Senso s 2007, 7, 1271-1286
senso s
ISSN 1424-8220
© 2007 by MDPI
www.mdpi.o g/senso s
Full Resea ch Pape
Haza ds o Seconda y B omadiolone In oxica ions E alua ed
using High-pe o mance Liquid Ch oma og aphy wi h
Elec ochemical De ec ion
Sona K izko a
1
, Mi osla a Beklo a
2
, Ji i Pikula
2,*
, Voj ech Adam
1
, Ales Ho na
3
and René Kizek
1
1
Depa men o Chemis y and Biochemis y, Facul y o Ag onomy, Mendel Uni e si y o Ag icul u e
and Fo es y B no, Zemedelska 1, CZ-613 00 B no, Czech Republic
2
Depa men o Ve e ina y Ecology and En i onmen al P o ec ion, Ve e ina y and Pha maceu ical
Uni e si y B no, Palackeho 1/3, CZ-612 42 B no, Czech Republic
3
Depa men o Food Enginee ing, Facul y o Technology, Tomas Ba a Uni e si y, T.G. Masa yka
275, CZ-762 72 Zlin, Czech Republic
* Au ho o whom co espondence should be add essed; E-mail:[email p o ec ed]
Recei ed: 28 June 2007 / Accep ed: 18 July 2007 / Published: 20 July 2007
Abs ac : This s udy epo ed on he possibili y o in oxica ions o non- a ge wild animals
associa ed wi h use o b omadiolone as he ac i e componen o oden icides wi h
an icoagula ion e ec s. A labo a o y es was done wi h ea hwo ms we e exposed o
b omadiolone-con aining g anules unde he condi ions speci ied in he modi ied OECD
207 guideline. No mo ali y o ea hwo ms was obse ed du ing he ou een days long
exposu e. When he ea hwo ms om he abo e es became a pa o he die o common
oles in he ollowing expe imen , no mo ali y o consume s was obse ed oo. Howe e ,
elec ochemical analysis e ealed highe le els o b omadiolone in issues om
ea hwo ms as well as common oles compa ed o con ol animals. The e we e de e mined
compa able le els o b omadiolone in he li e issue o common oles a e p ima y
(2.34±0.10 µg/g) and seconda y (2.20±0.53 µg/g) in oxica ion. The e o e, he isk o
seconda y in oxica ion o small mammalian species eeding on b omadiolone-con aining
Senso s 2007, 7
1272
ea hwo ms is he same as o p ima y in oxica ion h ough bai ed g anules. B omadiolone
bio-accumula ion in he ood chain was moni o ed using he newly de eloped analy ical
p ocedu e based on he use o a liquid ch oma og aphy coupled wi h elec ochemical
de ec o (HPLC-ED). The HPLC-ED me hod allowed o de e mine he le els o
b omadiolone in biological samples and is he e o e sui able o examining he
en i onmen al haza ds o his subs ance.
Keywo ds: an icoagulan oden icide; elec ochemis y, cyclic ol amme y; animal
issues; non- a ge o ganisms; Eisenia e ida; Mic o us a alis; Lepus eu opaeus
1. In oduc ion
Indus ial pollu an s come om a ious an h opogenic sou ces in o he en i onmen [1,2].
Ag icul u al pes icides including oden icides belong o such g oup o pollu an s [3-6]. Roden icides
a e used o con ol pes oden s ha may be ha bou ing zoono ic in ec ious agen s [7-11]. Howe e ,
oden icides may ha e also di ec o indi ec e ec s on non- a ge o ganisms [12,13]. De imen al
e ec s as well as mo ali y in bo h domes ic [14] and wild non- a ge animals [4,15] ha e been shown
a e use o an icoagulan oden icides. One o he so-called second-gene a ion an icoagulan
oden icide, b omadiolone, can be ound in li e s o p eda o s o small oden s such as he ox, ma en,
and bi ds o p ey a suble hal concen a ions [16-18]. B omadiolone ac s as he an agonis o i amin
K1 and inhibi s hus blood clo ing (Fig. 1). I may be cha ac e ized as ha ing a e y low LD50 alue
and high s abili y [19]. The e o e b omadiolone may pe sis in he en i onmen (pa icula in he soil),
bio-accumula e in an o ganism and cause acu e o ch onic in oxica ions [20]. In addi ion he in e ac ion
o b omadiolone and blood plasma p o eins is no clea ye . Recen ly, a s udy epo ing on he
in e ac ion o b omadiolone and albumin was published [21].
