EPA # 815-B-01-002

METHOD 531.2. MEASUREMENT OF N-METHYLCARBAMOYLOXIMES AND N-METHYLCARBAMATES IN WATER BY DIRECT AQUEOUS INJECTION HPLC WITH P OSTCOLUMN D ERIVATIZ ATION

Revision 1.0 September 2001
M.V. Bassett, S.C. Wendelken, and B.V. Pepich (IT Corporation) D.J. Munch (US EPA, Office of Ground Water and Drinking Water) L. Henry (American Water Works Service Company ) R.L. Graves - Method 531.1, Revision 3.0 (1989) T. Enge ls (Batt elle Colum bus La borator ies) D.J. Munch (US EPA, Office of Water) - National Pesticide Survey Method 5, Revision 2.0 (1987) D.L. Forest - Method 531, Revision 1.0 (1985)

TECHNICAL SUPPORT CENTER OFFICE OF GROUND WATER AND DRINKI NG WATER U. S. ENVI RONMENTA L PROT ECTI ON AGENCY CINCI NNATI, OHIO 45268

531.2 - 1MEASUREMENT OF N-METHYLCARBAMOYLOXIMES AND N-METHYLC ARBAMATE S IN WATER BY DIRECT AQU EOUS INJECTI ON HPLC WITH POSTC OLUMN DER IVATIZA TION 1. SCOPE AN D APP LICATION 1.1 This is a hi gh per formanc e liquid chromatog raphic ( HPLC)method ap plicable to the determination of certain N-methy lcarbamoyloximes and N-methy lcarbamates in finished drinking waters. The following compounds ca n be determined using this method:

Analy te Aldicarb Aldicarb sulfone Aldicarb sulfoxide Ca rb ar yl Carbofuran 3-Hydroxy carbofuran Methiocarb Me th om yl 1-Naphthol Ox am yl Propoxur Chemical Abstracts Servic e (CAS) Registry Numbe r 116-06-3 1646-88-4 1646-87-3 63-25-2 1563-66-2 16655-82-6 2032-65-7 16752-77-5 90-15-3 23135-22-0 114-26-1

1.2 Detection Limits are compound, instrument, and matrix dependent. The Detection Limit is defined as the statistically calculated minimum concentration that can be measured with 99% confidence that the reported value is greater than zero. (1) Experimentally determined Dete ction Limits for the above listed analy tes are provided in Se ction 17, T ables 2 - 4. The De tection L imit diffe rs from, a nd is lower than the Minimum Reporting Level ( MRL) (Sect. 3.15). The conce ntration range for targ et analytes in this method was eva luated between 0.2 ug/L and 10 ug/L

. Precisi on and ac curacy data and sample ho lding time data ar e prese nted in Section 17, Tables 5 - 8.

531.2 - 21.3 This metho d is restr icted to u se by or under the super vision of a naly sts skilled in HPLC analysis and the interpretation of HPLC chromatog rams. 2. SUMMARY OF METHOD 2.1 A water sample is filtered. Method analy tes are chromatographica lly separate d by injecting an aliquot (up to 100 0 uL)into a hig h perfor mance liq uid chromatographic (HPL C) system equipped with a re versed phase (C 18 ) column. After e lution from the colu mn, the analy tes are hyd roly zed in a postcolumn re ac ti on wi th 0. 07 5 N so di um hyd ro xi de (N aO H) at 80 to 10 0 ° C t o f or m m et hyl amine. The methyl amine is re acted with o-phthalaldehy de (OPA) and 2- mercaptoethano l (or N,N -dimethy l-2-mer captoet hyl amine) to form a hig hly fluorescent isoindole which is detected by a fluorescence detector. ( 2 ) Analytes ar e quantitated using the external standard technique. 3. DEFINITIONS 3.1 ANAL YSIS B ATCH – A se t of sample s prepar ed and a naly zed on the s ame instrument during a 24-hour period. An analy sis batch begins with a Continuing Calibration Check (CCC) at or below the MRL. Subsequent CCCs are analy zed every 10 sa mples, should alternate between medium and high conce ntrations, and must end the analysis batch. An a nalysis batch is limited to 20 field samples. Laboratory Reagent Blanks (L RBs), Laboratory Fortified Sample Matrices (LF SMs), L aboratory Fortifie d Sample Ma trix Duplica tes (L FSMDs), F ield Duplicates (FDs), and CCCs are not counted as samples. Required ba tch QC samples include: LRB, CCC, LF SM, and either a FD or a L FSMD. If a sample(s) in an a nalytical batch ne eds to be reloaded for analy sis within 48 hours of the start of the original analy sis batch, it may be consider ed part of the origina l analy sis batch

. The re analy zed sample (s) have the same QC requir ements as above

. Howeve r, reanaly sis of the L FSM, and F D or L FMD are not requi red if they were already analyzed and pa ssed QC requirements. Any newly prepa red samples would require a full set of QC samples. 3.2 SURROGATE ANALYTE (SUR) – A pure a nalyte, which c hemically rese mbles targe t analy tes and is extremely unlikely to be foun d in any sample. T his analy te is added to a sample aliquot in known amount(s) before filtration or other processing and is measured with the same proc edures used to measure other sample components. The purpose of the SUR is to monitor method performance with each sample. 3.3 LABORATORY REAGENT B LANK (L RB) – An aliquot of reagent water or other bla nk matrix tha t is treat ed exactl y a s a sample including exposure t o all 531.2 - 3glassware, equipment, solvents and reagents, sample preservatives, and surrog ates tha t are use d in the analy sis batch

. The L RB is used to deter mine if method analy tes or other inter ferenc es are present in the labor atory environment, the rea gents, or the app aratus. 3.4 LABORATORY FORTI FIED BL ANK (LFB ) – An aliquot of reagent water or other blank matrix to which known quantities of the method analy tes and all the preser vation compounds ar e added in the lab oratory . The L FB is analy zed exactly like a sa mple, and its purpos e is to dete rmine whe ther the methodolo gy is in control, and whet her the l aboratory is capab le of mak ing acc urate a nd preci se measure ments. For this dire ct inject ion method , a LF B is the sa me as a Continuing Calibration Check standard (CCC - Sect. 3.12). 3.5 LABORATORY FORTI FIED SAMPL E MATRIX (L FSM) – An aliquot of a preserved field sample to which known quantities of the method analy tes are added in the laboratory

. The LFSM is processed and a nalyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical re sults. The background concentrations of the ana lytes in the sample matrix must be determined in a separate aliquot and the measured value s in the L FSM corr ected f or backg round concentra tions. 3.6 LABORATORY FORTI FIED SAMPL E MATRIX DUPLICATE (L FSMD) – A second aliquot of the field sample used to prepare the L FSM, fortified, processed, and ana lyze d identic ally to the L FSM. The L FSMD is use d instead of the Fi eld Duplicate to access method precision when the occ urrence of target a nalytes are low. 3.7 LAB ORATORY DUPLICATES (L D1 and L D2) – Two aliquots o f the same sample taken in the laboratory and analyzed sepa rately with identical pr ocedures. Analyses of L D1 and LD2 indicate pre cision associated with laboratory procedu res, but n ot with sam ple colle ction, pr eserva tion, or storage p rocedur es. 3.8 FIEL D DUPLI CATES (FD1 and FD2) – Two separate samples collected at the same time and plac e under i dentica l circumstances, and trea ted exact ly the same throughout field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample collection, preservation, and storag e, as we ll as labor atory procedu res. 3.9 STOCK STANDARD SOLUTI ON (SSS) – A concentrated solution containing one or more method analy tes prepared in the laboratory using assayed reference materials or purchased from a reputable commercial source. 3.10 PRIMARY DIL UTION STANDARD (PDS) SOL UTION – A solution containing the analy tes prepared in the laboratory from stock standard solutions 531.2 - 4and dilute d as need ed to pre pare ca libration solutions a nd other needed a naly te solutions. 3.11 CALI BRATION STANDARD (CAL ) – A solution prepared from the primary dilution sta ndard sol ution and/ or stock standard solution, a nd the sur rogate analy tes. The CAL so lutions ar e used to c alibrate the instru ment resp onse with respect to analy te concentration. 3.12 CONTINUI NG CALI BRATION CHECK (CCC) – A calibra tion standard containi ng the me thod anal yte s and surr ogate( s), which is analy zed perio dically to verify the acc uracy of the exist ing cal ibration f or those a naly tes. 3.13 QUALITY CONTROL SAMPLE ( QCS) – A sample prepared using a PDS of methods analytes that is obtained fr om a source external to the laboratory and different from the source of calibra tion standards. The second source PDS and the surrog ate PDS ar e used to f ortify the QCS at a known con centra tion. The Q CS is us ed to ch ec k c al ib ra ti on st an da rd in te gr it y. 3.14 DETECTION L IMI T – The minimum concentration of an analy te that can be identifie d, measur ed and re ported with 99% confidenc e that the analy te concentration is greater than zero. This is a statistical dete rmination of precision (Sect. 9. 2.4), an d accur ate quan titation is n ot expecte d at this le vel. (1) 3.15 MINI MUM REPORTING LEVEL (MRL) – The minimum concentration that can be reported as a quantitated value for a target analy te in a sample following analysis. This defined c oncentration can be no lower than the conce ntration of the lowest continuing calibra tion stand ard for that ana lyte and can only be used if accept able qua lity control c riteria for this st andard a re met. 3.16 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided by vendors c oncerni ng a chemical’ s toxicity, health hazards, phys ical pro perties, fire, a nd reactivity data inc luding st orage , spill, and handling precau tions. 4. INTERFER ENCES 4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap wate r, rinse with tap w ater, f ollowed by r eage nt water

