Ga cía-Ruiz, And Øs, Vale a, Labo da, Ma ina 17
Ca men Ga cía-Ruiza),
Romµn And Øsb),
Jose Luis Vale ab),
Fe nando Labo dab),
Ma ía Luisa Ma inaa, b)
a) Depa amen o de Química
Analí ica, Facul ad de Química,
Uni e sidad de Alcalµ, C a.
Mad id-Ba celona Km. 33.600,
28871 Alcalµ de Hena es
(Mad id), Spain
b) Cen o de Tecnología de los
Alimen os y Se icios
Biosani a ios, Uni e sidad de
Alcalµ, C a. Mad id-Ba celona
Km. 33.600, 28871 Alcalµ de
Hena es (Mad id), Spain
Moni o ing he s e eoselec i i y o biodeg ada ion
o chi al polychlo ina ed biphenyls using
elec okine ic ch oma og aphy
A s udy o he biodeg ada ion o se e al chi al polychlo ina ed biphenyls (PCBs,
IUPAC numbe s 45, 88, 91, 95, 136, 144, 149, and 176) by a na u ally occu ing soil
bac e ium as well as he s e eoselec i i y o such p ocess has been pe o med using
Elec okine ic Ch oma og aphy wi h cyclodex ins (CD-EKC). In o de o achie e he
analysis o chi al PCBs, a CD-EKC sys em based on 50 mM 2-mo pholinoe hanesul-
onic acid (MES) bu e (pH 6.5) con aining 2 M u ea, 20 mM ca boxyme hyla ed c-
cyclodex in (CM-c-CD), and 10 mM b-cyclodex in (b-CD) was used. Fi s , he pe -
cen age deg ada ion o PCBs by he mic oo ganism used in his wo k was measu ed
o di e en incuba ion imes (22, 46, 71, 93, 165, 213, and 262 hou s). The esul s
showed a high deg ee o biodeg ada ion ( om 61% o 94%) o he eigh PCBs s ud-
ied a e 262 hou s o incuba ion wi h he mic oo ganism, deg ada ion being a ou ed
o he leas chlo ina ed congene s. Secondly, measu emen o he a opisome ic
a io o he PCB a opisome s du ing he biodeg ada ion p ocess e ealed no signi i-
can a ia ion o he a opisome ic a ios wi h he deg ada ion ime, indica ing a non-
s e eoselec i e deg ada ion o PCBs by he mic oo ganism.
Key Wo ds: Chi al polychlo ina ed biphenyls; S e eoselec i i y; Biodeg ada ion; Elec-
okine ic ch oma og aphy;
Recei ed: May 21, 2001; e ised: Oc obe 23, 2001; accep ed: Oc obe 25, 2001
1 In oduc ion
Polychlo ina ed biphenyls (PCBs) a e a oma ic com-
pounds ha cons i u e an impo an class o p io i y pollu-
an s classi ied by he U.S. En i onmen al P o ec ion
Agency (EPA) as ca cinogens [1, 2]. Biphenyls wi h bulky
a oms in o ho posi ions o he phenyl ings can be de ined
as molecules wi h axial chi ali y, in which o a ion abou
he axis is hinde ed by s e ic conges ion. In addi ion, i
such a o a ional ene gy ba ie is high enough o esis
acemisa ion a oom empe a u e, he a opisome s will
be con o ma ionally s able [3]. Among he 78 PCBs ha
possess axial chi ali y, only 19 a e s able owa ds acemi-
sa ion a oom empe a u e.
The enan iome s o chi al compounds may ha e di e en
biological and oxicological e ec s. The e o e, enan iose-
lec i e disc imina ion o chi al pollu an s in en i onmen al
samples may yield in o ma ion abou possible deg ada-
ion pa hways [4, 5]. The main ou es o he en i onmen-
al deg ada ion o PCBs a e mic obiological p ocesses
besides abio ic p ocesses [6].
To da e, he echnique mos widely employed o analyse
achi al o chi al PCBs has been gas ch oma og aphy
(GC) [7–13]. Ne e heless, capilla y elec opho esis (CE)
has expe ienced eno mous g ow h in he ield o chi al
sepa a ions because o i s lexibili y and high sepa a ion
e iciency [14]. Cyclodex ins (CDs) a e known o exhibi
s ong chi al ecogni ion owa ds se e al chi al PCBs
p esen in comme cial PCB o mula ions esul ing om
in e ac ions such as he o ma ion o inclusion complexes.
