Detection of Mutagens and BaPMO Inducers inRiver Water Using the Blue CottonAdsorption Technique

Smiljana Britvic,1 Branimir K. Hackenberger,2 Davorka Jaric,2 Sandra Stepic2

1Center for Marine and Environmental Research, Rudjer Boskovic Institute, Zagreb, Croatia

2Department of Biology, J. J. Strossmayer University, Osijek, Croatia

Received 26 November 2009; accepted 16 April 2010

ABSTRACT: Induction of Mixed Function Oxidase (MFO) activity and bioactivation potential were measuredin experimental carp and in native fish species from two rivers with different pollution level (Sava and Mrezn-ica). The experimental carp were intraperitoneally exposed to various water volume equivalents of Blue Cottonextracts from both rivers. The induction of MFOmeasured as a Benzo(a)pyrene monooxygenase (BaPMO) ac-tivity was increased up to 9.3-fold in experimental carp and up to 11.3-fold in native fish from Sava River,whereas the values from Mreznica River showed only a slight increase when compared with the control(highest increase of 1.8-fold in nose carp). Accordingly, bioactivation potential using modified Ames test washigher in both experiments with Sava River. Both measured parameters in experimental carp increased in adose-dependent manner in accordance to river volume equivalents. Different induction potential of native fishspecies comparable between two rivers confirmed the known possibility of their usage in biomonitoring stud-ies. These results give qualitatively a new support to the idea of using Blue Cotton extraction technique com-bined with induction of MFO activity and Ames test in fish as a good biomarker for assessing risk of exposureto mutagens/carcinogens such as Polycyclic Aromatic Hydrocarbons (PAHs), especially due to the simplicityand cost-effectiveness of these methods. # 2010 Wiley Periodicals, Inc. Environ Toxicol 27: 146–154, 2012.

Keywords: Ames test; benzo(a)pyrene monooxygenase activity; Blue Cotton; fish; MFO; bioactivationpotential

INTRODUCTION

Majority of surface waters receive large quantities of waste

water from various sources: industrial, agricultural, and

domestic. As those waters are further used for different pur-

poses they can become a serious public health and aquatic

ecosystem problem. Many compounds detected in those

wastewaters are known mutagenic or genotoxic carcinogens

(Ohe et al., 2004). Consequently, those compounds become

the components of complex environmental mixtures that

can have adverse health effects on humans and indigenous

biota (Dearfield et al., 2002). In numerous studies, munici-

pal river water mutagenicity was reported with its character

derived from PAHs including nitro- and amino-derivates

(Sakamoto and Hayatsu, 1990; Nagai et al., 2002; Umbu-

zeiro et al., 2004).

The effective concentrations of mutagens in water can

very often be below the detection limit of analytical instru-

ments. If the bioaccumulation index of majority of muta-

gens is taken under consideration, which is quite high, then

an importance of small quantities of mutagens detection in

the waters is understandable. Thus, in such research water

samples must be concentrated and proper extraction is of

great importance for both the chemical analysis and Ames

test. In many mutagenicity investigations, as a preconcen-

tration methods XAD-2 and XAD-7 resins, Blue Cotton,

Blue Rayon, and Blue Chitin have been used (Hayatsu

et al., 1983; Sayato and Hayatsu, 1990; Nagai et al., 2002;

Correspondence to: B.K. Hackenberger; e-mail: hack@biologija.unios.hr

Published online 1 June 2010 in Wiley Online Library

(wileyonlinelibrary.com). DOI 10.1002/tox.20625

�C 2010 Wiley Periodicals, Inc.

146

Umbuzeiro et al., 2004). Blue pigment has a ligand linked

copper phthalocyanine trisulphonate and is capable of

adsorbing aromatic compounds such as PAHs and heterocy-

clic amines (HCA) with two or more fused rings. The

adsorption capacity is strong, and the selectivity for adsorb-

ing this class of compound is high (Kobayashi and Hayatsu,

1984; Hayatsu et al., 1985). Additionally, the methods with

blue pigment show recoveries comparable to or better than

existing solid phase extraction methods, and are faster and

simpler than most of them (Skog, 2004). Therefore, they

have been extensively used in screening fresh and marine

water for the presence of mutagenic compounds (Kurelec

and Krca, 1989; Sakamoto and Hayatsu, 1990; Hayatsu,

1992; Sayato et al., 1993).

Besides the development of effective, economic, and

selective passive sampling techniques, a need for techni-

ques which fulfill the demands of monitoring trace and

ultra-trace bioavailable organic contaminants in water and

at the same time bridge the gaps between environmental an-

alytical chemistry and ecotoxicology approaches exists (Lu

et al., 2002).The coupling of bioassay assessments with

selective sampling technique yields more information than

either of these assessments individually.

