RAPID DETERMINATION OF TCOD IN SOIL AND SEDIMENT

USING GAS CHROMATOGSAPHY ADD TANDEM MASS SPECTROMETRY

M A R C H 1986

U . S. ENVIRONMENTAL PROTECTION AGENCY

REGION VII

80503 ATTACHMENT 1

TABLE OF CONTENTS

Page

. 1 . SCOPE AMD A P P L I C A T I O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

I I . S U M M A R Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

I I I . I N T E R F E R E N C E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

I V . S A F E T Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

V . APPARATUS AND M A T E R I A L S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

V I . R E A G E N T S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

V I I . C A L I B R A T I O N . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

V I I I . QUALITY CONTROL R E Q U I R E M E N T S . . . . . . . . . . . . . . . . . . . . . . . . . 11

I X . SAMPLE COLLECTION, PRESERVATION, AND H A N D L I N G . . . . . . . . 13

X . SAMPLE E X T R A C T I O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

X I . C L E A N U P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

X I I . GC/MS/MS A N A L Y S I S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

X I I I . METHOD P E R F O R M A N C E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

X I V . DATA R E P O R T I N G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Z2

T h i s method is for f" '̂ in the rap id de te rmina t ion »~\<;,J, / ,o-iei.i du i io ro-dib 'enzo-p-dioxin (2 ,3 ,7 .b i C D D ) in soil and sediment, wl.^.i 2,3.7,8-TCDO isknown to be the p r i n c i p a l or only t e t r ach lo rod ibenzod iox in isorer present.The method is not spec i f i c for the 2,3,7.8-TCDD isoiner. unless a cap i l la ryco lumn w h i c h separates that isomer f rom the other 21 TCDO isomers Is employed.The method is app l i cab le 1n the concentration range of 0.3-25 ug/kg.

The method employs a tandem quadrupole mass spectrometer (MS/MS) as thef i n a l detector. The spec i f ic i ty of detection inherent in such a systems i g n i f i c a n t l y reduces the need for sample c leanup . This , in turn, improvesp roduc t iv i t y and cos t -ef fec t iveness r e l a t ive to other h i g h resolution and lowr e s o l u t i o n GC/MS ana ly s i s techniques. The appara tus and methods described

-are des igned for use in a mob i l e laboratory, w h i c h permits on-site ana lyses .

The method is intended to be used when analy t ica l results are requiredr a p i d l y , such as when site c leanup operations are in progress. Since themethod is not isomer spec i f i c , f a l se pos i t ives , i n c l u d i n g isowrs other than2 , 3 , 7 , 8 - T C D O , may occur. But errors in th is regard would be on the s ide ofsa fe ty . E m p h a s i s in the method is placed on a v o i d i n g fa lse nega t ive s , asth i s is a more c r i t i ca l c o n s i d e r a t i o n when p u b l i c hea l th is to be protected.

T h i s method is restr ic ted to use only by or under the s u p e r v i s i o n ofa n a l y s t s experienced in the use of gas ch romatography / t r ip l e quadrup le massspectrometers and s k i l l e d i n the in te rpre ta t ion of mass spectra.

Because of the extreme t o x i c i t y of th i s compound, the a n a l y s t mustprevent exposure to h i m s e l f , or to others , by m a t e r i a l s known or b e l i e v e d t3c o n t a i n 2 , 3 , 7 , 8 - T C D U . Section I V of th is method conta ins g u i d e l i n e s andprotocols that s a r v e as niini.-.u.-, s a f e - h a n d l i n g s tandards i n a l i m i t e d accessl abo ra to ry .

A n a l y t e CAS Number

2 , 3 , 7 , B - T C D D 1746-01-6

I I . SUMMARY OF METHOD

F i v e (5 ) grams of anhydrous sod ium s u l f a t e is placed in a 10 rn'i serur.v i a l and the v i a l w i t h cap and septum is weighed . A p p r o x i m a t e l y 5 grams o' as o i l sample i s added and the v i a1 is re -weighea . The sample is s p i k e l wi^i n t e r n a l and su r roga te s t anda rds of i s o t o p i c a l l y l a b e l l e d 2 ,3 ,7 ,8 -TCD3. Thes a m p l e is m i x e d by s h a k i n g , and ext rac ted w i t h acetoni t r i le /c i ich1orornethaiei n the closed v i a 1 . An a l i q u o t of the extract is taken and , a f t e r separata".f rom a c e t o n i t r i I e , the d i c h l o r o m e t n a n e is used d i r e c t l y for G C / M S / M S a n a l y s i s .C l e a n - u p s h o u l d u s u a l l y not be necessary , but a c l e a n - u p procedure is i n c l u c ^ - afo r those samples w h i c h do not meet q u a l i t y a s s u r a n c e c r i t e r i a . Concsi i re t ; o"of the e x t r a c t may be done to lower the m i n i m u m de tec table concen t r a t i on .C a p i l l a r y c o l u m n G C / M S / M S c o n d i t i o n s are descr ibed w h i c h a l l o w for s e p a r a t i o nof TC33 f r o m th fc b u l k s amp le m a t r i x and measurement of TCDO i n the ex;ra: i .

/

Q u a n t i f i c a t i o n is based on the response of nat ive TCDO r e l a t i ve to the i s o t o p i c a l l vl a b e l l e d TCDD i n t e r n a l s tandard . Performance is assessed based on the resul ts fo rsurrogate s tandard recoveries, EPA performance e v a l u a t i o n samples , sp ike recoverytests, and method and f i e l d b l anks .

I I I . I N T E R F E R E N C E S

Method interferences may be caused by con taminan t s i n solvents , reagents ,gl 'assware and other sample p rocess ing hardware that lead to discrete a r t i f ac t sa n d / o r e leva ted backgrounds at the ions monitored. A l l of these mate r i a l smust be - r o u t i n e l y demonstrated to be free from interferences under the oc o n d i t i o n s of the ana lys i s by r u n n i n g laboratory method b l a n k s as described ,_i n Sect ion V I I I . "

m

The use of h i g h pur i ty reagents and solvents he lps to m i n i m i z e in te r fe rence r"'p r o b l e m s . P u r i f i c a t i o n of so lvents by d i s t i l l a t i o n in a l l - g l a s s systems may °be requi red . 0

M a t r i x in te r fe rences may be caused by con taminan ts that are co-extractedfrom the sample . The extent of mat r ix interferences w i l l vary cons iderab lyf r o m source to source, d e p e n d i n g upon the nature and d ivers i ty of the sample .2 , 3 , 7 , 8 - T C D O is of ten as soc ia t ed w i t h . o t h e r i n t e r f e r i n g ch lo r ina t ed compoundsw n i c h are at c o n c e n t r a t i o n s ' s e v e r a l magni tudes ' h igher than that of 2 ,3 ,7 ,8 -TCOO.

The use of a t r i p l e q u a d r u p o l e mass spectrometer as the detector servesto m i n i m i z e the i n f l u e n c e of many of these in ter ferents .

I V . SAFETY

Tne f o l l o w i n g safety pract ices are excerpted d i r ec t l y from EPA Method613, Section 4 ( J u l y 1982 v e r s i o n ) : See f o l l o w i n g page.

•» icc »'l • pofcni.ariictiin naiaro. ' 'Jm this i .awpoint. exposure to theceimicals mull be i«duc«d ie the•est possible level by whatever•ans available The laboratory R.pons'ble to' maintaining * current

ar enesi fir ol OSHA regulation!;aid'ng the t*(« hindling of the;micJl> apeofied in this method, Ar'ence file ol materiti dill hindling.•eti mould also be made available 10pe'sonnel involved in the chemicaliiysis. Additional icferencex 10oratory safety aie identified'* to'.•i;en( and 2.3.7.B-TCDD h»ve be«n"1'l.ea •t suspected human or

•mmalian cucmooens.

2 Each laboratory must develop •id safety p'og'am lor handling ol3.7.B-TCDD. Tht following labora-» p'a:tices •'e '(commended:

7 7 Contamination of the labora-Y will be minimised by conducting all.nipuiJiions in a hood.