Thus, cyclodex in-modi ied micella elec okine ic ch o-
ma og aphy (CD-MEKC) has been employed o achie e
he chi al sepa a ion o wel e chi al PCBs (IUPAC num-
be s 45, 84, 88, 91, 95, 132, 136, 139, 149, 171, 183, and
196) using sodium dodecyl sul a e (SDS) micelles wi h c-
cyclodex in (c-CD) [15] o mix u es o b-cyclodex in (b-
CD) and c-CD [16]. The use o CD-MEKC wi h bile sal s
(sodium chola e) mixed wi h SDS micelles and c-CD [17]
enabled he chi al sepa a ion o wel e PCBs (IUPAC
numbe s 45, 84, 88, 91, 95, 131, 135, 136, 139, 144, 175,
176). Unde he abo e-men ioned condi ions i was possi-
ble o sepa a e he a opisome s o eigh o nine PCBs in
mix u es in abou 35 min. Ne e heless, when bile sal s
alone [18] we e used in CD-MEKC only he indi idual
J. Sep. Sci. 2002, 25, 17–22
Co espondence: P o . Ma ía Luisa Ma ina, Depa amen o
de Química Analí ica, Facul ad de Química, Uni e sidad de
Alcalµ, C a. Mad id-Ba celona Km. 33.600, 28871 Alcalµ de
Hena es (Mad id), Spain.
E-mail: [email p o ec ed]
Fax: +34 91 8854971
Abb e ia ions: Polychlo ina ed Biphenyls, PCBs; Elec oki-
ne ic Ch oma og aphy, EKC.
O iginal Pape
iWILEY-VCH Ve lag GmbH, 69469 Weinheim 2002 1615-9306/2002/0101–0017$17.50+.50/0
chi al sepa a ion o PCB 84, 95, and 176 was possible.
Ne e heless, elec okine ic ch oma og aphy (EKC) using
a dual cyclodex in sys em has ecen ly been employed
o he apid chi al sepa a ion o hi een o he 19 PCBs
s able a oom empe a u e (PCBs 45, 88, 91, 95, 131,
132, 136, 139, 144, 149, 171, 176, 197) [19]. Unde hese
condi ions, he chi al sepa a ion o he a opisome s o up
o i e chi al PCBs was possible in less han 12 min.
The e o e, his sys em seems o be he mos app op ia e
in o de o check he chi ali y o a majo numbe o chi al
PCBs using he sho es analysis ime.
In his wo k, a na u ally occu ing soil bac e ium isola ed
om a soil wi h a his o y o con amina ion has been
employed o pe o m he biodeg ada ion o eigh chi al
PCBs (IUPAC numbe s 45, 88, 91, 95, 136, 144, 149, and
176) wi h di e en deg ees o chlo ina ion. In o de o
ob ain in o ma ion ela ed o he s e eoselec i i y o he
biodeg ada ion o PCBs by such bac e ia, a s udy on he
a opisome ic a io o he baseline esol ed a opisome s
has been pe o med du ing he biodeg ada ion p ocess
and he esul s a e epo ed he ein.
2 Expe imen al
2.1 Reagen s
All eagen s employed we e o analy ical g ade. 2-Mo -
pholinoe hanesul onic acid (MES), dime hyl o mamide
(DMF), and sodium hyd oxide we e supplied by Me ck
(Da ms ad , Ge many); b-cyclodex in (b-CD) and u ea
we e om Fluka (Buchs, Swi ze land); ca boxyme hyl-
a ed c-cyclodex in (CM-c-CD, deg ee o subs i u ion (DS)
ca. 3) was ob ained om Cyclolab (Budapes , Hunga y).
Wa e pu i ied h ough a Milli-Q sys em om Millipo e
(Bed o d, MA, USA) was employed o p epa e he solu-
ions. All solu ions we e il e ed p io use h ough 0.45 lm
po e size disposable nylon il e s om Scien i ic
Resou ces (Ea on own, NJ, USA).
Ace one, dichlo ome hane, and hexane o pes ipu e
g ade om sds (Peypin, F ance) we e used in glasswa e
cleaning and ex ac ion p ocess.