Se e al p oduc s con aining b omadiolone ha e been app o ed o use as bai ed g anules in he
Czech Republic. The g anules disin eg a e quickly in he ields o applica ion, hus he e is a isk o
bo h p ima y and seconda y in oxica ions o non- a ge o ganisms, because he ca ying subs ances o
g anules may a ac ea hwo ms. The insec i o ous and o he small mammalian species may possibly
become in oxica ed by his way (Fig. 2).
G ea a en ion has been paid o he su eillance o esidues o pes icides bo h in he en i onmen
and oods [22-27]. Se e al analy ical app oaches such as gas ch oma og aphy mass spec oscopy, hin
laye ch oma og aphy o immunochemis y [28,29] may be used o de e mine b omadiolone. Howe e ,
high pe o mance liquid ch oma og aphy (HPLC) coupled wi h a ious de ec o s ( luo escence o mass
de ec o s) is he mos commonly used ins umen o de ec b omadiolone [30-34]. These ins umen s,
howe e , su e om a ious eason, mos o all, high cos . Elec ochemical senso s and biosenso s
ha e supe io p ope ies o e he o he exis ing measu emen sys ems because hey can p o ide quick,
simple and low-cos on- ield de e mina ion o many biologically ac i e species and a numbe o
dange ous pollu an s [35-57].
Senso s 2007, 7
1273
Figu e 1. Au opsy o pheasan s killed a he end o he expe imen e ealed no ma ked pa hological
changes excep o occasional haemo hages wi hin he b eas muscles. The igu e shows a ypical
inding in animals poisoned wi h oden icides, so i indica es ha he pa hological changes we e ound
in opposi e o he ela i ely good heal h s a e o pheasan s [58].
Figu e 2. Scheme o he model expe imen simula ing he b omadiolone dis ibu ion wi hin he ood
chain.
THE RELATION BETWEEN BROMADIOLONE AND FOOD CHAIN – THE INFLUENCE OF THE RODENTICIDE
ON NON-TARGET ORGANISMS
Senso s 2007, 7
1274
Conside ing he abo e ac s, he aim o his wo k was o p opose a new me hod o b omadiolone
de ec ion. We op imized high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion
(HPLC-ED) o his pu pose and employed his app oach o de e mine he concen a ion o he a ge
molecule in issues o animals om wo ophic le els.
2. Expe imen al Sec ion
2.1 Chemicals
Ace ic acid, sodium ace a e, ace oni ile (HPLC g ade) and b omadiolone (Pes anal - analy ical
s anda d) we e pu chased om Sigma Ald ich (S einheim, Ge many). Me hanol was pu chased om
Fluka (S einheim, Ge many). Wa e o he p epa a ion o bu e s unde wen demine aliza ion by
e e se osmosis using he ins umen s Aqua Osmo ic 02 (Aqua Osmo ic, Tisno , Czech Republic).
Then, i was pu i ied using Millipo e RG (Millipo e Co p., USA, 18 MΏ). Wo king solu ions o he
b omadiolone s anda d we e p epa ed daily by dilu ing he s ock solu ion o 1 mg/ml concen a ion
wi h me hanol.
2.2 Roden icide-con aining p oduc
The expe imen was ca ied ou wi h a comme cially a ailable oden icide (wi h 0.005% o
b omadiolone; he eina e e e ed as PRODUCT), which is used commonly o he con ol o
common oles in ga dens, o cha ds, ineya ds, and non-ag icul u al land. The PRODUCT is no
app op ia e o la ge-scale use bu o he applica ion in he oles’ bu ows. The ecommended dose is
5-10 g anules pe open bu ow.
