8/2/2019 PAH Gingko

1/17

TRNG I HC KHOA HC T NHIN TP HCM

KHOA MI TRNG

LP 09KMT

QUAN TRC MI TRNG

nh gi kh nng quan trc sinh hcca cy Ginkgo i vi PAHs

GVGD: T Th Hin

Dch v tng hp: Nhm 4

Vn Hunh Thu i 0917013

inh Th Phng Chi 0917022

V Th M Chi 0917024

L Trng Ho 0917110

Nguyn Th Hi 0917123

L Th Nh Qunh 0917276

Nguyn Th Kim Thi 0917318

V Thu Trang 0917357

Nguyn Phng Trinh 0917364

8/2/2019 PAH Gingko

2/17

Ni dung...................................................................................................................................................2

Ni dung .....................................................................................................................................2

...................................................................................................................................................3

Tm tt.......................................................................................................................................4

1.Gii thiu .................................................................................................................................4

2. Mu v phng php ly mu ...............................................................................................6

2.1 Phng php ly mu ......................................................................................................62. 2 Phn tch PAH .................................................................................................................7

2.3 Phn tch thng k .........................................................................................................10

3. Kt qu v tho lun ............................................................................................................10

3.1 So snh nng PAH gia cc loi cy khc nhau .....................................................10

3.2 S phn tn ca PAHs trong l .....................................................................................11

3.3 nh hng ca v tr l trn cy vi s tch lu PAHs trn l .......................................13

3.4 Mi quan h gia nng PAH trong l cy ginkgo v khng kh ...............................14

3.5 S khc nhau theo ma ca PAHs trong l cy ginkgo v trong khng kh.................14

4.Kt lun .................................................................................................................................16

2

8/2/2019 PAH Gingko

3/17

3

8/2/2019 PAH Gingko

4/17

Tm tt

Cng dng ca l cy ginkgo nh mt cht quan trc sinh hc i vi cc loihydrocacbon thm a vng (PAHs) c nh gi. Chng ti iu tra nng PAH gia cc loi cy, nh hng ca v tr l trn cng mt cy, s phn b cc m,s tng quan gia l cy ginkgo v khng kh v nhng thay i theo ma. Trong 5loi c xc nh (Ginkgo biloba L.,Zelkova serrata Makino,Liriodendron tulipiferaL., Prunus yedoensis Matsum, v Magnolia kobus DC.), ginkgo tch t lng PAHsnhiu nht trong khng kh ven ng. Phn ln PAHs (~80%) c tch t trong phncht nn v hu ht phn cn li thm nhp vo m trong ca l. Nng PAH trong l

ginkgo gim khi cng ln cao v cch xa con ng, phn nh c ngun gc caPAH t hot ng giao thng. Nhng mu ly theo ma cho thy nng PAH trong lginkgo tng cng vi thi gian, do nh hng ca nhit v s tch ly qua thi gian

phi nhim di. Nng trong l ginkgo thu thp c t nhng con ng khc nhauch ra rng c s tng quan mnh m d nhn bit gia nng trong khng kh cthu thp bi cc dng c ly mu th tch ln (r2 = 0,68, P

8/2/2019 PAH Gingko

5/17

ng khng kh trong sut Th Vn Hi Bc Kinh nm 2008, v nhn thy rng ccphng php kim sot giao thng lm gim lng PAHs c khi lng phn t t252 n 300. V nng PAH trong khng kh dao ng theo ngy v theo ma donhiu nguyn nhn, vic quan trc thng xuyn c yu cu nh gi tnh trng

nhim ca chng. Tuy nhin, nhng gii hn v phng php hn ch vic quan trcd liu.

Thay vo , quan trc sinh hc s dng thc vt l mt phng php tip cnc trin vng, vi cc thun li sau: (1) d dng, kinh t v khng i hi k thut qucao i vi mu thc vt; (2) thc vt c th tch ly qua thi gian phi nhim di.Simonich v Hites (1994) nhn thy rng thc vt tch ly ng k PAHs. C ch tchly PAHs trong thc vt, v cc tc nhn kim sot ca chng c nghin cu nhsau. Ngi ta chng minh rng thc vt hp th cc cht hu c t bay hi nh PAHs

hu ht l t khng kh ch khng phi t, v c th d on cht lng khng kh.Bn cnh , s phn chia PAH gia khng kh v thc vt, s thay i v thi gian, s