The eigh PCBs s udied we e supplied by D . Eh ens o e
Re e ence Ma e ials (Augsbu g, Ge many). These com-
pounds and hei IUPAC numbe s (be ween pa en heses)
acco ding o he Ballschmi e nomencla u e [20] a e he
ollowing: 2,29,3,6- e achlo obiphenyl (PCB 45),
2,29,3,4,6-pen achlo obiphenyl (PCB 88), 2,29,3,49,6-pen-
achlo obiphenyl (PCB 91), 2,29,3,59,6-pen achlo obiphe-
nyl (PCB 95), 2,29,3,39,6,69-hexachlo obiphenyl (PCB
136), 2,29,3,4,59,6-hexachlo obiphenyl (PCB 144),
2,29,3,49,59,6-hexachlo obiphenyl (PCB 149), and
2,29,3,39,4,6,69-hep achlo obiphenyl (PCB 176).
2.2 Appa a us
A HP3D CE sys em (Hewle -Packa d, Waldb onn, Ge -
many) equipped wi h an on-column diode a ay de ec o
(DAD) and a HP 3D-CE Chems a ion so wa e was used.
Sepa a ions we e pe o med using uncoa ed used-silica
capilla ies o inne diame e (ID) 50 lm and ou e diam-
e e (OD) 375 lm, pu chased om Composi e Me al Se -
ices LTD (Wo ces e , England). The capilla ies had a
o al leng h o 58.5 cm and 50 cm o he de ec o . The
capilla y empe a u e was se a 458C and UV de ec ion
was pe o med a 230 nm.
Elec oly ic solu ions we e degassed in an ul asonic ba h
KM om Raypa (Ba celona, Spain). A 654 pH-me e om
Me ohm (He isau, Swi ze land) was employed o adjus
he pH o he sepa a ion bu e s.
A Büchi Ro a apo R-134 (Flawil, Swi ze land) and a
Pie ce Reac i-Vap model 18780 e apo a ing uni (Rock-
o d, Illinois, USA) we e used o o ally e apo a e he hex-
ane o he ex ac s.
2.3 P ocedu e
The s ain (MS3-02) used in his wo k was isola ed om
samples o soil o he Comunidad de Mad id. Fi s , he
deg ada ion ac i i y o PCBs by his s ain was checked
by analysing a comme cial mix u e o PCBs (A ochlo
1242) by gas ch oma og aphy [21]. This s ain has been
cha ac e ised and iden i ied by he Spanish Type Cul u e
Collec ion as Jonibac e sp.
The cul u e condi ions o he mic oo ganism we e as ol-
lows: 10 mL o modi ied phospha e-bu e ed mine al sal s
medium (PASM) wi h biphenyl we e inocula ed wi h he
cul u e s ain a 288C du ing 48 hou s. Then he cul u e
was cen i uged (4000 pm o 15 min) and he p ecipi a e
was washed h ee imes wi h a sodium chlo ide solu ion
(0.9% w/ ). Finally, a suspension o he mic oo ganism in
a solu ion o sodium chlo ide (0.9% w/ ) was p epa ed by
adding he mic oo ganism un il one uni o abso bance
was measu ed a 630 nm.
The samples we e p epa ed using he incuba ion medium
(10 mL o PASM) wi h 1 mL o a solu ion o hexane con-
aining a mix u e o wo PCBs (149–95, 88–136, 45–91,
144–176) a a concen a ion o 0.5 ppm o each PCB.
Hexane was e apo a ed a oom empe a u e p io o he
addi ion o 0.5 mL o he abo e-men ioned mic oo ganism
suspension. In he samples he mic oo ganism was inac i-
a ed by he addi ion o 70 lL pe chlo ic acid a e he co -
esponding incuba ion imes whe eas o he con ol sam-
ples he mic oo ganism was inac i a ed immedia ely.
A e inac i a ion o he bac e ium wi h pe chlo ic acid he
hyd ophobic PCBs we e ex ac ed wi h hexane
(4610 mL) and d ied wi h anhyd ous sodium sul a e. The
18 Ga cía-Ruiz, And Øs, Vale a, Labo da, Ma ina J. Sep. Sci. 2002, 25, 17–22
sol en was emo ed in a o a y e apo a o . The ex ac
was edissol ed wi h hexane (460.5 mL) and placed in 2-
mL ials. Hexane was hen e apo a ed o d yness unde a
ni ogen s eam. The inal ex ac con aining PCBs was
econs i u ed wi h DMF (50 lL) p io o CE analysis.
Ex ac ions we e pe o med in duplica e.