S.2 Tht etilue-ts of samplei:e's lot the ;» chromatog'aph «"d'

;6-"n; aJmps on the GC MS thouldis Th'c-^^ either a column ol•'vaie; ;"B'COB: o' be bubbledOus^ a Irao containing oil Or high-

mng alcohols

' 7 L—.'"? •>•<•; should Besc^ed in me'nanol of ethaiol •nd•; a;e3 i»'ih ultraviolet ligil with•i €";•"> g'eale' than 290 nm fo''«'a CJrt lUie F 40 BL lamsioij". *.(-•, I An*;y» liauid war.es •ndsase c* the to'ulioni when3.7 ,8 TCD3 can no longe' beectec

1 Co— Chemical U S A "•» ntuedlol'owing e'ecaulion* Irevnta7E' (01 tale handling ol

3 .7 .6 TC;3 in the laboratory:

^ 1 The follonin; ttaterrrnti one na-i(;;i"g a'e as ccmpier •a•.s-air on li-.t DBS i of a»a'lab>e•co'og cal inlo'mation ThecaiJtio^i fo' sa fe hangiing and u«enecacsarily ce^e^al in naluf aincea"ed. »ceci(<c fecomniendaiioni can

maoe only <o< the panicul*' opciuie1 CifCumxiancel of each individual• InQJiiiei aBout tpecific ose'anontatvt m«v be acs-'esseo to the OoweTiica'Comcany Atlitianct in•iwati^; the hea'th hazares of•ticuia* pian) conoitiont rrrv ba8'"i3 I'om cenrn contu'l'ma-a io^ fs and licm S fa le Oesarl-•^*i c' Mea'ih Of ol Labor, many ol

2 7.B TC3; i( aiiram«iY toiic to

"oef^4lo'•uJeu im yealk wniioul (h)ury (nani cal and biological laboftom.TechniQues used in handling radio-active and infecliout rmerrls •fapplicable to 2.3.7.B.TCDO.

4 3 . 1 . 1 Protective EQuipmBni;Throw-away platlic glovei. •0100 Oflab coat. tafety glasiet and lab hood•deguale (01 ladioactive work.

4 3.1.3 Training- Workeri mutt toutrained in the pioper method ofremoving of contaminated glovec •ndclothing without contacting theexenoi (urfacit.

4 3 . 1 . 3 Perional Hygiene; Thoroughwashing of hands and tofearms •hereach manipulation and belore brgakt(coffee, lunch, and thittl.

4 3 1 . 4 Confinement: Isolated workaiea. posted with xigns. aegregatttdg'sssware and tools, plastic-backedaDsorbent paper on benchicpt.

4 3 1 . 5 Waste-Good techniguBincludes minimising contamtnafdv«Js;e Plasiic bag liners ahould beused in was:e can* Janitors mult betrained in Kate handling of wsf

43 I S D'SDOSal of Wasies:2.3.7.8 TC3S decomposes above800 °C Lew-level waste luch •t theassorbent paoer. tissues, •nimalicmans and plastic givoes may beburned in a good incinerator. G'OISa-Jantities Imillig'amsl should Bepackaged securely and disposadthrouB'' ccmmercia! or governmentalchannels which are caoable of handlinghigh-level radioactive wastes oreitremely lone wastes Liauids ihouldbe allowec to evaporate in a goos hoodand in a disposable container. ResiOuafmay then be handlad as abov.

4 3 1.7 Oecontamination: P»r»onal—any mild saaD with plenty of acrubbingaction- Giassware. Tools.. •ndSurlaces-Chloroihene NU Solvnt(Tracemark of the Oow ChamicalCompanyl i< the least tone solventshown 10 be ef fect ive Satistaciory

nosing w»lh ChlorQthene. then washing

with any Odt'Otit ana —til Dith

it prudent 1C minimise »olvent watrsbecause they may >eQu<re aoeciaid'ssosai ihroush commercial aourcaswhich a'r e«oen»ive

4 3 1 8 LaunOry Clothing known to

be ec^t»minite3 ihouie be ditoordw-ih ihe o'ecautioni orscnoeo unoai"Duoott' o' Wai:es ' Lao coaf 01Other clothing worn in 2.3.7.B TC^S

^•**rw4«W(B ^kfrt.kll^W^' »k<k»llk U90%.

Persi Mho convy the bagi andlaundei the clothing ahouid be advisedol the harrd •nd (fined in properhandling. The clothing may e* out imo• washer without contact if thelaunderar know the problem. Thewasher should b« run through • cyclebelore being uied again lor otherClothing.

4. 3.1.3 Wipe Tettf A useful methodof determining cleanliness ol workaurlaces and tool it to wipe the surfacewith a piece of filter paper. Eitractionend analysis by gas chromatographycan achieve a limn of sensitivity of 0 1M per wipe. Leas than 1 ̂2.3.7.B-TCDO per sample indicatesacceptable cleanliness, anything highe*warrants funher cleaning More than10 pg on i wipe sample indicates an•cute ha;«irl and reouires promptcleaning before further use of theeguipment or work space and indicatesfunher mat unacceptable workpractices have been employed in thepast.

4 3 . 1 . 1 0 Inhalation Any procedurethat may produce airborne contam.nalion must Pe done with good ventilationGross losses to a vennla'ion systemmust not be allowed Handling of thedilute solutions normally uses in•naiyiiCJl and aninial work p'«irits noinhalation hazards ezceoi in case ol a"accident.

4 3 . 1 . 1 1 Accidents. Removecontaminated clothing immed;aie'»(•king precaut'ons not to co"tamina'eSkin or other articles Wash expose:skin vigorousiv and repeatee'y untiimedical attention is obtained

A'r HIS

^ ^>.V.' APPARATUS AND M A T E R I A L S

A l l g lassware is i n i t i a l l y c leaned w i t h aqueous detergent and then r insedw i t h tap water , d e i o n i z e d wate r , acetone, toluene and metnylene chloride.Other c l e a n i n g procedures may be used as long as acceptable method b l a n k s areobta ined.

E lec t ron ic b a l a n c e , c a p a b l e of w e i g h i n g at leas t 50 g, with an accuracyqf at least ^ 0.05 g.

Shaker , vortex-type or e q u i v a l e n t

C e n t r i f u g e . 4UUU rpm, capab le of h a n d l i n g 25 mm diameter v i a l s

C e n t r i f u g e tubes

liJ m1 serum v i a l s ; w i t h t e f l o n faced septa and a luminura caps (Chrompak102U^ and 1U213 or e q u i v a l e n t )

1 m1 serun v i a l s ; w i t h t e f lon faced septa and a l u m i n u m caps (Chrompak102J1 and 10211-or . e q u i v a l e n t )

C r i m p e r fo r 10 m1 serum v i a l (Chrompak 10233 or e q u i v a l e n t )

C r i m p e r f a r 1 m l s e rum v i a l (Chrompak 10231 or e q u i v a l e n t )

D i s p o s a b l e t e f l o n 0.45 micron f i l t e r s ( M i l l i p o r e SLHV025 H3, or e q u i v a l e n t )

5 (ill d i s p o s a b l e G la spak syr inges (Sargent We lch S-79401-B or e q u i v a l e n t )

18 gauge d i sposas l e syr inge needle (Sa rgen t W e l c h S-79'iJ2-G or e q u i v a l e n t )

D i s p o s a b l e pipe'.s, S 3/< inches x 7 mm o.d.

G l a s s woo l , s i l a n i z e d

N i t r o g e n blowdown appa ra tus

Gas chroinatograph - an a n a l y t i c a l system w i t n a11 r equ i red accessor iesi n c l u d i n g s y r i n g e s and a n a l y t i c a l c o l u m n s . The i n j e c t i o n port iiust be oesigie ifor c a p i l l a r y c o l u m n s and s p l i t l e s s i n j ec t i on .

T r i p l e q u a d r u p o l e mass spectrometer w i t h GC t r a n s f e r l i n e ana c l o w.d iscnargs ion source (TAoA* 60UU, S C I E X f , T h o r n h i l l , O n t a r i o , Canac: ; '

Compresse ' l Gases : Zero Grade A i r ( f r o m d i s t i ' I l a t i o n , not w a t e rh y d r o l y s i s )Ult ra Mign Purny NitrogenUltra High Purity Argon

C o l u m n : 15 m l o n g , w i d e bore f u s e a s i l i c a c a p i l l a r y (e;. U.3:mm 1 . D. )03-b l . U micro" f i l m t h i c k n e s s .