phn b khc nhau trong m, con ng hp thu PAHs (biu b bn ngoi v con ngkh khng), v s quang phn trn b mt l; tt c c nghin cu. Hn na, stch t PAHs gia cc loi cy khc nhau cng c iu tra. Quan trc sinh hcPAHs s dng l cy c tin hnh tm hiu v s phn b trong khng gian, tcng ca cc hot ng ca con ngi v s thay i theo ma. Mc d mt s nghincu chng minh tc ng ca giao thng i vi PAH trong khng kh th,nhng rt t nghin cu s dng cy ven ng quan trc PAHs trong khng kh ti

nhng con ng giao thng dy c. Nghin cu ny tp trung vo vic dng cy venng quan trc PAH trong khng kh ven ng. Bi v khng kh ven ng chunh hng ca giao thng nn vic quan trc khng kh th quan trng nh gi s

phi nhim ca con ngi i vi cc hp cht gy ung th ring bit. Chng ti chnginkgo (Ginkgo biloba L.) lm mc tiu v ginkgo l mt loi cy ven ng ph bin ng v c pht trin nhng nc gia 200 v 600 Bc v 200-500 Nam.

Mc tiu u tin ca nghin cu l nh gi cng dng ca ginkgo nh l mtloi quan trc sinh hc PAHs trong khng kh ven ng, v ti u hot ng quan

trc. Theo ui mc tiu ny, chng ti tp trung vo cc vn sau: (1) s tch tPAHs trong l cy ginkgo, so snh vi nhng loi cy ven ng ph bin khc, (2)PAH phn b gia cc m trong cy ginkgo, (3) s khc nhau v tch t PAH do chcnng ca l ty thuc vo v tr trn cy, (4) quan h gia PAH trong m cy ginkgo vkhng kh xung quanh, (5) kiu phn b theo ma ca PAH trong l ginkgo. Tt cchng l nhng tc nhn quan trng trong quan trc PAH trong khng kh s dng lcy. Tuy nhin, khng c nghin cu no trc y xc nh tt c cc kha cnh trni vi mt loi cy nht nh.

Mc tiu th hai ca nghin cu ny l nh gi hiu qu ca cc quy nhv pht thi diesel trn ng c gii thiu bi Chnh Ph Tokyo vo thng 10 nm

5

8/2/2019 PAH Gingko

6/17

2003, qua mt thi gian di (5 nm) quan trc s dng cy ginkgo. Cc phng tingiao thng dng du diesel khng p ng tiu chun mi v bi PM c th c thayth hoc trang b thm nhng b phn mi c thit b kim sot bi. Mc d hiu quca cc quy nh v carbon, PM v nito oxid c bo co, nhng hiu qu i vi

PAH th vn cha c bit r. V vy, chng ti thu thp mu l ginkgo hng nmtrong vng 5 nm so snh nng PAH trc v sau khi c cc quy nh. y lng dng u tin ca vic quan trc sinh hc PAHs s dng l cy.

2. Mu v phng php ly mu2.1 Phng php ly mu

Su kiu ly mu c tin hnh nh sau:

2.1.1 S so snh gia cc loi cy

so snh nng PAH gia cc loi cy, chng ta ly mu l t nm loi cyc l rng hng nm- ginkgo (Ginkgo biloba L.), Zelkova (Zelkova serrata Makino),cy tulip (Liriodendron tulipifera L.), cy anh o (Prunus yedoensis Matsum), vKobushi Magnolia (Magnolia kobus DC) - c ng i b v ng xe chy trm1(Fuchu trn tuyn ng Tokyo 133; bng 1) vo thng 8 nm 2000.

2.1.2 S phn phi trong m

Chng ta kim tra s phn phi m water-washed, wax, v phn bn trong(interior fractions) ca PAHs trong l cy ginkgo thu thp t ng xe chy trm 1vo thng 10 nm 2000.

2.1.3 S nh hng ca v tr l cy trn mt cy ginkgo

kim tra s nh hng ca l cy trn nng PAH, chng ti thu thpnhng l cy ginkgo pha trn ng i b 2,3,4 v 8m; v trn ng xe chy 2,4 v6m trm 2 (Fuchu trn tuyn ng Tokyo 133; bng 1) vo thng 8 nm 2002. Conng c 6 ln v cy bn cnh ng, ngang bng vi mt ng, khng c chng

ngi gii hn s lu thng khng kh.