The ex en o eco e y o he ex ac ion was checked o
PCB 91 as ollows: 1 mL o PCB 91 in hexane (4.8 ppm)
was added o 10 mL o PASM. A e e apo a ion o hex-
ane a ambien empe a u e he ex ac ion was ca ied ou
as desc ibed abo e. The inal ex ac was edissol ed in
150 lL o DMF con aining 200 ppm o PCB 84 as in e nal
s anda d. A calib a ion mix u e was p epa ed e apo a ing
o d yness 1 mL o a solu ion in hexane o PCB 91
(4.8 ppm) and adding 150 lL o he abo e in e nal s an-
da d solu ion. The ex en o eco e y anged om 77 o
75% when wo indi idual expe imen s we e injec ed in
duplica e.
The sepa a ion bu e was p epa ed dissol ing he app o-
p ia e amoun o MES, CD de i a i es, and u ea in wa e
o achie e a inal concen a ion 50 mM MES–2 M u ea–
20 mM CM-c-CD–10 mM b-CD p io o he addi ion o
wa e [19]. Finally, he pH was adjus ed o 6.5 wi h 1 M o
0.1 M sodium hyd oxide solu ion p io o making up he
inal olume.
Be o e i s use, a new capilla y was insed wi h 1 M
NaOH o 30 min, ollowed by a 30-minu e inse wi h
wa e . Be ween uns, he capilla y was condi ioned wi h
DMF o 4 min, 0.1 M NaOH o 4 min, and sepa a ion bu -
e o 4 min. Injec ions we e made by p essu e (50 mba
o 1 s o 10 mba o 1 s o eco e y de e mina ion). A
sepa a ion ol age o 20 kV was used and he de ec ion
ook place a 230 nm. This condi ioning me hod wi h DMF,
sol en used o dissol e he PCBs, was used o ob ain
good peak shapes and ep oducible e en ion da a. Two
injec ions we e pe o med o each sample.
2.4 Calcula ions
In his wo k, co ec ed a eas ha e been used in o de o
compensa e luc ua ions in elec opho e ic condi ions and
o ob ain a good ep oducibili y o da a. Co ec ed a eas
we e ob ained by di iding he aw a ea by he mig a ion
ime [22].
3 Resul s and discussion
Elec okine ic ch oma og aphy using a dual cyclodex in
sys em based on a 50 mM 2-mo pholinoe hanesul onic
acid (MES) bu e (pH 6.5) con aining 2 M u ea, 20 mM
ca boxyme hyla ed c-cyclodex in (CM-c-CD), and
10 mM b-cyclodex in (b-CD) a 20 kV and 458C, using an
on-column diode a ay de ec o a 230 nm o de ec-
ion [19], has been employed o moni o he biodeg ada-
ion o eigh chi al PCBs by a na u ally occu ing soil bac-
e ium and o s udy he s e eoselec i i y o such p ocess.
3.1 Biodeg ada ion o chi al PCBs
A s udy on he biodeg ada ion p ocess by he na u ally
occu ing bac e ium has been pe o med o eigh chi al
PCBs (IUPAC numbe s 45, 88, 91, 95, 136, 144, 149, and
176). These PCBs ha e been analysed in mix u es o wo
congene s (149–95; 88–136; 176–144; and 91–45)
baseline sepa a ed by a chi al sys em which ecognises
he a opisome s o each congene . Table 1 shows he
di e ence be ween he a e age mig a ion imes o he
a opisome s o each congene in each mix u e o wo
PCBs and he chi al esolu ion o each PCB o con ol
samples. I can be obse ed ha a opisome s o se en
PCBs a e baseline esol ed (Rs A1.53) whe eas a opi-
some s o PCB 176 a e only pa ially sepa a ed
(Rs = 0.47).
In o de o e alua e he ex en o he deg ada ion o PCBs
a di e en imes o incuba ion he o al peak a ea o each
congene (addi ion o he a ea o he peak o he i s -
mig a ing a opisome and he a ea o he peak o he sec-
ond-mig a ing a opisome ) was measu ed in he co e-
sponding elec ophe og am a di e en incuba ion imes.
J. Sep. Sci. 2002, 25, 17–22 Moni o ing he s e eoselec i i y o biodeg ada ion 19
Table 1. IUPAC numbe s (acco ding o Ballschmi e and Zell
nomencla u e [20]) and pai s o PCBs s udied in his wo k
oge he wi h he esolu ion o PCB a opisome s and di e -
ences o mig a ion imes o each pai o congene s consid-
e ed in he con ol samples (0 hou s o incuba ion).