V I . R E A G E N T S

Stock Standard Solutions

Stock standard so lu t ions correspond to three to luene solutions c o n t a i n i n aun labeUed 2,3.7.8-TCOO at v a r y i n g concentra t ions , and Ci7-2,3,7,8-TCDD( i n t e r n a l s tandard. CASEM 80494-19-5) at a constant concentrat ion. Theses o l u t i o n s also contain •"C'l4-2.3,7,8-TCDD (sur rogate compound , CASRN 85508-50-5) at v a r y i n g concen t ra t ions . These stock so lu t ions are to be used i np r e p a r i n g the c a l i b r a t i o n s t a n d a r d so lu t ions , and are to be obta ined f rom theQ u a l i t y Assurance D i v i s i o n , USEPA. E n v i r o n m e n t a l M o n i t o r i n g Systems Labora to ry( E M S L - L V ) , Las Vegas, N e v a d a . I f not a v a i l a b l e f r o m EMSL-LV, stock s t a n d a r d •^s o l u t i o n s may be prepared f rom commerc ia l ly a v a i l a b l e s tandards . However, "̂the accuracy of these so lu t ions must be checked against EPA s u p p l i e d s tandardinso lu t ions . .̂

The three stock s o l u t i o n s w i l l have the f o l l o w i n g concen t ra t ions of ,-,u n 1 a b e 1 l e d , i n t e rna l and sur roga te s tandards .

Stock Solution f\ (CCl)

U n l a b e l e d 2",3,-7.&,TCDO - 0.2 ng /u l^Ci? -2 ,3 ,7 ,8 -TCDD - 1.0 ng /u l-"ct4-2,3,7,8-TCDO - 0.06 ng /u l

Stock S o l u t i o n »2 ( C C 2 )

Uclaieled 2.3.7,8-7020 - 1.2 ng.'ul^C,2-2,3,7,8-TCOD - 1 .0 ng/ul•"CT^^^^-TCDO - 0 .12 ng/ul

Stock So lu t ion f3 (CC3)

y n l a a e l e d 2 ,3 ,7 ,8 -TCDO - 5.0 n g / u l^CI^^^^-TCOD - 1.0 n g / u l•"ct;;-2,3.7,8-TCOD - 0.2 n g / u l

NOTE: Store stock s o l u t i o n s i n 1 ml anoer m i n i - v i a l s under r e f r i g e r a t i o n .

C a l i b r a t i o n S t a n d a r d S o l u t i o n s

C a l i b r a t i o n s t andard s o l u t i o n s are prepared to s i m u l a t e the c o n d i t i o n sof s a n p l e a n a l y s i s as n e a r l y as p o s s i & l e . Three c a l i b r a t i o n s t andar i s o 1 u ^ i o " iare p repa red f r o m tne stock s tanaara s o l u t i o n s so as to c o n t a i n cons tantamounts of i n t e r n a l S t a n a a r a (5 u c / k g e q u i v a l e n t ) w i t n v a r i a b l e arnoJnis ofu n l a i e l e d s t a n d a r d (1 ,5 , and 25 u c / k g e q u i v a l e n t ) and su r roga te s t anca r j(0.3, 0.6, a n a 1.0 U Q . ' K g eq.i'i v a l e n i ) . Tne e q u i v a l e n t concen t ra t ions a'"ebased on lie use of 5-grai-. s a m p l e s , e x t r a c t i o n w i t h 5 inl of 2:1 a c e t o n i t n I e :d i c h l o r o m e t n a n e , and a f i n a l ex t r ac t v o l u m e of a p p r o x i m a t e l y 1.66 ml d i c n i o r c -metnane a ' t e r r emava l of a c e t o n l t n i e , as c a l l e d f o r i n the procedure .

Low Level

Add 750 ul of stock solut ion ,»! to a 5 ml volumetric f lask and bring tovolume with dichloronethane. Mix wel l . This solut ion-contains an equivalentconcentrauon of 1 ug/kg of 2 ,3 ,7 ,8 -TCDD, 5 ug/kg of "Ci7-2,3,7,8-TCDO and 0 3ug/kg of ^C\ ̂ -2,2,7 .S-fCW. a a i^ , .^

M e d i u m Level^

Add 750 ul of stock solut ion »2 to a 5 ml volumetric f lask and bring to ^3-volume with dichloromethane. Mix well. This solution-contains an equivalent ,r\concentration of 5 ug/kg of 2,3,7,8-TCDO, 5 ug/kg of "Cn-Z^^S-TCDD, and 0.6ug/kg of ^ 'C l4-2,3 ,7 ,8-TCDD. r"

High Level 0

Add 750 ul of stock solut ion ,i-3 to a 5 ml volumetric f lask and bring tovolume wi th dichloromethane. Mix w e 1 1 . This solution contains an eauivalentconcentration of 2 5 ' u g / k a Jof 2 ,3 ,7 ,8-TCDD, 5 ug/kg of ^Cn-Z^, 7,8-TCDD, and 1 .0ug/kg of -"C1 4-2,3,7,8-TCDO.

NOTE 1: Although the surrogate, ^C^-Z^^^-TCDO, is present in all threelevel cal ibration solut ions, only the high level solution is used for calculatingthe relat ive response factor for the surrogate.

NOTE 2: AH cal ibrat ion standard solutions must be stored in an isolatedrefrigerator and protected from light. Check these standard solutions frequentlyfor signs of evaporation.

Samole S o i k i n g S o l u t i o n

The sample spiking solution is also to be obtained from the QualityAssurance D i v i s i o n , U . S. EPA Environmental Monitoring Systems Laboratory( E M S L - L V ) , Las Vegas, Nevada. The spiking solution w i l l have the followingconcentrations of internal and surrogate standards.

^Ci^^^J.S-TCDD - 0.5 ng/ul^ct^-Z^J.S-TCDO - 0.1 ng/ul'CT^-Z^J.S-TCDO - 0.1 ng/ul

When 50 u1 of this solut ion is spiked in 5 g of soil, the resultingconcentrat ions in the soi l are 5 ug/kg and 1 ug/kg of internal and surrogatestandard, respect ively.

It is recommended tha; approximately 2 .5 -5 ml of the sp ik ing solut ion betransferred to a 5 ml serum v ia l and sailed wi th a septum and cap prior taeach d a y ' s work for use in sp ik ing saroles that day.

'NOTE: It is very imosrtant that no evaoora t ion of sample sp ik ing so lu t ionbe a l l o w e d to occur, since the accuracy of resul ts ars d i rect iv deDendent onthe aadi t ion of a known amount of internal standard.

/-^ r\F i e l d B l a n k S p i k i n g S o l u t i o n

The field blank spik ing solution is also to be obtained from the QualityAssurance Div-ision, U. S. EPA, Envi ronmental Monitoring Systems Laboratory[EMSL-LV), Las Vegas, Nevada. The spiking solution w 1 1 1 have the followingconcentrations of unlabelled, internal, and surrogate standards:

2 3,7,8-TCDD - 0.1 ng/ul"C^-Z.B.P.B-TCDD - 0.5 ng/ul•"(^-Z.B.T.S-TCDD - 0.1 ng/ul

ur̂When 50 u1 of this solution is spiked in 5 grams of soi'1, the resulting .̂

concentrat ions in the soil are 5 ug/kg of internal standard and 1 ug/kg eachof unlabelled and surrogate standard. Ln

c-NOTE: It is very important that no evaporation of field blank spiking 0

solution be allowed to occur, since the accuracy of results are directly 0dependent on the addition of a known amount of internal standard.

So1 vent

AH so1ven ts "should, be pesticide grade or equivalent. The followingsolvents w i 1 1 be needed:

Acetomtri leDicnioromethaneCyclohexaneTolueneBenzeneMethanol

S i l i c a Gel

Type 60, 70-230 mesh. Soxh te t extracted with dichloromethane for 24 hours,then ac t i va ted for 24 hours at 130°C.

Acid Alumina

AG 4, 100-200 mesh. soxhiet extracted with dichloromethane for 24 hours.then act ivated for 24 hours at 190°C.

Carbooack C

Celite 545

Sodium Sulfate

( A C S ) granular, anhydrous.

VII. CALIBS^TION AN3 LIMIT OF DETECTION DETERMINATIONS

Ca l ib ra t ion must be done using the internal standard technique. In fiscase, the internal standard is an isotope of the compound-;>f - interest , aid

metry. 'me tnree c'S r -auon sLaYioaro s o l u t i o n s aeioea ) n sect ion v i arerequi red.