. A final r inse with solvents may be nee ded. In place of a solvent rinse, non-volumetric glassware can be heated in a muffle furnace a t 400 o C for 2 hou rs. Volume tric glassware should not be heated above 120 o C. 4.2 Method interferences may be caused by contaminants, especially amines and ammonia, in solvent s, reag ents (inc luding r eage nt water ), sample bottles an d caps, and other sample processing hardwa re that lead to discrete artifacts and/or 531.2 - 5elevated baselines in the chromatogra ms. The samples or analy tical system may be conta minated f rom being handled w ith bare fingers. All items such as these must be routinely demonstrated to be f ree from interferences ( less than 1 / 3 the MRL for each target) under the conditions of the analysis by analy zing laboratory reagent blanks as described in Section 9. Subtract ing blank value s from sa mple results is not permitted. 5. SAFETY 5.1 The toxicity or carcinog enicity of ea ch reagent used in this method has not been precise ly d efined; each chemical c ompound should be tr eated a s a potent ial healt h hazard, a nd exposure to these c hemicals should be m inimized. The labora tory is responsible for maintaining a current awa reness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A refe rence file of MSDSs should also be made available to all personnel involved in the chemical analy sis. Additional references to laboratory safety a re available. (3-5) 5.2 Pure standard materials and stock standards of these compounds should be handled w ith suitabl e protection to skin a nd ey es. Care should be t aken not t o breathe the vapor s or inge st the mate rials. 6. EQUIPME NT AND SUPPLIES (All specifications are sugge sted. Brand names and/or catalog numbers are included for illustration only.) 6.1 SAMPLE CONTAINERS – Amber glass b ottles fi tted with PTFE (poly tetrafl uoroethy lene) lined scre w caps. 6.2 VIAL S – Screw cap or crimp top glass autosampler vials with PTFE fa ced septa, amber or clear. 6.3 VOLUMETRI C FLASKS – Class A, various sizes used for prepara tion of standar ds. 6.4 GRADUAT ED CYLIND ERS – Vario us sizes. 6.5 MICRO SYRINGES – Va rious sizes. 6.6 BALANCE – Analytic al, cap able o f accur ately wei ghing t o 0.00 01 g.

6.7 DISPOSABL E SYRINGES – 5 to 30 mL (B -D Cat.#: 309603, 309650 or equivale nt) size, us ed to filte r sample e xtracts be fore analy sis.

531.2 - 66.8 FILTERS – Disposable filters used to filter samples before analy sis (Millipore 0.22 um PVDF membrane, Cat. #: SLGV 013 NL or equivalent). 6.9 HIG H PERFORMA NCE LI QUID CHROMAT OGRAPHY ( HPLC) INSTRUMENTATI ON 6.9.1 HPLC SYSTEM – Capable of reproducibly injecting up to 1000-uL aliquots, and performing binary or ternary linear gradients at a constant flow rate near the flow rate used f or development of this method, e.g., 1.5 mL/min

. A terna ry

gradie nt was use d to achie ve the se paration picture d in Figure 1. However, a bina ry g radient may be used as long as the minimum resolution is achie ved as spe cified in Sect. 9.1 0. The use of a colu mn heater is strongly r ecommen ded. Dur ing method develo pment, the column was hea ted to 30 °C to help ins ure stab le reten tion times. Column temperature may be adjusted as long as column performance is not compromised. 6.9.2 POSTCOL UMN REACTI ON SYSTEM – Capable of mixing r eage nts into the mobile phase. Reactor should be constructed using polyetherethe rketone (PEEK) or PTFE tubing and equipped with two pumps capable of delivering 0.5 mL /min of each reagent; mixing tees; and two reaction coils. Postcolumn system manufac turers recommend different reaction coil temperatures f or the carbamate hy drolysis reaction. Therefore, the first reaction coil temper ature may r ange from 80 to 100 °C. Analyte sig nal can increase with temperature ove r this temperature range; however, analy sts should b e awar e that wit h some sy stems, ba seline no ise can also increase with increasing temperatures. If te mperatures greater than 95 °C are used, a slight backpressure must be maintaine d on the system to prevent boiling of the post column eluent. This may be accomplished by use of a restrictor placed in line after the de tector. The second reaction takes place at ambient temper atures. Method performance data were collected with two postcolumn reaction modules: the Waters Postcolumn Carbama te Sy stem and the Picker ing Mode l 5200 Car bamate System, both with a reactor te mperature of 80 o C. 6.9.3 HPLC DETECTOR – A fluorescence detector capable of excitation at approxima tely 340 nm and detection of emissi on energ y a t approxima tely 465 nm. Opt imum excita tion and e mission wa veleng ths may vary slightly for each sy stem. For the development of this method, Waters Model 474 and Model 2475 scanning fluorescence detectors were used. 6.9.4 ANALYTICAL COLU MN – For the development of this method, an HPLC “ carbamate” co lumn (3.9 × 150 mm) pa cked with 4 um d phase particles (Waters Cat. # : WAT035577) was used. Any column that p C 18 solid

531.2 - 7provides adequate resolution, peak shape, capa city, acc uracy, a nd precision (Sect. 9) may be used. 6.9.5 HPLC DATA SYSTEM - A computerized data sy stem is recommended for data acquisition and processing. A Waters Millennium software system was us ed to ge nerate all data c ontained in the Section 17 tab les. 7. REAGENTS AND STANDARDS 7.1 REAGENTS AND SOLVENTS – Reag ent grade or better chemica ls should be used. Unl ess other wise indic ated, it is intended that all r eage nts shall c onform to the specifications of the Committee on Analy tical Reagents of the American Chemical Society, wher e such specifications are available. Other grades may be used, provided it is f irst dete rmined tha t the rea gent is o f suffic iently high pur ity to permit its use without lessening the quality of the de termination. Reagents of lesser p urity may also aff ect instr ument per formanc e, such a s clogging of the post column reactor. 7.1.1 REAGENT WATER – Purified water which does not contain any measurable quantities of any target analy tes or interfering compounds greater than 1/3 the MRL for each compound of interest. 7.1.2 PRESERVED RE AGENT WATER – Reag ent wate r which has sample preservation reagents adde d in the same concentrations as the samples. To prepare 1 liter of preserved r eagent water, add a suff icient amount of potassium dihydrogen c itrate (Sect. 7.1.11.1) to yie ld a concentration of 9.2 to 9.5 g/L and sodium thiosulfate (Sect. 7.1.11.2) to yield a concentration in the range of 80 to 320 mg /L to a gradua ted bottle or volumetric flask. Fill to the 1 L mark with rea gent water. 7.1.3 ACETONI TRIL E (CH 3 CN, CAS#: 75-05-8) – High purity, de monstrated to be free of analy tes and interferences (HPLC grade or better). 7.1.4 METHANO L (CH 3 OH, CAS#: 6 7-56-1) – High pu rity , demonstr ated to be f ree of a nal ytes and in ter fer enc es ( HP LC grade or better). 7.1.5 SODIUM HYDROXI DE (NaOH, CAS#: 1310-73-2), 50% (w/w) solution (Fisher Cat. #: SS254-500 or equivalent). Due to the hy groscopic nature of NaOH pellets, a 50% (w/w) solution is used in this method for the preparation of reage nts. 7.1.6 POSTCOLUMN REAGENT #1 (hy drolysis solution) – Sodium hydroxide, 0.075 N. Dilute 4 mL of 50% w/w sodium hydroxide (NaOH)