The Ames mutagenicity test, with and without metabolic

activation by S9 mixture, has been frequently coupled with

blue pigment extraction technique for the detection of

mutagens in surface waters (White and Rasmussen, 1998;

Kataoka et al., 2000; Watanabe et al., 2002; Ohe et al.,

2003; Umbuzeiro et al., 2004) and drinking waters

(Schenck et al., 1998; Monarca et al., 2002) throughout the

world. As property of blue pigment is an adsorption of sub-

stances with planar polycyclic molecular structure, an

induction of cytochrome P450-dependent monooxygenase

(MFO) system in fish liver as a biomarker and bioindicator

of exposure to anthropogenic organic contaminants such as

PAHs, Polychlorinated Biphenyls (PCBs), several pesti-

cides and dioxins/furans could additionally be used (Buhler

and Wang-Buhler, 1998; Parrott et al., 2000). This can be

obtained with MFO-induct test, i.e., measurement of ben-

zo(a)pyrene monooxygenase (BaPMO) induction in the

carp liver after intraperitoneal (i.p.) application of sampled

water extracts (Kurelec et al., 1979). Previous studies were

conducted using hexane, XAD-2 or XAD-7 extracts of pol-

luted fresh and marine water samples, or dichloromethane

extracts of soil and waste from the municipal landfill. Those

extracts were i.p. injected into juvenile carp (Cyprinus car-pio) with consecutive measurement of MFO activity induc-

tion (Kurelec et al., 1982; Rijavec et al., 1981). The MFO

induct-test has been used in combination with the Ames

test (Kurelec et al., 1979, 1981) using liver homogenates

from carps i.p. injected with 3-methylcholanthrene (Protic-

Sabljic and Kurelec, 1983; Britvic et al., 1993).

The primary objective of this research was to investigate

the potential usage of the Blue Cotton adsorption technique

as the method for detection and concentration of MFO-

inducing lipophilic xenobiotics and premutagens in envi-

ronmental monitoring. For this purpose, two experiments

were conducted: (i) a laboratory experiment with juvenile

carp intraperitoneally exposed to Blue Cotton extracts

(MFO inducting experiment) and (ii) a field experiment

with native fish specimens from two investigated rivers

(Sava and Mreznica). Fish hepatic S9 was used to measure

MFO induction as the BaPMO activity and bioactivation

potential as the Ames-microsomal test in both experiments.

Additionally, BaPMO activity in S9 from juvenile carp was

measured with addition of a-naphtoflavone (ANF), a well

known inhibitor of aromatic hydrocarbon-inducible forms

of cytochrome P450.

MATERIALS AND METHODS

Sampling Sites

A volume of 20 L of water was collected on five adjacent

sublocations from Sava River at the power plant Zagreb II

cooling water pumping station and from Mreznica River

upstream from the city of Karlovac. A total of 100 L was

collected from each river. The quality of water from each

of these rivers was defined by its load of industrial and

domestic waste, expressed in population equivalents (p.e.)

(Sava river 2,400,000 p.e. and Mreznica river 2000 p.e.)

and according to physicochemical parameters (Sava river

III-V category and Mreznica river I-II category) (Vouk and

Malus, 2005; Environmental Health 1993). Recently,

Grung et al. (2007) identified several PAHs (alkylated

naphtalenes, acenaphtene, dibenzofuran, fluorine, alkylated

fluorine, and phenanthrene/anthracene) using reverse phase

HPLC fractionation technique in the wastewater effluent

from Zagreb which are released in the Sava River. The

water samples were transported to the laboratory and

extracted within 24 h with the Blue Cotton technique.

Experimental Organisms

For MFO inducting experiment, carp (Cyprinus carpio) 1year old, sexually immature, weighing 22 6 5 g were

obtained from the fish farm Draganici and were kept for up

to 3 months in laboratory basins with a flow of dechlori-

nated, well aerated tap water.

Native fish from Sava and Mreznica rivers were caught

by angling. Fish species caught were: Danubian roach

(Rutilus pigus virgo)(nSava 5 8, nMreznica 5 6), roach

(Rutilus rutilus)(nSava 5 6, nMreznica 5 5), chub (Leuciscuscephalus)(nSava 5 7, nMreznica 5 6), nose carp (Chondros-toma nasus)(nSava 5 8, nMreznica 5 5), and bleak (Alburnusalburnus)(nSava 5 8, nMreznica 5 6). All fish specimens used

in the experiment were sexually immature. The liver was sep-

arated immediately after the catch and transported in liquid

nitrogen to the laboratory and stored at2808C until analysis.