2.3 Bio es s
2.3.1 Toxici y o ea hwo ms – es in he a i icial soil subs a e
The es was ca ied ou acco ding o he modi ied OECD 207 guideline [59]. Glass bo les (1,000
ml) we e illed wi h 500 g o he a i icial soil es subs a e consis ing o pea (10 %), kaolin clay (20
%), and silica sand (70 %). The subs a e was mixed wi h 10 g anules o he PRODUCT.
Demine alised wa e was added a a le el co esponding o 35% o d y weigh o he subs a e. Fou
bo les wi h he PRODUCT and ou con ol bo les we e used in he expe imen . Ten labo a o y ea ed
indi iduals o Eisenia e ida we e weighed p io o he expe imen and placed on he su ace o he
subs a e in each bo le. Only adul indi iduals wi h he cli ellum we e used; he minimum a e age
weigh was 400 mg. The es was ca ied ou unde condi ions as ollows: empe a u e o 20±2°C and
con inual ligh ning (450-600 lx). The e alua ion o he es was pe o med a e 14 days. One hal o
con ol ea hwo ms and one hal o he ea hwo ms exposed o he PRODUCT we e ozen
immedia ely a e collec ing. The emaining ea hwo ms we e used in he ollowing expe imen wi h
common oles.
Senso s 2007, 7
1275
2.3.2 P ima y and seconda y in oxica ion o common oles
Nine common oles (Mic o us a alis) om he labo a o y ea ing we e used o expe imen ally
s udy he possibili y o p ima y and seconda y in oxica ion by b omadiolone. Voles we e kep
indi idually in 9 l glass aqua ia wi h wood sha ings as bedding. Th ee g oups o oles we e employed
in he expe imen las ed 5 days. The i s g oup consis ed o con ol animals we e ed wi h common
comme cially a ailable g anules o labo a o y mice. The second g oup o oles was ed wi h he
PRODUCT g anules o which hey ecei ed 0.086 g daily. The hi d g oup was ed wi h ea hwo ms,
which we e p e iously exposed o PRODUCT g anules o 14 days. D inking wa e was a ailable ad
libi um. To ensu e adap a ion o oles o ea hwo ms as a eed, he oles used in he expe imen we e
ed wi h ea hwo ms om he labo a o y ea ing o a pe iod o 7 days p io o he expe imen . A he
s a o he expe imen he a e age weigh o oles om he i s , second and hi d g oup was 16.6 g,
15.5 g and 15.6 g, espec i ely. A he end o he 5-days long expe imen , he weigh o animals om
he o e men ioned g oups was 17.3, 15.9 and 15.2 g, espec i ely. Voles we e weighed, eu hanized
and hen subjec ed o au opsy. No g oss pa hological lesions we e ound. The li e was collec ed om
each animal. The a e age weigh o he li e o animals om he o e men ioned g oups was 0.95, 0.93
and 0.94 g, espec i ely.
2.3.3 Ha es
Th ee ha e cada e s om hun ing g ounds on ag icul u al p emises in dis ic s o he eas o he ci y
o B no (Sou h Mo a ia, Czech Republic), whe e b omadiolone-con aining g anula bai s was used,
we e collec ed in No embe 2005 and used in ou expe imen s.
2.4 P epa a ion o biological samples
The samples o 0.5 g issue (ea hwo m, li e om he ea ed oles o li e and s omach con en o
ha es) we e homogenized wi h 0.5 ml o me hanol using a homogenize (Ul a u ax) o 15 min. The
homogena es was sonica ed a 150 W a 4 °C o 15 min. using K5 Sonica o (Slo akia) and
subsequen ly o exed o 15 min. using Vo ex–2 Genie (Scien i ic Indus ies, New Yo k, USA). The
mix u e was cen i uged o 20 minu es a 14,000 g a 4 °C using Uni e sal 32 R cen i uge (He ich-
Zen i ugen GmbH, Tu lingen, Ge many). Subsequen ly, he supe na an liquo was collec ed and
s o ed a –20 °C un il he nex analysis. P io o he analysis, he samples we e dilu ed 2 × wi h
me hanol.