531.2 - 8solution to 1 L with reage nt water

. The con centra tion of the hyd roly sis solution can dramatically e ffect the analy te response. Filter and degas with helium just before use. 7.1.7 PREPARATION OF THE OPA SOL UTION (to be used in pre paration of Postcolumn Reagent #2, Sect. 7.1.10) – Dissolve 100 +10 mg of ophthalde hyd e (OPA, CA S#: 643-79 -8) in 5 - 10 mL o f methanol. 7.1.8 PREPARATION OF THE OPA DI LUTENT (to be used in prepa ration of Postcolumn Reagent #2, Sect. 7.1.10) – Use one of the following formulations in the preparation of the preliminary OPA dilutent. Performance data presented in Section 17 wer e collected using the sodium tetraborate formulation

. Method pe rforman ce was c hecked with the boric acid for mulation a nd found to be equiva lent. OPA d ilutent is commercially a vailable. 7.1.8.1 Prepare d using so dium tetra borate : Dissolve 19.1 g of sodium tetraborate decahy drate (Na 2 B 4 O 7 � 10H 2 O, CAS#: 1303-96-4) in a 1 L volumetric flask. Bring the volume up to 1.0 L with reage nt water to make a 0.05 M solution. The sodium borate will dissolve in less tha n 2 hours if a stir bar is used. F ilter and degas prior to preparation of Postcolumn Reagent #2 (Sect. 7.1.10). OR 7.1.8.2 Prepared using boric acid: Dissolve 3.0 g of boric acid (H 3 BO 3 , CAS#: 10043-35-3) in a 1 L volumetric flask in approximately 800 mL of reag ent water. Add 1.2 mL of a 50% (w/w) NaOH solution. Bring the volume up to 1.0 L with re agent water. Filter and de gas prior to preparation of Postcolumn Reagent #2 (Sect. 7.1.10). 7.1.9 PREPARATION OF THE NUCLEOPHILI C SOLUTION (to be use d in preparation of Postcolumn Reagent #2, Sect. 7.1.10) – Use one of the following formulations in the preparation of the nucleophilic solution. Either one of these compounds reacts with OPA and the target methylamine to form the isoindole detected by the fluore scence detector. Both reagents have cha racteristic strong odors and should be handled in a fume hood. 7.1.9.1 Prepared using 2-mercaptoethanol ( CAS#: 60-24-2) : This compound is in liquid form and may be used dire ctly in the preparation of the derivatization solution, Postcolumn Reagent #2 (Sect 7.1.10). Use 1 mL of 2-mer captoethanol per liter of Postcolumn Reagent #2. OR 7.9.1.2 Prepared using N,N-dimethy l-2-mercaptoethylamine531.2 - 9hydrochloride ( CAS#: 13242-44-9) : Use approximately 10 mL of OPA dilutent (Sect. 7.1.8) to dissolve 2.0 + 0.2 g of N,Ndimethyl-2-merca ptoethylamine-hy drochloride. 7.1.10 FIN AL PREPARATI ON OF POST COLU MN REAGENT #2 (OPA derivat ization solu tion), – Stu dies have shown tha t this rea gent ma de with either of the two nucleophiles listed above (Sect. 7.1.9) is stable for a period of at least 36 hours. However, individual laboratory conditions vary an d d ai ly p re pa ra ti on of th is so lu ti on ma y be ne ce ss ar y. 7.1.10.1 Add the dissolved OPA (Sect. 7.1.7) to 1 L of either f ormulation of the OPA dilutent (Sect. 7.1.8.1 or 7.1.8.2), which has been filtered and degassed. 7.1.10.2 To the 1 liter flask, add either one of the 2 nucleophiles specifie d in Section 7.1.9 (7.1.9.1 or7.1.9.2). 7.1.11 SAMPLE PRESERVATION REAGENTS ( See also Sect. 8.1.1) 7.1.11.1 Potassium dihydrogen citra te (C 6 H 7 KO 7 , CAS #: 866-83-1) – Added to a djust sampl e pH to ~ 3. 8, which a cts as a b iocide to guard against potential deg radation of method analy tes by microorganisms and to preve nt chemic al degr adation. Oxamy l, 3-hy droxy carbof uran, ca rbary l, and met hiocarb all hy droly ze in neutral or basic waters even when he ld at refrigerated temperatures. 7.1.11.2 Sodium thiosulfate (Na 2 S 2 O 3, CAS#: 7772- 98-7) – Acts as a dechlor inating agent. Aldicar b and meth iocarb a re rapi dly degraded in waters that are not dechlorinated. 7.2 STANDARD SOLUTI ONS – Standard Solutions may be prepar ed from certified, commerc ially availab le solution s or from n eat comp ounds. Comp ounds use d to prepare solutions must be 96% pure or grea ter and the weight may be used without correction for purity to calculate the concentration of the stock standard. Solution concentrations listed in this section were used to develop this method and are included as an exam ple. Stand ards for sample fo rtifica tion generally should be p repare d in the sma llest volume that ca n be acc urately measure d to minimize the addition of organic solvent to aqueous samples. Even though stability t imes for standard so lutions are suggeste d in the following sections , laboratories should use standard QC practices to determine when Standard Solutions des cribed in this sectio n need to be replace d.

531.2 - 107.2.1 SURROGATE ANALYTE (SUR) STANDARD SOL UTION, 4- BROMO-3,5-DIMETHY LPHENYL N-METHYLCARBAMATE (BDMC, CAS #: 672-99-1) 7.2.1.1 SUR STOCK SOLUTION – May be purc hased as certified standard or prepared from neat mate rial. If prepar ing from neat material: accurately weigh approximately 25 to 35 mg of the neat SUR to the nearest 0.1 mg into a tared, 5-mL volumetric flask. Dilute to the mark with methanol. Stock solutions have been shown to be stable for 12 months when stored at -10°C or less. 7.2.1.2 SUR PRIMARY DIL UTION STANDARD – Prepar e the SUR Primary Dilution Standard (PDS) by adding enough of the SUR stock standard to a volumetric flask partially filled with methanol to make a final concentration near 10 ug /mL when filled to the mark with methanol. The PDS has been shown to be stable for 6 months when stored at -10 °C or less. 7.2.2 ANALYTE STANDARD SOL UTIONS – Obtain the analy tes listed in the table in Section 1.1 as neat or solid standards or as commercially prepared ampulized solutions from a reputable standard manufacturer. Prepa re the Analyte Stock and Primary Dilutions Standards as described below. 7.2.2.1 ANALYTE STOCK STANDARD SOL UTION – I f preparing from neat material, accurately weigh approximately 25 to 35 mg of pure mat erial to t he near est 0.1 mg into a tar ed, 5-mL volumetri c flask. Dilute to the mark with methanol. Repeat for each target analyte. 7.2.2.2 ANAL YTE PRI MARY DI LUTI ON STAND ARD (PDS) SOLUTI ON – Prepare the analy te PDS by dilution of the targe t analyte stock standa rds. Add enough of each target stock standard to a volumetric flask partially filled with methanol to make a final concen tration ne ar 10 ug /mL when filled to the mar k with methanol

. A seria l dilution of this PDS to ma ke a 1.0 u g/mL solution is useful for low level spiking. The PDSs have been shown to be stable fo r 6 months w hen store d at -10 °C or less.

7.2.3 CALI BRATION STANDARDS (CAL ) – At least 5 calibration concentrations will be required to prepare the initial calibration curve (Sect. 10.2). Prepare the calibration standards ove r the concentration range of interest from dilutions of the Analy te PDSs in preserved reagent 531.2 - 111 2 3 4 5 6 water (7.1.2), filtering the CAL standards in the same manner as the samples (Sect. 11.1.4). The lowest concentration of calibra tion standard must be at or below the MRL, which may depend on system sensitivity

. The calibration standards for the development of this method were prepared as specified below. Calibration standar ds must be prepared using preser ved rea gent wa ter (Sect. 7.1.2). An example of the dilu tions used to prepare the CALs use d to collect the data in Section 17 are shown in the table below. These standards may be also be used as CCCs. If stored, the aqueous standards must be stored in amber glass and re frigerated in the same man ner as the sample s.

PREPARATION OF CALIBRATION (CAL) CURVE STANDARDS

CAL Level Analy te PDS Conc.

(ug/mL) Vol. of Analyte PDS (uL) Vol. of 10 ug/ mL SUR PDS (uL) Final Vo l. of CAL Std.

