0 ~g/kg to one decimal; 1.0 ~g/kg and greater to two significant figures. Because negative bias exists in the extraction procedure, insecticides found in sediments and solids are considered minimum amounts. 9. Precision The recovery of pesticides from sediments and soils is mainly dependent on two factors: (l) the ability of the solvent to remove the pesticide from the solid, and (2) the amount of solvent reclaimed at each extraction step. Comparative studies of single and exhaustive extractions of soil samples taken from contaminated fields showed that the extraction technique described removed 90- 95 percent of the chlorinated pesticides. Dehydrated clay soils, however, proved slow to yield the pesticides unless they were premoistened. Apparently, the collapsed layers of certain dehydrated clays and the resulting agglomerates entrap the pesticides, and the addition of water prior to analysis helps to open the layers and separate the aggregation. It is imperative that sufficient solvent be reclaimed at each extraction to avoid low resultsO Removal of 90-95 percent of the desorbed pesticides may be expected if at least 75 percent of the solvent is recovered at each extraction step.