2.5 Elec ochemical measu emen s in he s a iona y sys em
Elec ochemical cyclic ol amme ic measu emen was ca ied ou using he AUTOLAB Analyse
(EcoChemie, The Ne he lands) connec ed o VA-S and 663 (Me ohm, Swi ze land). The h ee-
elec ode sys em was used; i consis ed o he ca bon pas e elec ode (pas e composi ion: 70% ca bon
powde and 30% mine al oil, w/w; he e lon body in which he diame e o he ac i e elec ode su ace
was 2.5 mm; he su ace o he elec ode was polished p io o each measu emen using a ine il a ion
pape acco ding o [60-62]); e e ence elec ode (Ag/AgCl/3 mol l
-1
KCl), and he auxilia y elec ode
om glassy ca bon. GPES so wa e (EcoChemie, The Ne he lands) was employed o p ocess aw da a

Senso s 2007, 7
1276
using Le el 4 Sa i zky and Golay il e . Expe imen s we e ca ied ou a oom empe a u e. The
measu emen was pe o med in he po en ial ange om 0.6 o 1 V using he ollowing pa ame e s: he
po en ial s ep o 2.4 mV, he scan a e o 0.3 V.s
-1
. Ace a e bu e (0.2 M, pH 4.0) was used as a
suppo ing elec oly e.
2.6 Elec ochemical measu emen s in he low sys em
The low sys em consis ed o he ch oma og aphic pump (Model 583 ESA Inc., Chelms o d, MA,
USA), Gua d cell (Model 5020 ESA, USA), eac ion loop (1m) o Flow Injec ion Analysis (FIA), and
he ch oma og aphic column Pola is (C18-A, 150 × 4.6 mm, wi h pa icle size o 3 µm; Va ian, Inc.)
and he elec ochemical de ec o . The de ec o consis ed o pa s as ollows: low ampe ome ic
analy ical cell (Model 5040, ESA, USA) con aining he e e ence (hyd ogen-palladium), auxilia y
ca bon and he wo king elec ode made o glassy ca bon, he Coulochem III con ol module. The
sample (5 µl) was injec ed manually using a 5 µl injec ion loop (Supelco, Belle on e, PA, USA). The
da a om he de ec o we e p ocessed using he CSW 32 so wa e. The isoc a ic mobile phase
consis ed o 0.2 M ace a e bu e (pH 4.0/ace oni ile, 40:60, / ). The de ec ion po en ial was
op imized; see in “Resul s” sec ion.
3. Resul s
3.1 De ec ion o b omadiolone using cyclic ol amme y
To employ HPLC wi h elec ochemical de ec ion o measu emen o b omadiolone we aimed on
s udy o he basic elec ochemical beha iou o he a ge molecule using cyclic ol amme y a he
ca bon pas e elec ode. The ol ammog ams wi h a dis inc oxida ion signal a po en ial o 836 mV due
o b omadiolone a e shown in Fig. 3A. Dependence o he peak heigh on he scan a e was loga i hmic
(y = 44.413 ln(x) - 78.08). In addi ion, he po en ial o he peak shi ed g adually o posi i e po en ials
(Fig. 3B). The dependence o he peak heigh on he concen a ion o b omadiolone was also
de e mined. The signal enhanced sha ply wi h he concen a ion in he gi en concen a ion ange up o
a concen a ion o 1 µg/ml, hen mo e g adually (Fig. 3C). In spi e o he ac ha he peak heigh o
b omadiolone enhanced wi h i s inc easing concen a ion, bu he dependence measu ed is no s ic ly
linea wi hin he in e al. Fo analy ical pu poses his dependence can be spli in o wo s ic ly linea
lines a a concen a ion o b omadiolone anging om 0.625 o 10 µg/ml (y = 600.53x + 67.114, R
2
=
0.9912); and a a concen a ion o b omadiolone anging om 20 o 300 ng/ml (y = 62.576x + 259.78,
R
2
= 0.9927, inse in Fig. 3C). The limi o de ec ion o b omadiolone measu ed on he ca bon pas e
elec ode by cyclic ol amme y was e alua ed as 5 ng/ml. I can be concluded ha CV is a me hod
sui able o undamen al s udying o elec ochemical beha iou o a a ge molecule; howe e , i can
no be used o analysis o eal sample due o ela i e low selec i i y. The e o e he expe imen al
esul s ob ained we e used in an op imiza ion p ocedu e o he de ec ion o b omadiolone using low
analysis.