Inject 1-2 ill of each of the c a l i b r a t i o n s tandard s o l u t i o n s and a c q u i r eselected react ion m o n i t o r i n g da ta for the f o l l o w i n g pa ren t - daughter ions:

m/z a 32U *257m/z = 322 -.259m/z = 328 *263 •m/z = 332 -*268

i

For s i m p l i c i t y i n subsequent sect ions, we w i l l refer on ly to the daugh te ri o n s , s ince q u a n t i t a t i o n is based on daugh te r ion response. s0

^t-R e l a t i v e response factors for u n l a b e l l e d 2,3,7,8-TCDD vs the i n t e rna l ^

s tandard for t r i p l i c a t e de te rmina t ions of each of the three c a l i b r a t i o n f~s tandard so lu t ions are ca lcula ted . ' 0

0E q u a t i o n I : R e l a t i v e Response Factor ( R R F s ) for 2 ,3 ,7 .8-TCDD

RRFs ' ( A s C , s ) / ( A ^ C s )

where A^ = the sum gf the area responses for the ions , m/z 257 and 259,co r re spond ing to the u n l a b e l l e d s tandard , 2 ,3 ,7 .8-TCDD.

A^ = the area response of-the ion m/z 268, c o r r e s p o n d i n g to thein t e rna l s t anda rd , --C^-2,3,7,8-TCDD.

C; = the concer.t.-.it-.on of the u r . l abe l l ed standari j , 2 ,3 ,7 .8-TCDD

C.j. = the concent ra t ion of the internal s tandard, C^-2,3,7,8-TCDO.

In the case of the u n l a b e l l e d 2.3,7,8-TCDD each of the c a l i b r a t i o ns t anda rd so lu t ions must be ana lyzed in t r i p l i c a t e , and the v a r i a t i o n of theR R F v a l u e s for each compound at each concentra t ion level must not exceed ID'.RSD. I f the three mean R R F v a l u e s for each compound do not d i f f e r by moret h a n + 10;, the R R F can be considered to be independent of ana ly te quant i tyfor the c a l i b r a t i o n concentrat ion range, and the mean of the three mean R R F ss n a i l be used for concentrat ion ca lcu la t ions . The overa l l mean is termed ac a l i b r a t i o n factor.

S i m i l a r l y , r e l a t i v e response factors for the surrogate s t andard vs thei n t e r n a l s t andard for the t r i p l i c a t e de t e rmina t ions of the h i g h leve l c a l i b r a t i o ns o l u t i o n are a l so ca lcula ted .

Eguaf ion I I : R e l a t i v e Response Factor ( R R F ^ ) for ^C^-Z^^.B-TCDD

R^ss - (Ass^s^isCss)

where A,c s the area response of the ,daughter i o n , m/z 263, c o r r e s p o n d i n g t3the sur roga te s t andard , - 'C l4 -2 ,3 ,7 .8 -TCDO.*

* Suotract O.Ulu^of any 257 response from the 253 response to correct forcontributions of 2 ,3 ,7 ,8 -TCOO to the 263 response.

^is = the area espouse of, the ion m/z 268, c^esponding to thei n t e r n a l s tandard , Ci2-2,3 .7 ,8-TCDD.

C^ = the concentra t ion of the surrogate s t anda rd , ^^-^^^.S-TCDD.

and C^ = the concentrat ion of the i n t e r n a l s tandard, ^C^-Z.B^.B-TCDO.

In the case of the surrogate s tandard. ^(^-Z.S.P.a-TCDD. the v a r i a t i o nof the three RRF va lues for the h i g h level c a l i b r a t i o n s o l u t i o n should notexceed 10; RSD. I f th i s is the case, the mean of the three RRFs sha l l be

.used for concentra t ion c a l c u l a t i o n s . The overal l mean is termed a c a l i b r a t i o nfactor .

The c a l i b r a t i o n factor for the u n l a b e l l e d 2 ,3 ,7 ,8-TCDD must be v e r i f i e d oneach work sh i f t of 8 hours or less by the ana lys i s of a low l eve l c a l i b r a t i o ns tandard . I f the R R F for the low leve l c a l i b r a t i o n d i f f e r s from the c a l i b r a t i o nfactor by more than 101, the en t i re c a l i b r a t i o n must be repeated and a newc a l i b r a t i o n factor de te rmined . The most recent ly ve r i f i ed c a l i b r a t i o n fac to rmust &e used in a l l c a l c u l a t i o n s . Th i s v e r i f i c a t i o n is only required for theu n 1 a 5 e l 1 e d s tandards . There is no need to check the surrogata c a l i b r a t i o nfac tor u n l e s s the surrogate recoveries appear biased or consis tent ly f a l l o u t s i d ethe 60-1401 control l i m i t s .

The theoret ical ra t io of the m/z 257 to 259 ions for n a t i v e 2 ,3 ,7 ,8 -TCDDis 1.02. However, i n p r a c t i c e t h i s ra t io w i l l d i f f e r f rom the theoret ical dueto the very low r e s o l u t i o n used in both a n a l y z i n g q u a d r u p o l e s for t h i s type ofa n a l y s i s . The r a t io must the re fo re , be de termined e m p i r i c a l l y as f o l l o w s :

E q u a t i o n I I I : ( R a t i o of n a t i v e TC33 daughter i ons )

R a t i o a ^257^259

where A^y ° Area response for ion m/z 257

^259 s Area response for ion m/z 259

Tne mean of the ratios ca lcu la ted for each of the n ine c a l i b r a t i o ns o l u t i o n s is used for comparison purposes for q u a l i t a t i v e i d e n t i f i c a t i o n of2 , 3 , 7 , 8 - T C D D .

It has been f o u n d that the sample s p i k i n g s o l u t i o n a l so g ives responsesfor tne 257 and 259 daucnte r ions cor responding to 2 ,3 ,7 .B-TCD3. Thesec o n t r i b u t i o n s rust be subtracted out for each sample . In order to de;e'"iinet h i s cor rec t ion fac to r , add 150 u l of the sample s p i k i n g s o l u t i o n to a 5 mlv o l u m e t r i c f l a s k and b r i n g to volume w i t i d i c n l o r o m e t h a n e . Twenty 1-2 u li n j e c t i o n s of t h i s s o l u t i o n must be made and the r a t i o of the area responsesfor the sum of tne m / z 257 and 259 i o n s vs the m/z 268 ion must be c a l c u l a t e d .Twenty s e p a r a t e r a t i o s must be dete^med.

r^ ^taJat-ion i»: mank >• ponse (b I o r - ^ a m p i e sp ik ing ^ Jiion

B = A b / A i s

where A^ = the sum of the area responses fo r the ions , m/z 257 and 259obtained w i t h the s p i k i n g s o l u t i o n

and A,s = The a rea response of the ion m/z 268, co r r e spond ing to thei n t e r n a l s t andard C^-2,3 ,7 ,8-TCDO present in the s p i k i n gsolution.

i

The correction factor for the blank contribution to sample response isthen calculated as the mean of the 20 blank responses.

^fEquation V: Correction Factor (C.F.) for Blank Contribution tr\

t—C.F. = ; B 0

n 0

where £ B = The sum of the individual blank responses determined byEquation IV.

n = Number of -repl icate measurements of the blank response (20 arerequired for initial determination).

L i m i t Of De tec t ion

The empirical limit of detection wi l l be calculated based on the variabil i tycf the S'ank responses. The blank responses correspond to those obtz'-'sdfro;n repeat injections of the (di luted) sample spiking solution. Each blankresponse must be converted to an equivalent concentration of 2,3,7,8-TCD3.

Equat ion VI : (Conversion of Blank Response to An Equivalent Concentration o*———————— 2.3,7,8-TCDD)

Co = A[, x Q,s 25 x Ab

A , ; x R R F ^ x W 5 x A, ; x R R F ^

where Cj, a equivalent concentration of 2,3,7,8-TCOD in blank (spik ingsolut ion) (in units of ug/kg or ppb)

A[) = the sum of the area responses of the ions m/z 257 and 259 forthe blank

A ,^ = the area response of the ion m/z 258, corresponding to theinternal standard '

RRF, = The re lat ive response factor previously determined for2.3,7,8-TCDD (Equation I)

Q , c s 25 n a n o g r a m s ( t h e w e i g h t of i n t e r n a l s t a n d a r d added to eacns a m p l e )

10

W = 5 grams (the weight of wet soil used for each s a m p l e )

The standard deviation of the blank responses (in concentration units)roust then be calculated.

Equa t ion V I I : (S tandard D e v i a t i o n of The B l a n k Responses )

' St, ° ^ Ct,2) - (S C^/h

m———— o^^

where Sp = s tandard d e v i a t i o n of the b l a n k responses ( i n u n i t s of u g / k g ) LHr-

CQ = b lank: response in concentra t ion u n i t s ( ca lcu la t ed u s i n g QEquat ion V I )

n = number of r ep l i ca te b l a n k results used (20 are r equ i red )

F i n a l l y , the l imi t of detection must be calculated from the standardd e v i a t i o n of the b l a n k . ''

E q u a t i o n V I I I : ( L i m i t of Detect ion Based on " W e l l - K n o w n " B l a n k ) *

LOD ' 2 t Sj,

where LOO = L i m i t of Detection

t = the 10; p o i n t of the t s t a t i s t i c for a double-sided tablewi th n-1 degrees of freedom (where n is equal to the numberof b l a n k resul ts used). NOTE: The LOD must be ca lcu la tedbased on at least 20 repl ica te b l a n k ( i .e . s p i k i n g s o l u t i o n )analyses . For n = 20, t = 1.72.