2.1.4 Mi quan h gia PAHs trong m cy ginkgo v trong khng khxung quanh

iu tra mi quan h gia nng PAH trong l ginkgo v trong mukhng kh, chng ta thu thp mu t trm 3 n trm 6 vo thng 10 nm 2001, trm7 vo thng 9 nm 2002 v t trm 8 n trm 14 vo thng 12 nm 2002 (bng 1). Mtthit b ly mu khng kh th tch ln (TFIA-2, Staplex, USA) c t cch mt t1m. Thit b ly mu ly 700 L/pht trong 24h. Mu ht (>0.3m) c gi li trn giylc si thch anh (mng lc Palflex 2500QAT-UP, 200mmx250mm). Bt polyurethane

6

8/2/2019 PAH Gingko

7/17

(PUF; Shibata 8013-0941A polyurethane cho dioxin, 5cm x 6cm) c thm vo phasau mng lc thu kh PAHs.

2.1.5 S khc nhau theo ma

Chng ta cng thu thp cc mu l ginkgo v mu khng kh trong chui thigian theo ma trm 3 (Fuchu, i hc Tokyo) t thng 6 n thng 11. Phng phply mu ging nh mc 2.1.4

2.1.6 Cc mu c ly hng nm

Cc mu l ginkgo c ly hng nm cc trm 3, 6 v trm 8 n 15, t2001 n 2006 nh gi nh hng ca cc quy nh v pht thi diesel i vi chtlng khng kh. Thng tin v mt giao thng c ly t Society of Traffic

Engineers ca Nht (2001, 2006).Trong tt c cc phng php, l cy c thu thp v tr ct l cch mt t

2-4 m gn ng xe chy, tr trng hp nhng ni c bit. Nhit khng kh cghi li cng lc . Cc giy lc c sy kh. L, giy lc kh v mu PUF c lutr 300C trong t ng cho n khi phn tch PAHs.

2. 2 Phn tch PAHPAHs c o bng phng php sc k kh vi thit b o khi ph (GC-MS),

s dng sc k ct silica gel hai bc (two-step silica gel column chromatography) cho

tt c cc qu trnh, d khc nhau v phng php chit (v d nh polytron, sonicationhay chit cht lng) cng nh ty chn cch lc (v d sc k bt u bng gel thmthu hay l s thy phn) c s dng ty thuc vo qu trnh. Cch chit v cngngh lm sch cho nhng phng php ring c m t c th nh sau:

2.2.1. So snh gia cc loi cy: phng php chit polytron (polytronextraction)

Mu l (~5 g) dng so snh gia cc loi cy c chit vi 120 mL

dichlromethane (DCM) v 30 g anhydrous sodium sulfate, trong mt Polytron RT2000ng ha (Kinematica). Phn DCM c tch ra bng cch ly tm ti 740 xg trong 10pht v kh nc cng vi anhydrous sodium sulfate. S chit c lp li hai ln, sau tt c dch chit DCM c kt hp li v lm giu vi 50 L surrogate chun (5

ppm vi mi dung dch anthracene-d10, p-terphenyl-d14, benzo(a)anthracene-d12,perylene-d12). Dch chit c c cn n khi cn 4 mL, v ly tm ti 740xg trong vng5 pht. Phn ni trn b mt c chit ra bnh thng qua len thch anh tch khicht rn, sau tip tc c cn, ri ha tan trong 0.5 mL MeOH/DCM (t l th tch l1:1), v tr thnh tinh khit nh sc k bt u bng gel thm thu (open-top gel

permeation chromatograph) cng vi cc ht Sephadex LH-20 (Amersham PharmaciaBiotech AB). 12.5 mL u ca MeOH/DCM (t l th tch1:1) b b i, v 10 mL sau

7

8/2/2019 PAH Gingko

8/17

c gi li l mu ca PAH. Mu PAH c c cn, sau c ha tan trong 1mL n-hexane/DCM (1:1 v:v) v mt mu tinh khit khc c to thnh nh sc k ctsilica gel hai bc (two-step silica gel column chromatography) vi 5% H2O- ct gelsilica bt hot v cng vi ct silica gel hot tnh. c miu t bi Zakaria et al. (2002)