(mL) Final Conc. of CAL

Std. (ug/L) Final Conc. of SUR

Std. (ug/L) 1.0 5.0 5.0 25 0.20 2.00 1.0 12.5 5.0 25 0.50 2.00 1.0 25.0 5.0 25 1.00 2.00 10.0 5.0 5.0 25 2.00 2.00 10.0 12.5 5.0 25 5.00 2.00 10.0 25.0 5.0 25 10.0 2.00
8. SAMPLE COLL ECTION, PRE SERVATIO N, AND STOR AGE 8.1 SAMPLE BOTTL E PREPARATION 8.1.1 Prior to sh ipment to the field, preser vatives li sted in Sec tion 7.1.11 must be added as dry solids to each amber bottle fitted with a PTFE lined screw cap. A 40 or 60-mL sample bottle is rec ommended. Add a sufficient amount of potassium dihydroge n citrate to yield a c oncentration in the sample of 9.2 to 9.5 g/L. Potassium dihy drogen citrate buffers sample pH to ~3.8 to prevent h ydr oly sis of oxamy l, 3-hy droxy carbof uran, ca rbary l, and methiocarb. Add sodium thiosulfate to yie ld a sample concentration in the range of 80 to 320 mg/L

. The concentration of 80 mg/L of sodium thiosulfate is adequate for dechlorination, but difficult to measure if a small (e.g., 40-mL size) sample bottle is chosen. Me thod performance was tested using up to 320 mg/L w ithout any adverse effect. Sodium thiosulfate eliminates the residual free chlorine in the samples, which

531.2 - 12rapidly deg rades aldicarb and methiocarb. 8.1.2 Grab samples must be collected in accordance with conventional sampling practic es ( 6 )

. Sample bottles must not be prerinsed with sample before collection. Doing so will wash out the preservatives added to the bottles prior to shipment. 8.2 SAMPLE COLL ECTION 8.2.1 When sampl ing from a cold wa ter tap, remove t he aera tor so tha t no air bubbles will be trapped in the sample. Open the tap, and allow the sy stem to flush until the water temperature has stabilized (usually about 3-5 minutes). Collect sa mples from the flow ing sy stem. 8.2.2 When sampling from an open body of water, fill a wide-mouth bottle or beaker with sampl e from a repres entative area, a nd care fully fill sample bottles fr om the con tainer. Sampling equipmen t, includin g automa tic samplers, must be free of plastic tubing, ga skets, and other parts that may leach interfering analy tes into the water sample. 8.2.3 If sampling water s high in colloidal iron, filtration of the sample may be necessary prior to preservation in the field to help prevent the precipitation of the iron in the reactor. 8.2.4 Fill sample bottles, taking care not to flush out the sample preservation reagents. Samples do not need to be collected he adspace free. 8.2.5 After collecting the sample, cap ca refully to avoid spillag e, and agitate by hand for 1 minute. K eep samp les seale d from co llection t ime until analy sis. 8.3 SAMPLE SHIPMENT AND STORAGE – All samples should be iced during shipment and must not exceed 10 o C during the first 48 hours after collection. Samples should be confirmed to be at or below 10 o C when they ar e received at the laboratory

. Samples stored in the lab must be held at or below 6 o C and protected from light until analysis. Samples should not be frozen. 8.4 SAMP LE AND EXT RACT HOLDING TIMES – Resu lts o f the s ample stora ge stability study of all method analytes indica ted that all compounds are stable for 28 days in water samples that are collected, dechlorinated, pre served, shipped and stored as described in Sections 8.1 - 8.3. Samples must be analy zed within 28 day s. 9. QUALITY CO NTROL 531.2 - 139.1 Quality control (QC) re quirements include the Initial Demonstration of Capabilit y, t he deter mination of the Dete ction Limit, and su bsequen t analy sis in each analysis batch of a Laborator y Reag ent Blank (LRB) , Continuing Calibration Check Standards (CCC), a Labora tory Fortified B lank (LFB) , a Laboratory Fortifie d Sample Ma trix (L FSM), an d either a La borator y F ortified Sample Ma trix Duplicat e (LFSMD) or a Field D uplicate Sample. Th is section details the specif ic requirements for each QC paramete r. The QC criteria discussed in the following sections are summarized in Section 17, Tables 10 and 11. These criteria a re considered the minimum acceptable QC criteria, and labor atories are encourag ed to institute additiona l QC practices to me et their s pecific needs. 9.2 INITIAL DEMONSTRATION OF CAPAB ILITY (I DC) – Requirements for the Initial Demonstration of Capability are described in the following sections and summarized in Section 17, Table 10. 9.2.1 INITIAL DEMONSTRATION OF L OW SYSTEM BACKGROUND – Befor e any field sam ples are analy zed, and a ny t ime a new set of re agents is used, it m ust be dem onstrate d that a la borator y r eage nt blank is reasonably free of contamination and that the criteria in Section 9.4 ar e met. 9.2.2 INITIAL DEMONSTRATION OF ACCURACY – Prior to the ana lysis of the IDC samples, verify calibration accuracy with the preparation and analy sis of a mid -level Q CS as defined in Sect ion 9.11. If the ana lyte recove ry

is not + 30% of the true val ue, the a ccurac y of the metho d is unacceptable. The source of the pr oblem must be identified and corrected. Afte r the accurac y o f the cali brat ion ha s bee n ver ifie d, pr epar e and anal yz e 4-7 replicate LF Bs (or CCCs in this method) fortified at 2 ug/L, or ne ar the mid-range of the initial calibration curve, ac cording to the procedure described in Section 11. Sample preservatives as described in Section 8.1.1 must also be added to these samples. The averag e recovery of the replicate values must be within ± 20% of the true value. 9.2.3 INITIAL DEMONSTRATION OF PRECI SION – Using the same se t of replicate data genera ted for Section 9.2.2, calculate the standard deviation and percent relative standard deviation of the r eplicate recoveries. The relative standard deviation (%RSD) of the results of the replica te analyses must be less than 20%. 9.2.4 DETECTION LI MIT D ETERMI NATI ON – Prep are and analy ze at least 7 replica te LF Bs at a c oncentr ation esti mated to b e near t he Dete ction Limit over at l east 3 da ys u sing the procedu re described in Se ction 11. T his fortification level may be estimated by sele cting a concentration with a 531.2 - 14signal o f 2 to 5 time s the noise level. The approp riate co ncentra tion will be depe ndent upon the sens itivity of the HPL C system being used. Samp le preser vatives a s descri bed in Section 8.1.1 m ust be add ed to thes e samples. Calculate the Detection Limit using the e quation Detecti on Lim it = St ( n - 1, 1 - alpha = 0.99) where t (n-1,1-alpha = 0.99) = Student's t value for the 99% confidence le vel with n-1 degrees of free dom n = number of replicates, and S = standa rd devia tion of re plicate a naly ses. NOTE: Calculated Detection Limits need only be less than 1 / 3 of the laboratory’ s MRL to be considered acc eptable. Do not subtract blank values when performing Detec tion Limit calculations. The De te ct io n Li mi t i s a st at is ti ca l d et er mi na ti on of pr ec is io n o nl y. (1) If the Det ectio n Limit repli cates a re fort ified at a lo w enou gh concentration, it is likely that they will not meet the precision and accuracy criteria for CCCs, and may result in a calculated Detection Limit that is higher than the for tified concentration. Therefore, no precision and acc uracy c riteria are specified. 9.2.5 MET HOD MOD IFICA TION S – The ana lyst is per mi tt ed t o m odi fy HP LC columns a nd conditi ons prior to the post column re action. T he analy st is also allowed to modify the surrogate standard. Each time such method modifications are made, the analy st must repeat the procedures of the I DC (Sect. 9.2). 9.3 Minimum Reporting Level (MRL ) – The MRL is the threshold concentra tion of an analyte that a laboratory can expect to accurately quantitate in an unknown sample. T he MRL should not b e establ ished at a n analy te conce ntration t hat is less than either th ree time s the Dete ction Limit or a c oncentr ation whic h would yield a response less than a signal-to-noise (S/N) ratio of five. Depe nding upon the study’s data quality objectives it may be set a t a higher concentration. Although the lowest cal ibration st andard mus t be at or below the M RL, the MRL must never be establishe d at a conc entrati on lower tha n the lowest calibration standard

.