The l i m i t of detection c a l c u l a t e d from equation V I I I should be less thanthe requi red l i m i t of detection of 0.3 ug /kg .

V I I I . Q U A L I T Y CONTROL R E Q U I R E M E N T S

The f o l l o w i n g q u a l i t y control ( Q . C . ) requirements are l i s t ed in theorder tha t they must be run. Requi rements 1 and 2 are to be run i n i t i a l l ybefore any other samples . Requi rements 3 through 7 are the Q.C. samples tobe i n c l u d e d w i t h each batch of real samples (requirement i»8) that i s run -inone 8-hour t ime pe r iod or on each s h i f t . The requirements 3 through 8 are tobe run i n the orasr as they appear i n the l i s t below on each s h i f t .

* Reference - Currie, Lloyd A. "Limits for Qua l i ta t i ve Detection andQuant i tat ive Determination" Anal , Chem., 40, 3, 585-593, 1968

11

'1. An i n i t i a l c a l i b r a t i o n must be per formed u s i n g c a l i b r a t i o n standards o l u t i o n s w i t h va r i ed (1,6, and 25 u g / k g e q u i v a l e n t ) n a t i v e TCOD and 5 ug /kge q u i v a l e n t i n t e r n a l s t anda rd . C a l i b r a t i o n for the sur rogate s t anda rd w i l lbe based only on the h i g h level s t andard (1 u g / k g e q u i v a l e n t ) . The cr i te r iagiven in Section V I I must be met or the c a l i b r a t i o n must be repeated.

2. I n i t i a l l y , 20 r e p l i c a t e d e t e r m i n a t i o n s of the s p i k i n g s o l u t i o n mustbe run and area responses for the sum of m/z 257 and 259 ions vs the m/z 268ion must be c a l c u l a t e d . Twenty separate rat ios must be de te rmined ( E q u a t i o nI V ) and used i n c a l c u l a t i n g the mean correction fac tor ( E q u a t i o n V ) .

03. A 1-point check v e r i f i c a t i o n u s i n g the 1 u g / k g e q u i v a l e n t na t ive m

TCDD and 5 ug /kg e q u i v a l e n t i n t e r n a l s t anda rd must be run once every 8 hours or ir\on every s h i f t . If the RRF v a l u e s f rom th i s c a l i b r a t i o n check d i f f e r by more r--than ^_ 10% from the p r e v i o u s l y determined mean re la t ive response factor ( R R F s ) , Qthe 3-point c a l i b r a t i o n must be repeated. The c a l i b r a t i o n check forthe surrogate is not necessary u n l e s s the surrogate recoveries appear b iasedand/or cons i s t en t ly f a l l ou t s ide the 60-140% control l i m i t s .

4. A laboratory "method b l a n k " must be run a l o n g w i t h each batch of 24or fewer samples. A method b l a n k is performed by e x e c u t i n g a11 of thespec i f i ed ex t rac t ion steps, except for the i n t r o d u c t i o n of a 5 g ram sample .The method b l a n k is a l so dosed w i t h the i n t e r n a l s t anda rd and surrogates t anda rd . Resu l t s fo r the method b l a n k must be c a l c u l a t e d the same way assamples . Th i s inc ludes correct ion for the s p i k i n g s o l u t i o n c o n t r i b u t i o n asi n d i c a t e d i n E q u a t i o n I X . A p o s i t i v e response > 0.3 u g / k g of n a t i v e TCDDfo l lowed by reinject ion. If s t i l l p o s i t i v e , re-extraction and r eana lys i s ofa l l related samples must be done.

5. "F ie ld b l a n k s " w i 1 1 be prov ided to monitor for poss ib le cross-contamina t ion of samples in the lab . The "f ie ld b l a n k " w i 1 1 consist ofuncontaminated soil (background soi l taken o f f - s i t e ) . A pos i t ive response^0.3 u g / k g na t i ve TCOD must be f o l l o w e d by re in jec t ion . If s t i l l pos i t ive ,a l l samples associated w i t h the f i e l d b l a n k s must be re-extracted and reana lyzed .

6. One sample , de s igna t ed by E P A , must be s p i k e d w i t h n a t i v e 2,3,7,8-TCODat a level of 1 u g / k g for each set of 24 or fewer samples. The F i e l d B l a n kS p i k i n g S o l u t i o n (Section V I ) shou ld be used to sp ike the des igna ted sample .The recovery must be 0.6 to 1.4 u g / k g or the ana lys i s stopped and a l l relatedsamples must be re-extracted and reanalyzed.

7. The laboratory w i l l be g iven per formance e v a l u a t i o n s a m p l e s by E'Ato run w i t h each batch of s amp le s . The resu l t s f rom these pe r fo rmance e v a l u a t i o nsamples w i l l be eva lua ted by EPA. If a resul t is not w i t h i n the acceptancec r i t e r i a set by EPA, a l l samples i n the batch associated w i t h that PE s a m p l emust be reanalyzed.

8. Each sample must be dosed w i t h 50 u l of the sample s p i k i n g s o l u t i o nc o n t a i n i n g i n t e r n a l s t a n d a r d ( e q u i v a l e n t to 5.0 u g / k g ) and su r roga t e s tancara( e q u i v a l e n t to 1.0 u g / k g ) . The su r roga te recovery must be 0.6 to 1.4 u g / k gor the sample must be r e a n a l y z e d .

12

'"-"• ^

' 9. The fo l lowing qual i tat ive requirements must be met in order toconf i rm the presence of native 2 ,3 ,7 ,8-TCOD:

a. The retention time must equal (wi th in 3 seconds) the retentiontime for the internal standard.

b. The 257/259 ratio must be wi th in the range ^ 10; of the valueof the ratio determined in Sect ion VII, (Equation III).

' c. The ion responses at 257 and 259 must be present and maximize' together. The signal to mean noise ratio must be 2.5 to 1 or better for bothdaughter ions. (Determine the noise level by measuring the random peak to ''va l ley signal present on either side [within 20 scans] of the 2,3,7,8-TCDD L

retention window. The 2,3,7,8-TCOO signal must be at least 2.5 times larger '-than this.) r

<d. For those samples giving non-detect results, the result must be <

less than the 0.3 ug/kg required limit of detection. Otherwise the analysismust be stopped and interferences identified and corrected until the 0.3ug/kg required limit of detection is met.

e. For 'each sample, the internal standard must be present with atleas t a 1U to 1 signal to noise ratio based on the m/z 268 ion response.

I X . SAMPLE C O L L E C T I O N , P R E S E R V A T I O N AMD H A N D L I N G

The procedures for sample collection, shipping and handling w i 1 1 bespeci f ied by the EPA Regional Of f ice responsible for the monitoring exercise.The sampling team will be provided with an 8 ounce g lass jar. and 30-300 gramsof soil wil l be collected. When received in the laboratory, the sample shouldbe thoroughly mixed in the jar for a minimum of 3 minutes, using a stainlesssteel spatula. The spatula should be used to break up large clumps of soilwhi le mix ing to achieve a homogeneous sample.

A 5 gram aliquot sample should be taken and placed in a pre-weighed 10 mlserum v i a 1 containing approximately 5 grams of anhydrous sodium sul fa tetogether with a Tef lon-faced septum and cap (The entire vial, Na^SO^, septumand cap is pre-weighed and labelled). The 5 gram aliquot sample should berepresentat ive of the entire sample. Thus, large stones or other particleswhich are uncharacteristic of the sample, should not be included in thealiquot.

Samples may be stored under ambient conditions as long as temperatureextremes (below freezing or aoove 9U°F) are avoided. Samples must be protectedfrom ligit to avoid photodecomposition.

A 1 1 samples must be extracted and completely analyzed within 2^ hours.Extracts ni'JSt be held for 6 months prior to d isposa l .