Senso s 2007, 7
1277
Figu e 3. The elec ochemical analysis o b omadiolone using cyclic ol amme y. (A) Cyclic
ol ammog ams o b omadiolone (1 µg/ml) a scan a e o 5, 50, 150, and 300 mV/s. (B) Dependence
o he heigh and posi ion o po en ial o he b omadiolone signal on he scan a e. (C) Va ia ion in he
heigh o he b omadiolone signal on he concen a ion a a scan a e o 300 mV/s. Pa ame e s o
measu emen : 0.2 M ace a e bu e (pH 4.0), in he po en ial ange om 0.6 o 1 V, po en ial s ep: 2.4
mV, expe imen s we e ca ied ou a oom empe a u e.
3.2 De ec ion o b omadiolone using low injec ion analysis wi h glassy ca bon elec ode
To make he analysis o b omadiolone-con aining samples apid and sui able o ou ine, a new
app oach o he de ec ion o b omadiolone using Flow Injec ion Analysis (FIA) was p oposed. The
elec ochemical esponse o b omadiolone was s udied a a cons an low a e o he mobile phase (0.5
ml/min) and a a ying wo king elec ode po en ial. The de ec ion po en ial o 840 mV was selec ed
based on he hyd odynamic ol ammog am (no shown). To ob ain he maximum esolu ion o he
b omadiolone peak and he maximum in ensi y o he signal, he e ec o an o ganic componen o he
mobile phase (ace oni ile) was s udied (Fig. 4A). The inc eased po ion o he o ganic phase was
ound bene icial o he de ec ion o b omadiolone. This ac is likely o be associa ed wi h he
physicochemical p ope ies o b omadiolone as an non-pola compound (mo e soluble in o ganic
sol en s) al hough ou p e ious s udies indica ed a signi ican dec ease in he elec ochemical signal in
he p esence o he inc eased po ion o he o ganic componen in he mobile phase [58,63-66]. The
low a e o he mobile phase also a ec s he elec ochemical signal. Va ia ions in he b omadiolone
0
100
200
300
400
500
600
700
800
900
0 2 4 6 8 10 12
y = 44.413Ln(x) - 78.08
R
2
= 0.9801
0
50
100
150
200
0 100 200 300 400 500
0.72
0.76
0.80
0.84
0 100 200 300 400 500
Scan a e (mV/s)
Po en ial (V)
0.6 0.7 0.8 0.9 1.0
500 nA
Po en ial (V)
Cyclic ol ammog ams
5 mV/s
50 mV/s
150 mV/s
300 mV/s
Scan a e
B omadiolone signal
Peak heigh (nA)
Scan a e (mV/s)
A
B
C
O
OH
O
CH CH
2
CH B
OH
y = 600.53x + 67.114
R
2
= 0.9912
0
100
200
300
0 0.1 0.2 0.3 0.4
B omadiolone concen a ion (µ
µµ
µg/ml)
Peak heigh (nA)
B omadiolone concen a ion (µ
µµ
µg/ml)
Peak heigh (nA)
Senso s 2007, 7
1278
signal wi h he low a e anging om 0.1 ml/min o 2.0 ml/min we e de e mined. The b omadiolone
signal inc eased a a low a e up o 0.8 ml/min. Then i did no change (no shown).
Figu e 4. The analysis o b omadiolone using high pe o mance liquid ch oma og aphy wi h
elec ochemical de ec ion. (A) The e ec o he o ganic componen in he mobile phase (ace oni ile)
on he b omadiolone peak heigh . (B) Ch oma og am o b omadiolone a a concen a ion o 50 µg/ml,
e en ion ime o he peak o 9.5min. (C) Dependence o he heigh o he signal on he b omadiolone
concen a ion. Expe imen al condi ions we e as ollows: ch oma og aphic column Pola is C18-A, 150
× 4.6 mm, diame e : 3 µm (Va ian, Inc.). The sample was injec ed manually. Isoc a ic mobile phase
consis ed o 0.2 M ace a e bu e (pH 4.0)/ace oni ile (40:60, / ) and he de ec ion po en ial o 840
mV we e used. The low a e o he mobile phase was 0.8 ml/min.