2.2.2 Phn phi m: chit ln lt (Tissue distribution: successiveextraction)

PAHs trong l ginkgo c tch ra t water-washed, wax, v phn bn trong(interior fractions). Nm n mi l (~5g) c khuy trong 1 L nc ct 20 0Ctrong 10 pht, 3 ln. Nc c lc qua mng lc si thy tinh (glass fiber filter)(GF/F, Whatman). Dch lc tip theo s qua mt hp (cartridge)ODS (Sep-Pak PlustC18; Milipore) c chun b vi n-hexane trong DCM, MeOH v nc ct. PAHsc tch ra vi 30 mL DCM. Mng lc thy tinh c chit soxhlet (Soxhlet-extracted) cng vi DCM trong 12h. Phn chit c t dch lc v mng lc thy tinhc kh nc v kt hp vi water-washed. Nhng l cy sau c sy kh vc chit trong 400 mL n-hexane/DCM (1:1 v/v) bng ultrasonication trong 2h, vkh nc vi anhydrous sodium sulfate to ra phn sp wax fraction. Nhng lc chit xut bng ultrasonication sau c chit bng Polytron ng nht(Polytron homogenizer) nh trn to nn interior fraction. Tt c dch chit uc c inh vi surrogate chun , c cn v lm tinh khit bi sc k ct silica gel hai

bc (two-step silica gel column chromatograph) nh phn trn.

2.2.3. Ultrasonic extraction

i vi quy trnh m t trong 2.1.3 (nh hng v tr l), 2.1.4 (So snh viPAHs trong khng kh), v 2.1.5 (bin thin theo ma), ultrasonic extraction c pdng. Khi lng (~5g) ca l ginkgo c chit trong 400 mL n-hexane/DCM bngultrasonication trong 2h, khng ra trc bng nc. Dch chit c kh nc vianhydrous sodium sulfate, lm giu vi surrogate chun (surrogate standards), v lmtinh khit bi hai bc ct sc k ct silica gel (two-step silica gel columnchromatograph) v sc k thm thu gel (open-top gel permeation chromatograph).

2.2.4 Chit sut cht lng p lc (PFE)

Cc mu c ly cng chui thi gian hng nm ca l ginkgo (khong 4g)c ng kh v chit sut bi PFE vi DCM 175oC trong 1 h thng chit sutdung mi gia tc (ASE 200, Dionex). Dch chit c lm giu vi dung dch surrogatechun, v c tinh ch bng cch thy phn. Dung dch chit sut c t trong ngthy tinh v cc dung mi c bc hi di mt dng kh nito. ng c thmvo 1M KOH trong MeOH, sau ng c bt kn v c phn huyer trong mi

trng kim 80o

C trong 2 gi. Sau thm nc v hexane phn hy mu, PAHc chia gia hexane v nc/MeOH trong lp hexane. PAH c tinh ch bng sc

8

8/2/2019 PAH Gingko

9/17

k ct silica gel hai bc v sc k thm thu gel (open-top gel permeationchromatography) nh trn.

2.2.5 Mu khng kh

PUFs v giy lc c chit Soxhlet (Soxhlet-extracted) vi DCM trongkhong 12 gi. Dch chit c lm giu vi the surrogate chun, c c c v tinhch bng ct sc k silica gel 2 bc (two-step silica gel column chromatography) nhtrn.

2.2.6 Php o GC-MS

Tt c cc phn PAH c lm sch c c c v tim vo trong GC-MS(Hewlett Packard 5890 Series II, vi mt u d 5972A). 19 loi PAH vi t 3-7 vng

phenanthrene (Phe), anthracene (Anth), 3-methylphenanthrene (3-MP), 2-methylphenanthrene (2-MP), 9-methylphenanthrene (9-MP), 1-methylphenanthrene (1-MP), fluoranthene (Fluo), pyrene (Py), benz(a)anthracene (B(a)A), chrysene (Chry),

benzo(b)fluoranthene (B(b)F), benzo(j)fluoranthene + benzo(k)fluoranthene (B(j/k)F),benzo(e)pyrene (B(e)P), benzo(a)pyrene (B(a)P), perylene (Pery), indeno(1,2,3-cd)pyrene (IndP), benzo(ghi)perylene (B(ghi)P) v coronene (Coro)dwere cxc nh trong quan trc ion c chn lc (selected-ion monitoring mode) bi GC-MS,c m t trong Zakaria et al. (2001).