9.4 LABORATORY REAGENT B LANK (L RB) – This is a direct injection method without a conventional extraction. An LRB is required w ith each analy sis batch (Se ct. 3.1 ) of sam pl es t o de ter mi ne a ny ba ckg rou nd s yste m co nt ami nat io n. If within the retention time window of any a nalyte, the L RB produces a peak that 531.2 - 15would prevent the determination of that analy te, determine the source of contamination and eliminate the interference bef ore processing samples. Background contamination must be reduce d to an acceptable level before proceeding. Backg round from method analy tes or contaminants that interfere with the measurement of method analy tes must be below 1 / 3 the MRL. I f the target analy tes are detected in the L RB at concentrations equal to or gre ater than this level, then all data for the problem analy te(s) must be considered invalid for all samples in the analysis batch. 9.5 CONTINUI NG CALI BRATION CHECK (CCC) – A CCC is prepared in the same manner as the initial calibration solutions (Sect. 7.2.3), using preserve d reagent water (Sect. 7.1.2) and filtering in the same manner as the samples (11.1.4). It is analy zed during an analysis batch at a required frequency to confirm that the instrument meets initial calibration criteria. See Section 10.3 for concentration requirements, frequenc y require ments, and acceptance criteria. 9.6 LABORATORY FORTI FIED BL ANKS – For this direct injection method, a CCC is the same as an LFB. Consequently , the analysis of an L FB is not required. 9.7 SURROGATE RECOVERY – The surr ogate standard is fortif ied into a ll samples , blanks, L RBs, and LFSM s and L FSMDs pri or to samp le filtra tion. They are als o added to t he calibration c urve and calibra tion chec k standar ds. The su rroga te is a means of assessing method performance from preparation and filtration to final chromat ographic meas urement

. 9.7.1 When surrogate recovery from a sample, blank, or CCC is less than 70% or greater than 130%, che ck (1) calculations to locate possible errors, (2) standard solutions for degradation, (3) contamination, and ( 4) instrument perfor mance. If those ste ps do not re veal the cause of the probl em, reanaly ze the sample. 9.7.2 If the reana lysis meets the surrog ate recovery criterion, report only data for the reanaly zed sample. 9.7.3 If the sample reanal ysi s fails the 70-130% r ecover y criterion , the ana lys t should che ck the ca libration by r einjecti ng the mo st rece ntly accept able calibration standard. If the calibration standard fails the criteria of Section 10.3, recalibration is in order per Section 10.2. I f the calibration standard is acceptable, preparation (which include s spiking with surrogate and filtration) and analy sis of the sample should be repeated provided the sample is still within the holding time. I f this samp le reanaly sis also fa ils the rec overy criteri on, report all dat a for tha t sample a s suspect due to surrogate recovery

.

531.2 - 169.8 LABORATORY FORTI FIED SAMPL E MATRIX (L FSM) – Analysis of an LFSM is requir ed in eac h analy sis batch and is use d to deter mine that t he sample matrix does not adversely aff ect method accuracy

. precision is accomplished by analysis of a F ield Duplicate (Sect. 9.9), however, infrequent occurrence of tar get analy tes would hinder this assessment. occurrence of targ et analytes in the sa mples is infrequent, or if historical trends are unavaila ble, a se cond L FSM, or L FMSD, must be prepa red and a naly zed from a duplicate of the field sample. sis batches that contain LFSMDs will not require the analysis of a Field Duplicate. a variety of different sample matrices are analy zed regularly , for example, drinking water from groundwater and surface water sources, method performance should be established for each. LFSM data should be documented for all routine sample sources for the laboratory. 9.8.1 Within each analysis batch, a minimum of one field sample is fortified as an LFSM for ever y 20 samples proce ssed. d by spiking a sample wi th an appr opriate amount of the appr opriate Analy te PDS (Sect. 7.2.2.2). matrix backgroun d conce ntration, if known. Use histor ical data or rotate through a range of c oncentrations when selecting a fortify ing concentration. mple that has already been analy zed aids in the sele ction of a ppropria te spiking levels. 9.8.2 Calculate the (R) for each analy te using the equation

where A = measu red conc entration in the fortified s ample B = measured concentration in the unfor tified sample, and C = fortification concentration. 9.8.3 Analyte re coveries may exhibit a matrix bias. above their native concentration, recove ries should range between 70 - 130%. tion at the MRL, 50 to 150% recoveries ar e acceptable. I f the accuracy of any ana lyte falls outside the desig nated range , and the l aboratory perfor mance f or that a naly te is shown to be in control in the CCCs, the recove ry

is judge d to be mat rix biased. The resu lt for that a naly te in the u nfortifi ed sample is labele d suspect /matrix to inform the data user that the results are suspect due to matrix effec ts. 9.9 FIELD DUP LICAT E OR LABO RAT ORY FOR TIFIE D S AMP LE MA TR IX Assessment of method If the
Analy If Over time,

The LFSM is prepare Select a spiking concentration at least twice the
Selecting a duplicate bottle of a sa

percent recovery

For samples fortified at or For LFSM fortifica

531.2 - 17DUPLI CATE (FD or LF SMD) – Within each analysis batch, a minimum of one Field Duplicate (FD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD) must be analy zed. Duplic ates che ck the pr ecision a ssociate d with samp le collection, preservation, storage, and labor atory proc edures. If targe t analytes ar e not routinely observed in fie ld samples, an LFSMD must be analy zed rather than a FD. 9.9.1 Calculat e the re lative pe rcent di ffere nce (RPD) for dupli cate measurements (FD1 and FD2) using the equation

9.9.2 If an L FSMD is analyzed instead of a Field Duplicate, calculate the relative percent difference (RPD) for duplicate LFSMs (L FSM and LFSMD) using the equa tion

9.9.3 RPDs for FDs and duplicate LF SMs should fall in the range of ± 30% for samples f ortified at or above their native c oncentr ation. Gr eater variabili ty may b e ob ser ved whe n LFS Ms are spi ked nea r th e MR L. At th e MR L, RPDs should fall in the range of 0 to 50% for samples fortified at or a bove their native concentration. I f the accuracy of any ana lyte falls outside the designated range, a nd the laboratory performance for that analy te is shown to be in control in the LFB, the r ecovery is judged to be matrix biased. The result for that analy te in the unfortified sample is labeled suspect/ matrix to inf orm the da ta user that the re sults are suspect d ue to matrix eff ects 9.10 RESOLUTI ON CHECK – The resolution of peaks in a calibration standard or CCC near the mid level of calibration must be monitored in each analy tical batch. During the deve lopment of this method , the 2 ug /L level was mon itored. Cl osely eluting peaks that are not baseline resolved must have a resolution (R s ) of 1.0 or greater using the equa tion (7)

531.2 - 18where t 1 and t 2 = retention times of the first and second adjacent pea ks W 0.5,1 and W 0.5,2 = widths of the adja cent pea ks at half height. Resolution must be monitored once for every 24-hour analy tical batch and may be monitored at any time during the 24-hour period. It is rec ommended that resolution be checked prior to sample analy sis, especially if the sy stem in use has a history of re solution problems. If a resolution check fa ils, all samples must be reanaly zed after the problem is corrected, including the QC samples. 9.11 QUALITY C ONTROL SAMPLE (QCS) – Du ring the analy sis of the I DC (Sect. 9.2), each time that new analy te standard solutions (Sects. 7.2.2.1 and 7.2.2.2) are prepared, or at least quarterly , analyze a QCS from a sour ce different from the source of the calibration standards. The QCS is fortified into preser ved reagent water (Sect. 7.1.2) and analy zed as a CCC. The acceptance criteria is the same as the CCC criteria at mid-level; the calculated amount for eac h analyte must be + 30% of the true val ue. I f measur ed analy te conce ntrations are not o f acce ptable accuracy, check the entire analy tical procedure to locate and corre ct the problem source. 10. CALIBRATI ON AND STAN DARDIZ ATION 10.1 After in itial cal ibration i s succes sful, a Con tinuing Ca libration Check (CC C) is require d at the be ginning and end o f each a naly sis batch , and aft er ever y te nth sample (Sect. 10.3). Initial calibration should be re peated each time a major instrument modification or maintenance is performed. 10.2 INITIAL CALI BRATION 10.2.1 Establish HPLC operating parame ters equivalent to the suggested conditions in Section 17, Table 1, including the postcolumn reactor. The system is calibrated using the external standard technique. A fluorescence detector was used with the excitation and emission wavelengths optimized at 340 and 465 nm, respectively

. Other HPLC conditions may be used as long as all QC requirements in Section 9 are met. 10.2.2 Prepare a set of at least 5 calibration standar ds as described in Section 7.2.3. The lowest concentration calibration standard must be at or be low the MRL, which may depend on system sensitivity