13

^ ^' C A U T I O N : A l t h o u g h the sample and s tandards are sealed throughout the

e x t r a c t i o n procedure, there is a lways the p o s s i b i l i t y of leakage and b reakage( e s p e c i a l l y du r ing the sample s p i k i n g and c e n t r i f u g i n g steps). The analysts h o u l d , the re fore , be f u l l y protected by w e a r i n g p l a s t i c gloves and ' laboratoryjacket (a face protector is o p t i o n a l ) . See Section I V for de ta i l s on s p e c i f i csafety requirements.

1. Prepare ex t r ac t ion solvent by m i x i n g two vo lumes acetoni tn ie wi th onevo lume d ich lo ro f f i e thane . M i x so lven t s thoroughly .

2. W e i g h the sample v i a 1 and determine the net w e i g h t of sample ( to 3s i g n i f i c a n t f i g u r e s ) .

3. Add 50 ul of the sample sp i t t ing so lu t ion ( c o n t a i n i n g both in t e rna land surrogate s t anda rds ) . The so lu t ion win con ta in 0.5 ng /u l of i n t e rna ls tandard and 0.1 ng /u l of surrogate s tandard. Add the 50 u1 so lu t ion d i r e c t l yto the so i l , s p r e a d i n g it over severa l sites on the su r face of the so i l .

4. Attempt to mix the so i l and s o d i u m s u l f a t e by s h a k i n g . ( E x t r e m e l ywet s a m p l e s may not m i x w e l l , but DO NOT open the v i a l to s t i r the c o n t e n t s . )A d d i t i o n a l anhydrous s o d i u m s u l f a t e s h o u l d be added i f needed.

5. P ierce the septum w i t h a d i s p o s a b l e needle and l eave the needle i np l a c e to vent the contents w h i l e the ex t rac t ion so lvent is in t roduced .

6. Add 5 m1 of the 2:1 acetoni t r i le : d i ch lo rome thane ex t rac t ion so lven tu s i n g a 5 m1 s y r i n g e arnj d i s p o s a b l e needle. R e t a i n the syr inge for so lven ta d d i t i o n s o n l y .

N O T E : A d d i t i o n a l ex t rac t ion so lven t can be added i f the analyst judgest h i s necessary to ach ieve e f f i c i e n t ex t rac t ion on a p a r t i c u l a r sample .

7. Renove the sy r inge and both needles (they should be t reated asthough c o n t a m i n a t e d ) . Dispose of botn needles .

8. Snake the v i a l v i g o r o u s l y on a vortex m i x e r for 2 m i n u t e s .

9. C e n t r i f u g e the v i a 1 and contents at 4UOO rpm for 2 minu tes . Removec a r e f u l l y so as not to d i s t u r b the sed.iment.

10. Insert a needle th rougn the septum so that it just b r eaks the s u r f a c eof the septum i n s i d e the v i a l . U s i n g a c lean d i s p o s a b l e syr inge and needle,w i t h d r a w a p p r o x i m a t e l y 1 ml of the e x t r a c t ; NOTE: The other needle th rough theseptum serves to e q u i l i b r a t e the pressure upon w i t h d r a w a l of the ext rac t .

11. I n v e n t the s y r i n g e and w i l h a r a w the p l u n g e r to remove the e x t r a c tf rom the needle . D i spose of the need le ( i t is c o n t a m i n a t e d ) .

12. Place a 0.45 m i c r o n d i s p o s a b l e Te ' lon f i l t e r on the s y r i n g e and in.e:the ex t rac t in to a c l ean 10 m1 serum v i a l c o n t a i n i n g 9 m1 d i s t i l l e d wa te r .Dispose of the s y r i n g e a n d the f i l t e r .

l-

13. U s i n g a T e f l o n Y~>ed septum and an a l u m i n u m cap^*\cover ana cr imp thev i a 1 c o n t a i n i n g the wa te r dxtract tn- ix ture .

14. M a n u a l l y snake the v i a l v igorous ly for about one minute .

15. C e n t r i f u g e the v i a l to separate the dichloromethane phase f rom thew a t e r / a c e t o n i t r i l e phase. The dichloroinethane phase w i l l appear as a smallbubb le at the bot tom of the v i a l .

16. Prepare a m i n i a t u r e dry ing tube as fo l l ows :

a. P l u g the t i p of a d i sposab le p ipe t wi th a s m a l l amount of s i l a n i z e dglass wool .

b. Add approx imate ly 1/2 cm anhydrous sodium su l f a t e .

17. W i t h a d i sposab l e syr inge and needle , remove the d i c h l o r o m e t h a n e phasef r o m the v i a l (step 15) as completely as possible.

IB. T r a n s f e r the d ichloro ine thane phase through the d r y i n g tube i n t o a c l e a n1 m l se rum vi a l .

19. R i n s e the dryi-ng tube w i t h one-half ml d i ch lo rome thane , and c o l l e c ti n the same 1 m1 serum v i a l .

20. U n d e r a s t ream of n i t r o g e n , evaporate the solvent gent ly u n t i l tnev o l u m e of s o l u t i o n r e m a i n i n g i n the serum v i a l is 0.05-0.1 m 1 .

21. Seal the 1 ml serum v i a l w i t n a T e f l o n l ined septum and cap. Labe l t-ev i a 1 a p p r o p r i a t e l y .

X ; . C L E A N U P

The need f o r c l e a n u p is i nd i ca t ed when a p a r t i c u l a r extract does not mes:tne Q: c r i t e r i a fo r the coe lu t ion of a l l f o u r monitored ions , sur roga te r ecove r ,or the r a t i o A^/A^g. Two c l e a n u p procedures are g i v e n b e l o w .

A . M o d i f i e d O p t i o n A C l e a n u p

1. P l u g the t i p of a d i sposab le pipet w i t h a s m a l l amount of s i l a m ; e ag l a s s wool.

2. P lace a p p r o x i m a t e l y a 1 cm l a y e r of s i l i c a gel over the g l a s s w o a l .

3. P l a c e a p p r o x i m a t e l y a one -ha l f cm layer of a n h y d r o u s s o d i u c s ' jKat iOver tne s i l i c a ge1.

4 . P l u g the t i p of a second d i s p o s a b l e p ipe t w i t h a s m a l l amo.in; o'si lam;e3 g l a s s woo l .

5. P l a c e a p p r o x i m a t e l y 0.5 cm a c i d a l u m i n a over the s i l a n i z - e a g l a s s ».o:'.

6. P l a c e a p p r o x i m a t e l y U . 5 cm anhyd rous s o d i u m s u l f a t e over tie a l u r - i n i .

15

^ ^7. A r r a n g e the two caurr .ns sa t ha t the s i l i c a eel c o l u m n w i l l e1uts onto

t h e a l u m i n a cslu-;.", and t h e a l u m i n a c s l u m n a n p p i n g s w i l l be c s i l e c t e di n a v i a l .

8. R i n s e the two c o l u m n s w i t h 0.5 m1 cyc lohexane and d i s ca rd the e l u a t e .

9. Open the v i a 1 con ta in i ng the ext ract ana add 1 ml cyclohexane to theextract.

10. U n d e r a s t r eam of n i t r o g e n , c a r e f u l l y evapora t e the d i c h l o r o m e t h a n e fro."the extract v i a l ( the v o i u m e of the r e ' - a i n i n g s o l u t i o n s h o u l d he jus t ,-1.under 1 m l ) . .

11. T r a n s f e r the e n t i r e c o n t e n t s of the extract v i a l onto the s i l i c a c o l u m n , Ln

arranged as s p e c i f i e d i n step 7. i^~0

12. When the s o l u t i o n jus t reaches the s u r f a c e of the s o d i u m s u K a t e l a y g r 0i n the s i l i c a gel c o l u m n , add 0.5 nil cyclohexane.

13. Repeat step 12 a second t i m e . A l l o w the s o l u t i o n to d r i p c o m p l e t e l ya f t e r the second a d d i t i o n of cyc lohexane .

1-i. D i s c a r d the s i l i c a ge1 c o l u m n .

15. R i n s e the a l u m i n a colur-.n w i t h an a d d i t i o n a l 1 m1 c y c l o h e x a n e . D i s c a r dthe a c c u m u l a t e d e l u a t e s i n the v i a l benea th the c o l u m n .

16. P l a c e a dean 1 a\ se rum v i a l under the a l u m i n a c o l u m n .

17. E1uts the a l u m i n a c o l u r n w i t h three success ive por t ions of 0.5 m1 eachof 15i; by v o l u m e d i c h l o r o m e t h a n e i n c y c l o h e x a n e , c o l l e c t i n g the e l u a i ai n the c l e a n v i a 1 .

18. W i t h gentle heat ing ana under a stream of nitroce", evaporate the solve":until the volume in the v i a 1 is 0.05-0.1 m 1 .