PCBs IUPAC
numbe
Chi al
esolu ion a)
Pai s o
PCBs b)
29– 19c)
45 2.67 149–95 2.09
88 1.62
91 2.32 88–136 0.22
95 4.16
136 3.72 176–144 0.76
144 1.91
149 1.53 91–45 1.79
176 0.47
a) Mean alue o ou de e mina ions calcula ed as
Rs = 1.18( 2– 1)/(w1+w2) whe e 1and 2a e he mig a-
ion imes o he wo a opisome s o a PCB and w1,w2
co espond o middle high wid h o he co esponding
peaks.
b) Pai s o PCBs s udied ollowing he mig a ion o de .
c) 29– 19= [( 3+ 4)/2] – [( 1+ 2)/2].
1and 2a e he mig a ion imes in minu es o he a op-
isome s o he i s -mig a ing PCB.
3and 4a e he mig a ion imes in minu es o he a op-
isome s o he second-mig a ing PCB.
As an example, Figu e 1 shows he elec ophe og ams
ob ained du ing he biodeg ada ion o a mix u e o PCBs
149 and 95 a h ee di e en incuba ion imes (0, 94, and
262 hou s). Using as e e ence he esul s ob ained om
con ol samples analyses (% o deg ada ion = 0) he
deg ee o deg ada ion o PCBs was calcula ed o wo
indi idual expe imen s injec ed by duplica e o he a e -
aged incuba ion imes o 22, 46, 71, 93, 165, 213, and 262
hou s. Table 2 shows he deg ee o deg ada ion calcu-
la ed o he eigh chi al PCBs s udied a he abo e-men-
ioned incuba ion imes. The biodeg ada ion p ocess has
been ollowed un il a deg ada ion deg ee highe han 61%
o all chi al PCBs was eached ( om 61 o 94%). I could
be obse ed ha he deg ada ion a e is di e en depend-
ing on he s udied PCB. As an example, a an incuba ion
ime o 48 hou s he di e ence o he deg ee o biodeg a-
da ion be ween he mos deg aded congene s and he
leas deg aded congene s is close o 40% while a 262
hou s his di e ence is abou 33%. In gene al, i can be
obse ed ha he deg ada ion is a ou ed o he leas
chlo ina ed PCBs. A compa ison o he deg ee o deg a-
da ion ob ained a 46 and 262 hou s o all PCBs s udied
is shown in Figu e 2. I can be seen ha a low incuba ion
ime (46 h) he highes deg ee o deg ada ion co e-
sponds o he leas chlo ina ed PCBs ( ou and i e chlo -
ine a oms) whe eas he lowes deg ees o deg ada ion
a e ob ained o PCBs 149 and 176 wi h six and se en
chlo ine a oms, espec i ely.
3.2 S udy o he s e eoselec i i y o he
biodeg ada ion p ocess
The in es iga ion o he s e eoselec i i y o he biodeg a-
da ion p ocess has been ca ied ou measu ing he a op-
isome ic a io o se en o he eigh chi al PCBs s udied
due o he low chi al esolu ion ob ained o he PCB 176
20 Ga cía-Ruiz, And Øs, Vale a, Labo da, Ma ina J. Sep. Sci. 2002, 25, 17–22
Figu e 1. Elec ophe og ams ob ained du ing he biodeg a-
da ion o a mix u e o PCBs 149 and 95 a h ee di e en
incuba ion imes (0, 94, and 262 hou s). Sepa a ion bu e :
50 mM MES (pH 6.5), 2 M u ea, and 20 mM CM-c-CD
wi h10 mM b-CD. Injec ion by p essu e, 50 mba o 1 s.
Capilla y, 58.5 cm (50 cm o he de ec o )650 lm i.d. Tem-
pe a u e, 458C. Applied ol age, 20 kV (l97 lA); UV de ec-
ion a 230 nm.
Table 2. Deg ada ion, in %, o he PCBs analysed a di e en incuba ion imes.
Incuba ion PCB 45 PCB 88 PCB 91 PCB 95 PCB 136 PCB 144 PCB 149 PCB 176
ime (h) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%)
22 38.2 (0.0) 62.3 (0.0) 5.5 (0.0) – 63.7 (0.0) 71.7 (0.0) – 52.9 (9.6)
46 71.5 (1.7) 68.0 (2.7) 47.9 (1.5) 60.1 (0.0) 57.0 (6.3) 69.0 (3.5) 31.7 (2.8) 42.4 (7.9)
71 82.5 (2.2) 85.7 (2.2) 57.4 (7.1) 61.9 (4.8) 76.4 (5.6) 74.7 (12.2) 30.5 (7.3) 71.7 (0.0)
93 88.7 (0.0) 84.4 (8.3) 52.1 (3.1) 84.8 (7.9) 73.5 (9.2) – 61.2 (15.0) –
165 – 87.8 (3.6) 63.6 (5.7) 87.1 (10.3) 71.9 (11.8) – 67.0 (13.9) –
213 80.9 (0.1) 92.3 (1.6) 60.2 (13.5) 94.1 (1.6) 83.1 (4.2) 78.5 (0.0) 77.1 (9.9) –
262 86.8 (2.6) 93.1 (0.2) 72.2 (0.8) 94.4 (1.1) 77.0 (3.4) 74.3 (0.0) 76.5 (2.7) 61.3 (0.0)
Figu e 2. Compa ison o he deg ee o biodeg ada ion
ob ained a 46 and 262 hou s o he eigh chi al PCBs s u-
died.