. It is recommended that at least four of the Calibration Standards are at a conc entration greater than or equal to the MRL. 10.2.3 Genera te a cal ibration c urve for each an aly te by plotting the peak response 531.2 - 19(area is recommended) against analyte conc entration. The instrument used during method development y ielded linear curves for the targ et analytes over the concen tration r ange o f intere st. However, data may be fit with either a linear r egres sion (re sponse vs. concen tration)or quadr atic fit (response vs. concentration). Alternately , if the ratio of the analy te peak area to concentration (or response f actor) is relatively constant (%RSD < 30%) an a verag e respon se fact or may be used to calcula te analy te concentration. 10.2.4 When quan titated us ing the i nitial ca libration curve, e ach cal ibration p oint, except the lowest point, for each analy te should calculate to be 70-130% of its true value. The lowest calibration point should calculate to be 50-150% of its true value. F ailure to meet this c riteria may indicate future d ifficult y in meeting CCC QC requirements during the analy sis batch. 10.3 CONTINUI NG CALI BRATION CHECK (CCC) – Minimum daily calibration verification is as follows. Verify the initial calibration at the beginning and end of each g roup of a naly ses, and a fter ev ery tenth samp le during analy ses. (I n this context, a “sample” is considered to be a field sample. L RBs, CCCs, LFSMs, and LFSMDs are not counted a s samples. The beginning CCC each day must be at or below the MRL in order to v erify instrumen t sensitivi ty prior to an y a naly ses. Subsequent CCCs should alternate between a medium and high concentra tion standard. 10.3.1 Inject an aliquot of the appropr iate concentration calibration solution and analyze with the same c onditions used during the initial calibration. 10.3.2 Calculate the concentration of each ana lyte and surrogate in the check stand ard. T he calc ulate d amou nt for each an alyte for m edium and hi gh level CCCs must be ± 30% of the true value. The calculated amount for the lowest calibration point for each analy te must be within ± 50% of the true value. If these conditions do not exist, then all data for the problem analyte must be conside red invalid, and remedial action should be taken which may re quire recalibration. Any field or QC samples that have been analyzed since the la st acceptable calibration verification should be reanaly zed after adequate calibration has bee n restored. 11. PROCEDURE 11.1 SAMPLE PREPARATION 11.1.1 Samples a re pres erved, c ollected and store d as pres ented in Se ction 8. All field and QC samples must contain the preservatives listed in Section 8.1.1. Measure a 25-mL a liquot of sample into a volumetric flask. 531.2 - 20Alterna te volume s are al lowed as long as t hey are det ermined using a g lass volumetric flask or pipette. When the volumes of SUR and analy te PDS added to the field and QC samples are kept to a minimum, as described below, no volume adjustment is necessary

. 11.1.2 Add an aliquot of the SUR PDS (Sect. 7.2.1.2) to all samples and mix by capping and inverting the sample. If the SUR PDS volume is less than 1% of the total volume, no volume adjustment is necessary

. For the development of this method, the addition of 5.0 uL of a 10 ug /mL SUR PD S t o a 2 5-m L sam pl e re sul ted in a SU R co nce nt rat io n of 2.0 ug/ L. 11.1.3 If the sample is an L FSM, LFSMD or L FB/CCC, add the necessary amount of analyte PDS. If the PDS volume is less than 1% of the total volume, no volume ad justment is necessa ry . Cap and i nvert ea ch sample to ensure all components are properly mixed. 11.1.4 Filter samples prior to filling appropriate autosampler vials (F ilters as specified in Section 6.8 are recommended.). 11.2 ANALYSI S OF SAMPLES 11.2.1 Establish operating conditions as summarized in Table 1 of Section 17 for the HPLC sy stem, including the postcolumn reactor. HPLC c onditions and columns should be optimized prior to the initiation of the IDC. Confirm thatresolution of target peaks meets the require ments of Section 9.10. Rete ntion times are gi ven for i llustration in Table 2. Specif ic retention times will depend on the column and chromatographic c onditions chosen a nd may be modifie d by the user as long as adequa te sensiti vity and peak resolution are achieved. 11.2.2 Load filled autosampler vials into the HPL C autosampler. Start the injection sequence. For the development of this method, the injection volume ranged between 200 and 1000 uL and depended on system sensitivity. 11.2.3 Determine optimal excitation and emission wavelengths (if necessary ). Complete the IDC requirements descr ibed in Section 9.2. 11.2.4 Establish an appropriate retention time window for each tar get and surrogate to identify them in the QC and field samples. This should be based on measurements of actual retention time variation for each compound in standard solutions analyzed on the HPLC over the course of time. Plus or minus three times the standard deviation of the retention time for each compound while establishing the initial calibration and 531.2 - 21completing the IDC ca n be used to calculate a sugg ested window size; however, the experience of the analy st should weigh heavily on the determination of the appropriate retention window size. 11.2.5 Calibrate the system by either the analy sis of a calibration curve (Sect. 10. 2) or b y confirm ing th e init ial ca libra tion is st ill v alid b y analyzing a continuin g calibration check as d escribe d in Section 10.3. B egin analyzing fie ld and QC samples at their appropriate frequency by injecting the same size aliquots under the same conditions used to analy ze the initial calibration. Confirm that the system mee ts the resolution check criteria as described in Section 9.10 once for every 24 hour analytica l batch. 11.2.6 The analyst must not extrapolate bey ond the established calibration range. If an analy te peak area exceeds the range of the initial calibration curve, the extract may be diluted with pre served reagent water (Sect. 7.1.2). Acceptable surrogate pe rformance (Sect. 9.7) should be determined fr om the undiluted sample extract. Any dilutions will also affect analyte MRL

. 12. DATA ANA LYSIS AND C ALCULATION 12.1 Identify the method analytes in the sa mple chromatogram by comparing the retention time of the suspect peak to the retention time of an analy te peak in a calibra tion stand ard. Surr ogate r etention times shoul d be conf irmed to be within acceptance limits (Sect. 11.2.3) even if no targ et compounds are detected. Surrogate concentrations need to be c alculated and determined to be within QC limits (Sect. 9.7). 12.2 Calculate the analy te concentrations using the initial calibration curve g enerated as described in Section 10.2. Quantitate only those values that fall between the MRL and the highest calibra tion standard. Samples with target analy te responses that exceed the highest standard require dilution and rea nalysis (Sect. 11.2.5). 12.3 Adjust the calculated concentrations of the detecte d analytes to re flect the initial sample volume and any dilutions perf ormed. 12.4 Prior to reporting the data, the chromatogr am should be reviewed for any incorrect peak identification or poor integration. 12.5 Analyte conc entrations are reported in ug/L (usually to two significa nt figures). 13. METHOD PERFORMANCE 13.1 PRECISION, ACCURACY, AND DE TECTION L IMI TS – Detection Limits are presented in Section 17, Tables 3-5 and were ca lculated using the formula present 531.2 - 22in Section 9.2.4. De tection L imits were evaluat ed using two comme rcially availab le postco lumn systems and two diffe rent fluo rescen ce dete ctors. Sin gle laboratory pr ecision and accuracy are presented for three wa ter matrices: reagent water (Table 6); chlorinated (finished) surf ace water (Table 7); and c hlorinated (finished) ground water (Table 8). 13.2 SAMPLE STORAGE STABI LI TY STUDIES – Chlorinated (finished) g round water samples, fortified with method analy tes at 2.0 ug/L, were preserved and stored as required in Section 8. The averag e of triplicate analyses, conducted on day s 0, 2, 7, 14 , and 28 da ys a re pres ented in Se ction 17, T able 9. T hese dat a document the 28-day sample holding time. 14. POLLUTION PREVENT ION 14.1 For information about pollution prevention that may be applicable to laboratory operati ons, cons ult “L ess is Be tter: L aboratory Chemical Manag ement for Waste Reduction” available from the American Chemical Society ’s Department of Government Relations and Science Policy , 1155 16th Street NW, Washington, D.C., 20036. 15. WASTE MANAGEMENT 15.1 The ana lyti cal proc edures d escribe d in this met hod generate r elativel y small amounts of waste since only small amounts of reagents and solvents are used. The matrices of concern are finished drinking water. However, the Agency require s that laboratory waste ma nageme nt pract ices be c onducted consiste nt with all applicable rules and regulations, and that labora tories protect the air, water, and land by minimizing and controlling all re leases from fume hoods and bench operations. Also, compliance is required with any sewage discharge permits and regulations, particularly the hazardous waste identification rules and land disposal restrictions. For further informat ion on waste management, c onsult “The Waste Management Manual for Laboratory Personnel” also available from the American Chemical Society at the addr ess in Section 14.1.