19. Seal the serum v i a l w i t h a t e f l o n l i n e d septum a n d cap. Label the v i a 1a p p r o p r i a t e l y . N O T E : I f it is a p r i o r i k n o w n tha t the second step ofc l e a n u p is r e q u i r e c , evapora t e the s amp le i n stacs 18 to jus t be low1 ml and i m n e a i a t e l y proceed w i t h a second c l e a n u p as d e s c r i b e d be low.

Q p t i e n D C l e a n u p

A l l s a m p l e s i n d i c a t i n g the p resence of oiler T C D D isoi-ers or w h i c h c c n t c i ncoripounos co-e lemnc -ust 5e c l e a n e d u p u s i n g O p t i o n 0.

1. I n a d v a n c e , presa ' -e a m ' x f J r e of 3.6 c C a r f a c s s c k C w i t h 15.4 c C e ' ^ t s5"5. A c t i v a t e tl-e ;-mtJ-e at l;;]^ for 6 h o u r s .

2. P 1 u c the t ip cf a c i s : csa ; l e p 'pet w i th a sidl ar-s'-int o' silc'";sd c l a s sw o o f .

3. P lace 2 c~i layer o' fe ca rpooac^ -Ce l its m i x t u r e over the c l a s s wooip 1 u c , •JSI - IG S u C t i c n 13 S 3 C < fe cclunn.

4. Rinse the coiumn^&equ'eniid i ly ><ii..-i i. »;i i.uii)c!ic_t .... ".^•.i". u.^i.imrie-methanol-beniene 5 :20:5 by volume), 1 m1 cycl ;xane-dicnloro;-,etnane1 : 1 by volume), and f inal ly 2 m1 cyclohexane. Col lect the eluate in av i a 1 and discard tne eluate.

5. D i l u t e the extract w h i c h has been c leaned up by the M o d i f i e d O p t i o n Aprocedure to 1 ml w i th cyclohexane.

6. Maintaining a discard v ia1 under the column, introduce the extract' onto the column.

7. A f t e r the solvent has drained, r inse the column success ive ly with 2 n'lcyclohexane, 1 ml cyclohexane-dichloromethane mixture ( 1 : 1 by vo1u;-.s)and 1 ml dichloromethane-methanol-benzene mixture (75 :20 :5 by volun°). lr\

ur\8. Al low the column to drain completely and discard the accumulated dual's. Ln

r--9. P lace a clean serum v ia1 under the column. 0

o10. Elute the dioxin from the charcoal wi th 2 ml toluene.

1 1 . Kitn gentle hea-ting and under a stream of nitrsgen, concent ra te theextract to a volume of O.U5-0.1 ml.

12 . Seal tne serum v i a 1 w i th a Te f l on lined sestun and cap. Label app^o:^•l 2:3'

XI I . GC/MS/MS ANALYSIS

1 . Tab le 1 summarizes the 1 5 IT. 03-5 gas chromatscraphic cap ' i i a rycolun" and operating conditions. The 15 m OB-5 coiunn has been used forchro-iatoaraphy which is not isomer spec i f ic (no valley is observed betwee" thel ,2,3,s- fCD3 and 2 ,3 ,7 ,5 -TCD3 isoners).

2. Standards and samples must be analyzed under idenficsl MS/'P'Sconc'. t 'ons. Selected React ion Monitoring (SRM) scans are uses, us inc a s:i'time to g ive at least f ive points per chromatographic peak . Reconmenae.;MS/MS condi t ions are given in Tab le 2.

3. Ver i f y the Cal ibrat ion of the system daily as descr ibed in Sec t ionV I I . The volume of ca l ibrat ion standard injected should be aoproximately t-.esails as a 1 1 sample inject ion volumes. The reauirements descr ibed in Sect io"V 1 I 1 , Par ts 93 and 9c trust be met for all cal ibrat ion stanaaras.

<. Inject a 1 to 2 u1 a l iquot of the sample ex t rac t .

5. The presence of TC03 is qual itati v e 1 y confirmed if tne cri'.s'-'.s ofSect ion V: l l , Part 9, are acmevec.

6. For auanti ta; ion, measure t~e area res^sise o*' the f / z 2;' ini ;:rp e a < S for 2 ,2^ ,8 -TC33; tne m/z 26= peai. far *-€,'-;, 3 , 7 .S-TC30, anc tie n- ;2 fc2 pea< for •"Cl ^ -2 ,3 ,7 ,3 - 'C33. C a l c u l a t e the concentrat ions of na fve a":sur rogate s tanaaras using tie fo l lo 'ng equations:

r^- • ^( ( A , / A , , ) - C . F . ) ( Q ^ )

R R F s x W

where C; = The concen t ra t ion of n a t i v e 2 ,3 ,7 ,8 -TCDD in ug/kg

A^ s the sum of the area responses for the ions , m/z 257 and 259

A , ^ s the area response for the ion m/z 268r

C.F. = correction.factor for spiking solution (blank) previously determined ,-,(Equation V) \0

in

0,5 = quan t i ty ( i n nanograros) of ^C^-2.3.7.8-1'000 added to the s a m p l e before Ln

ex t rac t ion i~~-0

R R F ^ = R e l a t i v e response fac tor for 2,3,7,8-TCDD c a l c u l a t e d p r e v i o u s l y 0( E q u a t i o n I )

W = w e i g h t ( i n grains) of wet so i l or sediment sample.

I n e v a l u a t i n g the r e s u l t s , a d i s t i n c t i o n must be made between q u a n t i t a t i v emeasurement and q u a l i t a t i v e i d e n t i f i c a t i o n of 2 ,3 ,7 ,8-TCDD. The f o l l o w i n g stepsmust be f o l l o w e d in the t rea tment of a l l sample resul ts :

1. C a l c u l a t e the concen t ra t ion of na t ive 2,3,7,8-TCDD u s i n g equat ion I X .

2. D e t e r m i n e i f a l l of t.*ie q u a l i t a t i v e i d e n t i f i c a t i o n c r i t e r i a are met.

3. If a11 q u a l i t a t i v e i d e n t i f i c a t i o n c r i t e r i a are met, report theconcen t r a t i on found by equa t ion I X , regard less of concentrat ion.

4. I f the q u a l i t a t i v e i d e n t i f i c a t i o n c r i t e r i a are not met, and thec o n c e n t r a t i o n c a l c u l a t e d by e q u a t i o n I X is less than the required l i m i t ofde t ec t i on of 0.3 u g / k g , report the concen t ra t ion as less than 0.3 ug /kg ( i .e.<0.3 u g / k g ) .

5. I f the q u a l i t a t i v e i d e n t i f i c a t i o n c r i te r ia are not met , and theconcen t ra t ion c a l c u l a t e d by e q u a t i o n I X is greater than the requ i red l i m i t ofde tec t ion of 0.3 u g / k g , the extract must be reinjected. I f the q u a l i t a t i v ei d e n t i f i c a t i o n c r i t e r i a are s t i l l not met and the r e su l t is s t i l l greater than0.3 u g / k g , the extract must be c leaned up or the sample r e a n a l y z e d u n t i l as a t i s f a c t o r y r e su l t is o b t a i n e d , ( i . e . pos i t i ve r e s u l t or n e g a t i v e r e s u l tb e l o w 0.3 u g / k g ) .

N O T E : I n r epo r t i ng r e s u l t s for sample a n a l y s i s , a c o m p a r i s o n is made w i t hthe r e q u i r e d l i m i t of de tec t ion . Tne l i m i t of de tec t ion based on the b l a n k( E q u a t i o n V I I I ) m i g h t a l s o be u sed , but i n t e r f e r e n c e s may be present anai n t r o d u c e f a l s e p o s i t i v e s i n some cases. .However, as e x p l a i n e d i n Sect ionV I I , the e m p i r i c a l l i m i t of de tec t ion based on the b l a n k must be less t h a nthe r e q u i r e d l i m i t of d e t e c t i o n of 0.3 u g / k g .

IS :

Equation X : (Calcu lat ion of concentration of surrogate standard, C 1 < -——————— 2,3 ,7 ,8 -TCDD)

A S S " °is

A,^ x Ri^ss x W

where C.^ = .the concentrat ion of surrogate standard Cl4-2,3,7,8-TCDOin ug/kg.

ASS = the area response for the ion m/z 263* ^

A , ^ = the area response for the ion m/z 268

Q^ = quantity (in nanograms) of ^C^-^.B.P.S-TCDD added to the r"sample before extraction. 0

RRF^ = Re la t i ve response factor for ^(^^.B.P.S-TCDD calculatedpreviously (Equation II).