3.3 High pe o mance liquid ch oma og aphic analysis o b omadiolone
Unde he expe imen al condi ions op imized using FIA (wo king elec ode po en ial 840 mV,
mobile phase 0.2 M ace a e bu e (pH 4.0)/ace oni ile (40:60, / ), he low a e o he mobile phase
0.8 ml/min) high pe o mance liquid ch oma og aphy coupled wi h elec ochemical de ec o was used
o de e mine he con en o b omadiolone in eal samples. The signal o b omadiolone was obse ed
wi h he e en ion ime o 9.5 min (Fig. 4B). In addi ion, he calib a ion dependence o b omadiolone
a a concen a ion ange om 0.3 o 50 µg/ml was de e mined. The s ic ly linea calib a ion cu e was
ob ained: y = 3.5x + 0.9; R
2
= 0.9997, wi h a ela i e s anda d de ia ion o abou 4 % (Fig. 4C). When
we p oposed he echnique o de e mina ion o b omadiolone, he oxic e ec s o he a ge molecule
on a ious expe imen al models we e in es iga ed.
15
y = 3.5319x + 0.9243
R
2
= 0.9997
0
40
80
120
160
200
0 20 40
B omadiolone concen a ion (µ
µµ
µg/ml)
Peak heigh (µA)
0
10
20
30
40
50
60
70
80
90
100
30% 35% 40% 60%
Ace oni ile con en in mobile
phase (%, / )
Peak heigh (%)
7 9 11 13
50 µA
b omadiolone 50 µg/ml
Re en ion ime (min)
RT 9.5 min
B
AC
Re en ion ime (min)
8.5 9.5 10.5
con ol
b omadiolone posi i e
300 nA
Re en ion ime (min)
8.5 9.5 10.5
con ol
b omadiolone posi i e
300 nA300 nA
50 µA
Senso s 2007, 7
1279
3.4 B omadiolone exposu e
3.4.1 Ea hwo ms
Du ing he 14-days long expe imen , all ea hwo ms exposed o b omadiolone su i ed. A he
beginning o he expe imen hei a e age weigh was 430 mg. Howe e , he weigh o con ol
ea hwo ms was 293 mg while ea hwo ms exposed o he PRODUCT g anules weighed 344 mg a he
end o he expe imen ,. The dec ease in he weigh o con ol ea hwo ms (mo e han 30 %) was caused
by s a a ion since he subs a e con ained no sui able nu ien s. Ea hwo ms exposed o he
PRODUCT g anules los only 20 % o hei o iginal weigh . They we e assumed o ha e ecei ed
g anules con aining b omadiolone. Using he p oposed app oach o homogeniza ion and ex ac ion, he
samples o ea hwo ms (Eisenia e ida) we e p epa ed and subsequen ly analyzed by HPLC-ED. The
a e age le el o b omadiolone was 1.7 ± 0.2 µg pe ea hwo m, which co esponds o 3.3 ± 0.9 µg pe
g o ea hwo m weigh (Fig. 5). No signal due o b omadiolone was de e mined in samples om
con ol ea hwo ms.
Figu e 5. The con en o b omadiolone in he issues om expe imen al animals. No b omadiolone
signal was de e mined in samples om he con ols ( oles, ea hwo ms). The le els o b omadiolone in
ea hwo ms we e ecalcula ed ela i e o he weigh o ea hwo ms. The le els o b omadiolone in
oles we e ecalcula ed o he weigh o he li e . Fo o he de ails see in Fig. 3 and “Expe imen al
sec ion”.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
B omadiolone con en (µg/g)
Con ol
Eisenia e ida
Mic o us a alis
(p ima y exposu e)
Mic o us a alis
(seconda y exposu e)
Biological samples
Lepus eu opaeus
(li e )
Lepus eu opaeus
(s omach con en )
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
B omadiolone con en (µg/g)
Con ol
Eisenia e ida
Mic o us a alis
(p ima y exposu e)
Mic o us a alis
(seconda y exposu e)
Biological samples
Lepus eu opaeus
(li e )
Lepus eu opaeus
(s omach con en )
Senso s 2007, 7
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