531.2 - 2316. REFEREN CES 1. Glaser, J.A., D.L. Foerst, G.D. McK ee, S.A. Quave, and W.L. B udde, "Trace Analy ses for Wastewaters ," Environ

. Sci

. Technol . 1981, 15, 1426-1435. 2. Moye, H.A ., Sh errer, S.J ., and St. J ohn, P .A., “ Dynamic Labe ling o f Pest icide s for H igh Performance Liquid Chromatog raphy: Dete ction of N-Methylcarbamates and oPhthalaldehyde,” Anal. Lett. 1977, 10, 1049. 3. "OSHA Safety and Health Standards, General I ndustry," (29CRF1910)

. Occupational Safety and He alth Administration, OSHA 2206, (Revised, Jan.1976). 4. “Carcinogens-Working with Carcinog ens,” Publication No. 77-206, Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute of Occupational Safety and Health, Atlanta, Georgia, Aug ust 1977. 5. “Safety In Ac ademic Chemistry Laboratorie s,” 3rd E dition, Ame rican Che mical Soc iety Publication, Committee on Chemical Safety, Washington, D.C., 1979. 6. ASTM Annual Book of Standards, Part II , Volume 11.01, D3370-82, "Standard Practice for Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986. 7. Snyder, L .R.; Kirkland, J.J.; Glajch, J.L. Practical HPLC Method Development, 2 nd ed.;Wiley: New York, 1997; pp 22-25.

531.2 - 2417. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA* TABLE 1. INSTRUMENT METHOD CONDITIONS

Time (min) %Water %Methanol %Acetonitrile

initial 88.0 12.0 0.0 5.30 88.0 12.0 0.0 5.40 68.0 16.0 16.0 14.00 68.0 16.0 16.0 16.10 50.0 25.0 25.0 20.00 50.0 25.0 25.0 22.00 88.0 12.0 0.0 30.00 88.0 12.0 0.0
Instrum ent Method Conditions Column: Waters Ca rbamate 3.9 × 150 m m packed with 4.0 um C 18 stationary phase. Postcolumn Reactor: Reaction coil set at 80 o C, flow rate for Postcolumn Reagent #1 and #2 = 0.5 mL/min (each) f or Waters unit, 0.3 mL/min for the Pickerin g unit. Fluorescence Detector: 340 nm excitation, 465 nm emission with a 18 nm band width; Gain = 10 0; Attn. = 1 6; Respons e = Standa rd; 16 uL flow cel l. HPLC: A ternar y g radient comprise d of wate r, metha nol, and a cetonitr ile with a flow of 1.5 mL/min as shown in the table above.

*Instrum entatio n, when spec ified, do es no t constitute endo rsem ent. B rand name s are inc luded for illustratio n only.

531.2 - 25TABLE 2. RETENTION TIME DATA *
Analyte Retent ion Time (min.) STD DEV %RSD Aldicarb sulfoxide 4.36 0.0092 0.21 Aldicarb sulfone 5.07 0.0089 0.17 Ox am yl 5.74 0.0095 0.17 Me th om yl 6.53 0.0077 0.12 3-Hydroxy carbofuran 9.82 0.013 0.13 Aldicarb 11.5 0.013 0.11 Propoxur 14.3 0.020 0.14 Carbofuran 14.8 0.024 0.16 Ca rb ar yl 17.0 0.026 0.16 1-Naphthol 18.6 0.019 0.10 Methiocarb 21.8 0.015 0.07 BDMC (SUR ) 22.3 0.015 0.07 *Retention time data is calcu lated fr om preci sion and a ccurac y da ta result s presen ted in Table 6 and the calibration curve used to quantitate the da ta. Retention times may differ depending on the chromatogra phic conditions and columns used.

531.2 - 26TABLE 3. DETECTION LIMITS IN REA GENT WATER USING THE WATERS POSTCOLUM N CARBAM ATE SYSTEM AN D THE WATERS M ODEL 474 DETECTOR
Analyte Fortification Level (ug /L) Detection Limit a (ug/L) Signal to No ise Ratio Aldicarb sulfoxide 0.20 0.059 8:1 Aldicarb sulfone 0.10 0.057 3:1 Ox am yl 0.20 0.065 10:1 Me th om yl 0.20 0.050 10:1 3-Hydroxy carbofuran 0.20 0.029 18:1 Aldicarb 0.20 0.026 9:1 Propoxur 0.20 0.037 6:1 Carbofuran 0.20 0.043 9:1 Ca rb ar yl 0.20 0.045 13:1 1-Naphthol 0.20 0.063 10:1 Methiocarb 0.20 0.061 11:1

a Detection Limits were deter mined by analy zing 7 replicates over 3 days using the conditions outlined in Table 1 with a 1000-uL injection.

531.2 - 27TABLE 4. DETECTION LIMITS IN REA GENT WATER USING THE PICKE RING MO DEL PCX5 200 POSTC OLUMN SYSTEM AND THE WATERS MOD EL 474 DETECTO R
Analyte Fortification Level (ug /L) Detection Limi t a (ug/L) Signal to Noise Rat io Aldicarb sulfoxide 0.20 0.056 13:1 Aldicarb sulfone 0.20 0.026 15:1 Ox am yl 0.20 0.045 9:1 Me th om yl 0.20 0.045 11:1 3-Hydroxy carbofuran 0.20 0.041 11:1 Aldicarb 0.20 0.042 7:1 Propoxur 0.20 0.040 11:1 Carbofuran 0.20 0.058 7:1 Ca rb ar yl 0.20 0.065 22:1 1-Naphthol 0.20 0.034 9:1 Methiocarb 0.20 0.036 5:1

a Detection Limits were deter mined by analy zing 7 replicates over 3 days using the conditions outlined in Table 1 with a 250-uL injection.

531.2 - 28TABLE 5. DETECTION LIMITS IN REA GENT WATER USING THE WATERS POSTCOLUM N CARBAM ATE ANALYS IS SYSTEM AND THE WATERS MOD EL 2475 DETEC TOR
Analyte Fortification Level (ug /L) Detection Limit a,b (ug/L) Signal to Noise Rat io Aldicarb sulfoxide 0.20 0.038 14:1 Aldicarb sulfone 0.20 0.033 9:1 Ox am yl 0.10 0.044 4:1 Me th om yl 0.20 0.054 24:1 3-Hydroxy carbofuran 0.20 0.038 7:1 Aldicarb 0.20 0.049 12:1 Propoxur 0.20 0.061 10:1 Carbofuran 0.20 0.050 12:1 Ca rb ar yl 0.10 0.043 9:1 1-Naphthol 0.20 0.115 3:1 Methiocarb 0.20 0.055 5:1

a Detection Limits were deter mined by analy zing 7 replicates over 3 days using the conditions outlined in Table 1 with a 200-uL injection. b Th es e d at a w er e c ol le ct ed at Am er ic an Wa te r W or ks Se rv ic e C om pa ny.

531.2 - 29TABLE 6. PRECISIO N AND AC CURACY OF LOW AN D HIGH LEVE L FORTIFIED REAG ENT WATER a

Analyte Concentr ation = 0.2 0 ug/L (n=7) Concentr ation = 10 ug /L (n=7)

Mean % Recovery Relative Standard Deviation (%) Mean % Recovery Relative Standard Deviation (%) Aldicarb sulfoxide 112 6.2 106 1.8 Aldicarb sulfone 92 9.5 106 2.6 Ox am yl 101 8.6 106 2.2 Me th om yl 101 6.5 106 2.9 3-Hydroxy carbofuran 105 6.8 108 1.2 Aldicarb 95 7.4 106 1.3 Propoxur 109 5.9 109 2.0 Carbofuran 112 6.7 110 2.2 Ca rb ar yl 112 7.0 107 2.1 1-Naphthol 113 12.6 108 3.1 Methiocarb 105 5.9 107 1.5 BDMC (SUR ) b 108 4.3 101 2.3

a Data obtained using conditions in Table 1 using a 1000-uL injection. b Su rro gat e co nce nt rat io n i n al l s amp les was 2.0 ug/ L.