2.2.7 m bo cht lng v kim sot cht lngNng PAHs c iu chnh bng cch thu hi dung dch surrogate chun

c lm giu ngay sau khi chit. T l thu hi ca surrogate chun l hn 65% cho ttc phng php. S lp li ca phng php chit xut v cc k thut tinh ch linquan c s dng trong cc phng php ring, c kim tra bng phn tch lp limu ginkgo trc khi phn tch mi quy trnh. lch chun ca tng loi PAHs trongmu chit sut l

8/2/2019 PAH Gingko

10/17

2.3 Phn tch thng kMt php phn tch phng sai (ANOVA) c thc hin nh gi s bin

i trong nng PAH gia cc loi cy khc nhau. S khc bit gia cc nhm cyc nghin cu thng qua th nghim ca Tukey. Tng quan tuyn tnh c c

lng nh gi mi tng quan gia nng PAH trong l cy ginkgo v trongkhng kh, v gia nng PAH trong l cy ginkgo v mt giao thng tngng. H s tng quan th bc Spearman c c lng nh gi mi tng quangia nng PAH v thi gian tri qua, v gia h s phn b KL (t l nng

PAH trong l cy ginkgo [ng mg lipid-1] so vi trong khng kh [ng m-3] v hm snghch o ca nhit . Th nghim t-test c s dng nghin cu s khc nhaugia logKL v hm s nghch o ca nhit gia thng 6 v thng 9. Kim nh t chocc bin s theo cp (paired t-test) vi tng quan Bonferroni c s dng khost s chnh lch gia nng PAH v t s L/H (t s gia PAH ca phn t c khi

lng thp low-molecular-weight (LMW) (nh, Phe-Py) vi PAH ca phn t c khilng ln high-molecular-weight (HMW) (nh, nng B(a)A-Coro) trong sut nhiunm. Phn mm IBM SPSS Statistics 19 c s dng cho php phn tch thng kny.

3. Kt qu v tho lun3.1 So snh nng PAH gia cc loi cy khc nhau

Hnh 1 so snh nng PAH trong l ca nm loi cy c em ra nghin

cu. Nng PAH trong cy ginkgo ( iu kin trng lng kh c bn) cao hnhn so vi nhng loi cy khc (P

8/2/2019 PAH Gingko

11/17

ca cy ginkgo c th quy cho b mt gn sng ca l cy ginkgo - lm tng din tchtip xc b mt v gi li cc mnh nh. Nhng kt qu trn cho thy rng cy ginkgotch ly nhiu PAHs hn so vi nhng loi khc, cho thy rng n l mt cng c bch i vi vic quan trc sinh hc PAHs trn ng xe chy .

Hnh 1: So snh nng PAH gia cc loi cy.

(a) Nng da trn trng lng kh ca l(b) Nng da trn lng lipid trong l

(c) Nng da trn din tch b mt ca l

Cc gi tr c ly trung bnh i vi kt qu t ng i b v ngxe chy i vi tng loi cy.Thanh sai s biu hin s chnh lch gia nhngkt qu ly t ng i b v ng xe chy. Cc k t trn hnh ch th schnh lch ng k gia nhng loi cy khc nhau (P

8/2/2019 PAH Gingko

12/17

(desorption) t bi vo lp sp. LMW PAHs thng th kh (hnh 2b) Possanzini etal., 2004; Araki et at., 2009 ). LMW PAHs trn lp sp c gii thch do s phn chia(partitioning) ca kh PAHs trn lp sp cutin.. Tuy nhin, HMW PAHs ch th kh vv vy, HWM trn lp sp khng th xc nh bi phn t kh PAHs , nhng bi s

khuch tn ca HMW PAHs t by vo trong lp cutin.Mi phn t HMW PAHs kthp vi mt i phn t (v d nh coronene) trong nc sch nhiu hn khi kt hpvi nhng phn t c khi lng nh hn (v d benzofluoranthenens), mc d 2 s kthp my l nh nhau trong mu kh. Cng nh vy, s chuyn ho ca PAHs t bi volp sp ph thuc vo tnh khng a nc v ln phn t, cho thy s khc nhau cacc loi cht hu c tm thy trong bi v lp sp. t phn t PAHs k nc khi lng

phn t nh (lower molecular weight compounds) c chuyn vo trong lp sp mtkhi c nhiu phn t k nc khi lng phn t ln (higher molecular weightcompounds) cn li trong bi.