M = Weight ( in grams) of wet soil or sediment sample.

* Subtract 0.0108 of any 257 response from the 263 response to correct forcontributions of any 2,3,7,8-TCDD to the 263 response.

Native 2,3,7,8-TCDO contains an innate quantity of 3 7C^4-2,3,7,8-TCDD.Except at nign concentrations of native 2,3,7,8-TCOD, this contribution istoo small to signif icantly af fect the calculated concentration of surrogate: ' /C^4-2,3,7,8-TCDD. The theoretical correction is calculable on the basis ofisotope distribution and amounts to 1.08'; of the m/z 257 peak. (This correctionshould be checked at low resolution by analyzing about 20U pg/ul of unlabelled2,3,7,8-TCDD.) On this bas is , the correction to the area count of the surrogate,is made as fo l lows:

A263 = ^63 - 0-0103 A;,^

C a l c u l a t e the a n a l y t i c a l percent recovery of the surrogate s tandard .

Surrogate amount measured* ( n a n o g r a m s ) X 100A n a l y t i c a l = 5 ngPercent Recovery

* NOTE: The amount measured is equal to the concentration found byequation X mult ipl ied by the weight of soil used for the sample (i.e., C;; x

U ) .

X I I I . M E T H O D P E R F O R M A N C E

The required detection limit for this method is 0.3 ug/kg. For certainsamples , this detection limit may not be achievasle because of interferences.These samples require cleanup as described in Section XI. This method hasbeen compared w i t n the EPA- IF3 GC/MS Method for 2 ,3 ,7 ,8 -TCDD and founo t3 beapp l i cab le to analyses of so i l s where 2,3,7,S-TCDD is U.e only tetraci loroisomer known to be present.

19

0

r^ ^TABLE I

OPERATING CONDITIONS FOR DS-5 GAS CKROMATOGRAPHY COLUMN

C O L U M N

Length

I. 0.

F i l m T h i c k n e s s

2 .3 ,7 ,8 -TCDD R. T. ( a p p r o x . )

C a r r i e r gas

lmt ia '1 Tempera ture

I n i t i a l Time

Spl i t i e s s Tim.e

Prograin R a t e

F i n a l T e m p e r a t u r e

S p 1 i t F low

SepUni Purge F l o w

C a p i l l a r y Head P ressure

T r a n s f e r L i n e Tempera ture

20

DB-5

15 m

0.32 mm

1.0 m i c r o n

5-6 n i in .

NZ

15U°C

l .U m i n .

1.0 mi n .

2 0 ° C / m i n .

240°C

20 m l / m i n .

0.6 m l / m i n .

8 ps i

240°C

Ins t rument

Ion Source

CI Reagen t Gas

Reagent Gas F low

Source Tempera tu r e

D i s c h a r g e Curren t

Q l R e s o l u t i o n

03 R e s o l u t i o n

C o l l i s i o n Energy ( L A B )

Col 1 i s i o n Gas ' • - _,

C o l l i s i o n Gas T h i c k n e s s

Ions Morito'-ert"

--^

TAGA® or TAGA» 600UE

Townsend/g low d i scha rge CI

Zero grade a i r ( H ^ and He f r ee )

35 + m l / i n i n .

200°C

-1 mA0'inin

3 amu at SO; peak height at m/z = 32U (singlli* f.0

3 amu at 50; peak h e i g h t at iti/z = 320 (sing^g I-

55eV [(OR + G R ) / 2 - R 2 ] or 55eV (OR - K ^ )

Ar

400 x 10^2 nio1ecu1es/cm2

320 257 (nat ive-TCDD)322 259 (native-TCDO)32d 263 (surrogate standard)332 26B (internal standard)

21

X I V . DATA • R E . P U R l l N b ^ ^

Repor t a l l da ta i n u n i t s of mic rograms per k i l o g r a m of wet so i l . Usethree s i g n i f i c a n t f i g u r e s at concen t ra t ions above 1 u g / k g and 2 s i g n i f i c a n tf i g u r e s at c o n c e n t r a t i o n s below 1 u g / k g . The data package must i n c l u d e thef o l l o w i n g i n f o r m a t i o n :

1. I n d i v i d u a l and mean response factor for the three-point c a l i b r a t i o nof u n l a b e H e d 2 ,3 ,7 ,8 -TCDD. (Based on H i g h level standard only) .

2. I n d i v i d u a l and mean response factors for the i so top ic surrogates t anda rd (based on h i g h l eve l s tandard on ly) .

03. The i n d i v i d u a l ra t ios of the sum of areas 257 and 259 ions to the v£>

268 i o n fo r 20 r e p l i c a t e measurements of the b l ank (i.e., sample s p i k i n g y,s o l u t i o n ) , and the mean Correc t ion Factor based on these rat ios . r—

4. The e m p i r i c a l l i m i t of detection based on the 20 b lank measurements. 0

0

5. The d a i l y or s h i f t v e r i f i c a t i o n of the mean response factors.

6. The percent accuracy i.e., ( a n a l y t i c a l percent recovery) for thesur roga te s t andard . -.

7. The r e su l t fo r the method b l a n k .

8. The percent recovery of na t ive TCD3 f rom the s p i k e d . s a m p l e .

9. The resu l t for the P£ saapis

10. The resul t for the f i e l d b l a n k .

11. The data f i l e n a m e (to f a c i l i t a t e data r e t r i eva l ) .

12. The s ample i d e n t i f i c a t i o n number (as ass igned by the f i e l d s a m p l i n gt e a m ) .

13. A n a l y t i c a l date and t ime .

14. The area responses for ions 257, 259, 263, and 268.

15. The observed response r a t i o of ions 257/259 for the sample .

16. The calculated value for nat ive 2 ,3 ,7 ,8 -TCDD. (Values above orbelow 0.3 ug/kg are to be reported only if qua l i ta t i ve ident i f i ca t ion cr i te ' - iaare met.)

17. I f no 2 . 3 , 7 , 8 - T C D O was detected, report "not detected" or N . O . andtie 0.3 u g / k g r e q u i r e d de t ec t i on l i m i t .

18. The mass cn romatoa rams fo r a l l s a m p l e s and s tandards . I n c l u d e bcthlie r e a l - t i m e d i s p l a y da t a a n d reduced data s h o w i n g l i m i t s of i n t e g r a t i o n .I n c l u d e any computer genera ted response t a b l e s .

^ ^19. The w e i g h t of the o r i g i n a l wet sample B i i q u o t .

20. Documenta t ion on the source and his tory of the na t ive and l a b e l l e d2,3 ,7 .8-TCDD s tandards used. ;

21. Any other s u p p o r t i n g documenta t ion . An example of the required•data format f o l l o w s :

2 3

" I D S I O N 10H ION B L A N K . »SnVE1: 257 ;;? ;i5 RESPOXSt CONC

ffl,

? ?;i/l d

0- 0L *̂ X

: s

" ss^" 5

0 0 7 5 6 3

*=« Cl <:n f

n a.3: r^ t->n as

)

^ Sl™60TE COIIC ;,: 1 STt CDSC i;; ' ' BF N A T I V E

RF SUBROSxIEIM BAI10 (VEF.A6ECCBRECTIOII FACTOfi

^3 Sr.O EPd (»;LTSIS IIE1 MT ION I O N ION IOH lltTID SifflB U6/t:EnBFLE I EAHFLE I S"KFL£ t D"TE • T I B E EF.BBS 257 2S9 263 2iB 257/259 ACC TCCi

)

)

g

g ,

a m

fn

"I»» C3n mn 3»

0 0 7 5 6 5

Calcu la t ions :

• Note: The equiva lent concentrat ions of 2.3,7,8-1X00 ( l a s t co lumn) are ca lcula ted u s i n g E q u a t i o n VI '-.' '.<

C,, " A(, ' 0|s

A,., ° R R F , • W

Other ca l cu l a t i ons required are:

1. E q u a t i o n V: Correct ion Factor (C.F.) for B l a n k C o n t r i b u t i o n

C. F. • tB " ___________n

t. E q u a t i o n V I I : (Standard Oevla t lon of the B l a n k Responses)

2 l - I r r. l2/-S,, = ( FC^ ) - (l:Ct,)'-/n _____________

,——————rH—————it

3. Equation VI11: (L imt t pf Detection based on "Well Known" B l a n k )

LOO ° 2 '• t • S,, ' ______________

•.5(̂ *0 0 7 5 6 6