531.2 - 30TABLE 7. PRECISIO N AND AC CURACY OF LOW AN D HIGH LEVE L FORTIFIED CHLORIN ATED SURF ACE WATER a

Analyte Concentr ation = 0.2 0 ug/L (n=7) Concentr ation = 10 ug /L (n=7)

Mean % Recovery Relative Standard Deviation (%) Mean % Recovery Relative Standard Deviation (%) Aldicarb sulfoxide 113 7.0 104 2.8 Aldicarb sulfone 104 5.5 106 1.4 Ox am yl 107 6.4 104 2.2 Me th om yl 110 9.8 104 1.6 3-Hydroxy carbofuran 128 3.9 107 1.1 Aldicarb 123 2.7 105 1.5 Propoxur 128 6.0 106 2.1 Carbofuran 140 5.6 105 2.5 Ca rb ar yl 112 9.7 106 0.9 1-Naphthol 113 12.1 101 1.3 Methiocarb 104 13.3 107 1.1 BDMC (SUR ) b 108 2.1 96 3.9

a Data obtained using conditions in Table 1 using a 1000-uL injection. b Su rro gat e co nce nt rat io n i n al l s amp les was 2.0 ug/ L.

531.2 - 31TABLE 8. PRECISIO N AND AC CURACY OF LOW AN D HIGH LEVE L FORTIFIED CHLORIN ATED GROU ND WATER

Analyte Concentr ation = 0.2 0 (n=7) Concentr ation = 10 ug /L (n=7)

Mean % Recovery Relative Standard Deviation (%) Mean % Recovery Relative Standard Deviation (%) Aldicarb sulfoxide 111 7.3 106 1.1 Aldicarb sulfone 98 9.2 106 0.9 Ox am yl 99 8.4 105 1.2 Me th om yl 99 10.2 105 1.4 3-Hydroxy carbofuran 107 3.0 108 0.4 Aldicarb 100 6.3 105 0.6 Propoxur 112 6.1 107 0.8 Carbofuran 112 4.1 107 1.6 Ca rb ar yl 119 5.1 108 1.3 1-Naphthol 109 8.2 109 1.2 Methiocarb 105 3.9 107 1.0 BDMC (SUR ) b 109 2.0 97 4.3 ug/L

a Data obtained using conditions in Table 1 using a 1000-uL injection. b Su rro gat e co nce nt rat io n i n al l s amp les was 2.0 ug/ L.

531.2 - 32TABLE 9. SAMPLE HOLDING TIME DATA FOR CHLORINATED GROUND WATER SAMPLES FORTIFIED WITH METHOD ANALYTES AT 2.0 ug/L

Analyte % Recover y for Samp les For tified at 2. 0 ug/L Day 0 Day 2 Day 8 Day 15 Day 28 Aldicarb sulfoxide 94 97 93 93 96 Aldicarb sulfone 93 99 97 98 98 Ox am yl 97 97 101 103 101 Me th om yl 96 95 99 98 97 3-Hydroxy carbofuran 96 99 96 95 98 Aldicarb 95 100 96 92 93 Propoxur 95 98 97 98 99 Carbofuran 96 99 97 97 100 Ca rb ar yl 96 97 98 94 100 1-Naphthol 96 97 99 95 98 Methiocarb 95 98 96 94 97 BDMC (SUR ) b 99 105 100 99 99

a Data obtained using conditions in Table 1 using a 500-uL injection. b Su rro gat e co nce nt rat io n i n al l s amp les was 2.0 ug/ L.

531.2 - 33TABLE 10. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREME NTS

Method Reference Requirem ent Specificat ion and Frequency Accept ance Cri teria

Sect. 9.2.1 Initial Demonstration of Low System Background Analyze LR B other IDC steps. Demonstrate that a ll target analytes are below 1/3 the intended MRL or lowest CAL standard, and that possible interference from reagents and glassware do not prevent the iden tification and quantitation of m ethod analytes. prior to any

Sect. 9.2.2 Quality Control Sample (QCS ) Second source standar d, used to fortify preserved reagent water (Sect. 7.1.2). Analyze as a CCC after initial calibration but prior to the analysis of th e IDA samples. Verifies initial calibration accuracy, recovery must b e within + 30% of true value.

Sect. 9.2.2 Initial Demonstration of Accuracy (IDA) Analy ze 4- 7 replic ate LFBs/CCCs at midrange co ncentration. Mean r ecovery + 20% of true value.

Sect. 9.2.3 Initial Demonstration of Precision (IDP ) Calculate averag e recovery for replicates used in IDA. %RSD must be < 20%.

Sect. 9.2.4 Detecti on Lim it Determination Over a period o f three days, prepare a min imum of 7 replicat e LFBs fortifie d at a concentration estimated to be near the Detection Lim it. Analyze t he repl icate s throu gh all steps of the analy sis. Calcula te the D etection Limi t using the equation in Sect. 9.2.4. Data from Detection Limit replicates are not required to meet meth od precision and accuracy criteria. f the Detecti on Lim it replic ates are forti fied a t a low en ough concentration, it is like ly that they will not m eet precision and accuracy criteria. fortified

I

531.2 - 34TABLE 11. QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method Reference Requirem ent Specification and Frequency Accept ance Cri teria

Sect. 8.3 and Sect. 8.4 Sample and Extract Holding Times Properly preserved samples m ust be shipped at or below 10 0 C and may be held in the lab at or below 6 0 C for 28 days. Samples should not be froz en. Do not report data for samples that hav e not be en prop erly preserved or stored, or that have exceeded the ir holding time. Sect. 9.4 Laborat ory Reagent Blank (LRB) Incl ude a LRB wit h each a nalysi s batch (up to 20 sam ples). Analyze prior to analyzing samples and determ ine to be free from interferences. Demonstrate that a ll target analytes are below 1/3 the intended MRL, and that possible interference from reagents and glassware do not prevent the identification and quantitation of metho d analytes. If targets e xceed 1/3 the MRL, results for all subject analytes in the a nalyti cal batch are invalid. Sect. 9.5 and Sect. 10.3 Continuing Calibration Check (CCC) Verify initial calibration by analyzing a low level (at the MRL or below) CCC prior to analy zing samp les. CCs are then injected after every 10 samples and after the last sample, rotating concentrations to cover the calibrated range o f the instrument. Recovery for each a nalyte must be 70- 130% of the true value for all but the lowest level of calibration. The lowest calibr ation level CCC must be 50- 150% of the true value. Results that are not brack eted by acc eptable CCCs are invalid. C

Sect. 9.7 Surrog ate Standards The surrogate, 4- bromo- 3,5- dimithyl phenyl –meth ylcarb amate is adde d to all field and QC sam ples. Surrogate recovery must b e 70-130% of the true value. Samples that fail criteria,must be reported as suspect due to surro gate rec overy.

531.2 - 35Method Reference Requirem ent Specification and Frequency Accept ance Cri teria

Sect. 9.8 Laborat ory Fortified Samp le Matrix (LFSM) and Laborat ory Fortified Samp le Matrix Duplica te (LFSMD) With each analy sis batch (Sect. 3.1), a mini mum of one LFSM is extracted and analyzed. duplicate LFSM , or LFSMD, should be extracted when occurre nce of t arget analy tes is low. ield Du plicate analy sis is not required for analy sis batches containing an LFSMD. Recoveries not w ithin 70- 130% (50-150% a t the MRL) of the fortified amou nt may indicate a ma trix effe ct. If an LFS MD is analyzed instead of a La borator y Duplicate , target RPDs should be within + 30%. If all CCC s meet acceptance criteria, and LFSMD do not, samp le is designated suspect/m atrix. A

F

LFSM or

Sect. 9.9 Field Duplicates Analy ze at l east one duplica te with each analysis batch (20 sampl es or le ss). Labora tory Fortifie d Sam ple Matri x Duplicate may be substituted for a Field Duplicate when the occurre nce of t arget analy tes is low. RPDs should be w ithin + 30%. If all CCC s meet acceptance criteria, and Field Duplicates do not, samp le is designated suspect/matrix. A

Sect 9.10 Resolution Check Monitor once for every 24 hour analysis period. Closely eluting peaks that are not baseline resolved must have a resolut ion of > 1.0 or greate r using the equ ation in Sect. 9.10 Sect. 9.11 Quality Control Samp le Analyze at least quarterly or when preparing n ew standards, as well as during the IDC. Same acceptanc e criteria as a mid-level CCC.

Sect. 10.2 Initial Calibration Use external standard calibratio n technique to generate a calibration curve with at least five standards. When each calibration standard is calculated using the calibration curve, the results should be 70- 130% of the true value for all but the lowest standard. est standard should be 50- 150% of the true value. The lowest CAL standard concen tration must be as low or lower than the intended MRL. The low

531.2 - 36FIGUR E 1. SAMP LE CHROMAT OGRAM O F METHOD531.2 ANA LYTES

Rea gen t wa ter , sa mp le p res erv ati ves add ed, for ti fie d at 2.0 ug/ L.

531.2 - 37531.2 - 38