T l nh hn ca lp bn trong cho thy s phn phi qua kh khng t hnqua lp cutin. N khc vi cch tnh ca Tao v Hornbuckle (2001). Tuy nhin, T lnh hn ca phn bn trong c quan st trn cc cy ven ng Bc Kinh (Wang etal.,2008). T l HMW PAHs th nh hn LMW PAHs, cho thy s hp thu ca bi mncha HMW PAHs qua kh khng th khng quan trng.

Ni tm li, S hp thu tip theo cho thy s quan trng ca lp cutin bnngoi i vi kh v cc phn t PAHs, v i vi s di chuyn ca HMW PAHs n

lp cutin. Do , lp cutin l mt phn quan trng quan trc PAHs. V vy, nhngnghin cu sau y ca chng ti s tp trung vo lp sp, cng vi lp water-wash.

12

8/2/2019 PAH Gingko

13/17

Hnh 2: phn b ca PAHs trong (a) l v (b) khng kh.

3.3 nh hng ca v tr l trn cy vi s tch lu PAHs trn l bn ng, tng nng PAHs trong l ginkgo cao nht l cch mt t

2m v gim t 69 ng mg-lipid-1

xung cn 42 ng mg-lipid-1

khi tng chiu cao (hnh 3),phn nh ngun gc ca PAHs l t cc phng tin giao thng. Kt qu ny ph hpvi bn bo co trc v s gim nng PAHs trong khng kh khi ln cao (Tao et al.,2007). Tng nng PAHs trong nhng mu trn ng i b cng c xu hng gim,t 49 ng mg-lipid-1n 37 ng mg-lipid-1 khi tng chiu cao. Tng nng PAHs trong lginkgo ng xe chy c khuynh hng cao hn ng i b, iu ny ph hpvi s pht hin trc rng nng PAH gim khi cng xa mt con ng (Yang etal., 1991). Trong nghin cu hin ti, l ginkgo trn ng i b c dng nh gis phi nhim PAHs trong khng kh i vi ngi i b.

13

8/2/2019 PAH Gingko

14/17

Hnh 3: So snh tng nng PAHs trong l ginkgo nhng v tr khc nhau.

3.4 Mi quan h gia nng PAH trong l cy ginkgo v khng kh

Ni chung, tng nng PAH trong l cy ginkgo c thu thp t nhng conng khc nhau biu din mi quan h mnh m v quan trng vi nng PAHtrong khng kh thu thp c bi dng c ly mu khng kh th tch ln (r2=0.68,P

8/2/2019 PAH Gingko

15/17

ph thuc vo thi gian r rng i vi tng nng PAH trong khng kh (p=0.04,P>0.20) v cng khng c s ph thuc thi gian r rng cho tng nng PAH phakh hay pha ht. S thay i theo thi gian ca tng nng PAH trong l cy ginkgo gi ra s lin quan n nhit khng kh. Simonich v Hites (1994b) nhn ra rng

s phn chia ca PAHs gia khng kh v l cy ph thuc vo s nghch o ca nhit (nh 1000/T) H s phn chia KL c ngha tng quan vi 1000/T (p=0.59,P 0,20 nm 2001 - 02 so vi 2004 v2006). LMW PAHs th c nhiu trong kh thi xe chy bng diesel, trong khi HMW

PAHs c nhiu trong kh thi xe chy bng xng (Rogge et al., 1993; Okuda et al.,2003; Boonyatumanond et al., 2007). V nng PAH c th b nh hng bi thigian ca nm v nhit hng thng, khc bit trong nhng nm qua (c bit vo nm2003), chng ta s dng t s L/H lm gim s tc ng tch ly v nhit trongiu tra s thay i ti lng trong kh thi ca xe chy bng diesel t nm 2001 -02n nm 2006. Vo nm, t s L/H gim ng k t nm 2001 -02 n nm 2005 2006(Hnh 6d; P> 0.20 nm 2001-02 so vi 2003; P =0.07 nm 2001 -02 so vi 2004; P