(see Table 1). The a opisome ic a io has been calcu-
la ed compa ing he peak a eas o he i s -mig a ing a o-
pisome and he second one in each case. Table 3 dis-
plays he esul s ob ained o he a opisome ic a io o
he se en chi al PCBs along he incuba ion pe iod. In gen-
e al, i can be obse ed a sligh de ia ion o he a opiso-
me ic a io alues om he expec ed alue o a acemic
mix u e (a opisome ic a io = 1) which could be a ibu ed
o expe imen al e o s. Al hough o PCB 149 a e y sligh
inc ease in he a opisome ic a io was obse ed wi h
inc easing incuba ion imes (a opisome ic a io inc eased
om 1.04 o 1.13) hese a ia ions could be conside ed as
lying wi hin he expe imen al e o . In conclusion, esul s
ha e e ealed no signi ican s e eoselec i e deg ada ion
by he soil bac e ium unde he expe imen al condi ions
employed in his wo k.
4 Conclusions
In his wo k we ha e s udied he applica ion o elec oki-
ne ic ch oma og aphy (EKC) using a dual cyclodex in
sys em o moni o he biodeg ada ion p ocess o eigh
chi al PCBs (45, 88, 91, 95, 136, 144, 149, and 176) du -
ing mo e han 250 hou s and o s udy he s e eoselec i i y
o his p ocess o each congene .
A high deg ee o deg ada ion ( om 61% o 94%) by he
na u ally occu ing soil bac e ium used in his wo k has
been achie ed o all he PCBs s udied, he deg ada ion
a e being highe o he leas chlo ina ed congene s
(PCBs 45 and PCB 88).
No s e eoselec i e biodeg ada ion has been obse ed o
he se en chi al PCBs whose a opisome s ha e been
baseline sepa a ed (PCBs 45, 88, 91, 95, 136, 144, 149).
Acknowledgmen s
The au ho s hank he Comunidad Au ónoma de Mad id
(Spain) o p ojec s 07 M/0640/1997 and 07 M/0049/
1998.
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iod (A opisome ic a io = co ec ed a ea o he i s -mig a ing a opisome /co ec ed a ea o he second-mig a ing a opisome ).
Incuba ion PCB 45 PCB 88 PCB 91 PCB 95 PCB 136 PCB 144 PCB 149
ime (h) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%) (RSD,%)
0 1.05 (1.8) 1.01 (0.8) 1.02 (0.0) 1.01 (5.9) 1.05 (1.8) 1.05 (1.8) 1.04 (1.6)
22 – 1.04 (0.0) 1.07 (0.0) – 1.00 (0.0) 1.10 (2.4) 1.04 (0.0)
46 1.07 (7.9) 1.02 (1.4) 1.06 (0.0) 1.09 (0.0) 1.03 (4.7) 1.08 (2.7) 1.03 (3.4)
71 1.04 (3.1) 1.03 (2.9) 1.03 (3.1) 1.09 (2.8) 0.99 (5.1) 1.04 (0.2) 1.11 (0.0)
93 1.05 (0.0) 1.05 (5.6) 1.05 (2.5) 1.05 (0.0) 1.03 (0.2) – 1.09 (3.0)
165 – 1.02 (6.4) 1.05 (0.9) 1.11 (0.0) 1.05 (0.2) 1.01 (5.6) 1.11 (4.2)
213 0.99 (6.0) 1.02 (7.3) 1.05 (2.8) 1.09 (0.0) 0.97 (1.6) 1.06 (0.7) 1.13 (5.1)
262 1.02 (2.0) 1.02 (0.0) 1.03 (4.1) 1.11 (0.3) 1.00 (0.9) 1.04 (0.0) 1.13 (2.3)

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