147BLUE COTTON COUPLED WITH MFO INDUCTION AND AMES TEST

Environmental Toxicology DOI 10.1002/tox

The livers of both native fish and carp were used for mea-

surement of BaPMO activity and for the preparation of S9

fraction in the Ames Salmonella assay. Livers were homoge-

nized with Potter-Elvehjem homogenizer (Sartorius, Goetin-

gen, Germany) in sucrose buffer (0.05 M Tris, 0.25 M sucrose

in 1% KCl, pH 5 7.6) at a tissue weight/buffer volume ratio

of 1:5, centrifuged at 90003 g for 20 min at 48C (S9 fraction)

and resulting homogenates were stored at2808C until use.

Preparation of Blue Cotton WaterConcentrates

Blue Cotton fibers were washed and tested for mutagenicity

activity with the TA100 strain and when the extracts

showed negative results they were used in the experiment

(Kummrow et al., 2003).

Water samples from Sava and Mreznica rivers were

passed through a set of five glass columns (200 mm 3 20

mm) with 2 g Blue Cotton each (a total of 10 g of Blue Cot-

ton per sample) at a flow rate of 70 mL/min at room tem-

perature. Afterwards, the Blue Cotton was carefully

removed from columns and washed with 50 mL of distilled

water per gram in order to remove the nonadsorbed com-

pounds and solids. The water was removed with aspiration

and the residual water was wiped out with a paper towels.

Then the adsorbed materials were extracted by gently shak-

ing the Blue Cotton in 50 mL/g Blue Cotton of methanol-

concentrated ammonia (50:1, v/v) for 1 h (Kira et al.,

1989). The eluants were combined and reduced to the vol-

ume of 6 mL in a rotary evaporator and further used for i.p.

application in carp (MFO inducting experiment).

Measurement of BaPMO Activity

In a MFO inducting experiment, an aliquot of the Blue Cot-

ton extract (equivalent to 1, 3, and 6 L of river water) was

evaporated to dryness and redissolved in 4.0 mL of corn

oil. The extracts prepared in this way were i.p. applicated to

experimental carp on a body weight basis (20 mL/kg). For

a positive control carp were i.p treated with 50 mg/kg of

3-methylcholanthrene (3-MC) and for the negative control

only corn oil was administered. Seven specimens were used

per treatment (C1, C2, 1, 3, and 6 L equivalents); all treat-

ments were done in triplicate. After 3 days, carp were sacri-

ficed and liver was prepared for determination of BaPMO ac-

tivity by measuring 3-hydroxy-benzo(a)pyrene (3-OHBaP)

concentration. BaPMO activity was determined in liver post-

mitochondrial fractions (S9) according to the method of

Nebert and Gelboin (1968). The inhibitory effect on the

BaPMO activity was measured in each sample in parallel af-

ter addition of 1024 M ANF in the reaction mixture. The

activities were expressed as pmol of BaPOH per mg of pro-

tein per min. Proteins were determined according to the

method of Lowry et al. (1951).

The livers from native fish specimens of both rivers

were extracted and S9 fraction was prepared (at least five

specimens per species). BaPMO activity was determined in

liver postmitochondrial fractions (S9) according to the

method of Nebert and Gelboin (1968). The activities were

expressed as pmol of BaPOH per mg of protein per min.

Proteins were determined according to the method of

Lowry et al. (1951).. For the negative control intact experi-

mental carp was used.

Ames Test

The potential of liver postmitochondrial fractions (S9) from

the experimental carp induced with i.p. injection of Blue

Cotton extracts or from natural fish specimens for bioacti-

vation of precarcinogen BaP was measured with a modified

Ames test (Britvic and Kurelec, 1986). Bioactivation poten-

tial was expressed as the net increase of the number of

revertants activated from BaP (10 lg/plate) with S9 (2 mg/

protein/plate) over the spontaneous number (125 6 6) of

Salmonella typhimurium TA100 revertants. Plates were

incubated upside down at 378C and colonies (His1 rever-

tants) were counted with automatic colony counter after 48

h. Each sample was tested in triplicate and all data were

presented as averages of revertants numbers. As positive

controls, methyl methanesulphonate (MMS) 625 lg/platewas used to monitor the sensitivity of the bacterial strain

TA100 and BaP 10 lg/plate for the activity of carp liver

S9. Additionally, for a positive control carp were i.p treated

with 50 mg/kg of 3-methylcholanthrene (3-MC) and for the

negative control only corn oil was administered. There was

no positive and negative control for native fish species. Val-

ues of mutagenicity ratio (MR) were obtained by dividing

the number of revertant colonies in sample plates by the

spontaneous number of revertants. A MR [2 with a clear

dose response was defined as mutagenic, whereas a MR\2

was defined as a lack of mutagenicity.

Statistical Analysis

Prior to comparison among various groups of fish and corre-

lation analysis, the data were tested for normality (Shapiro-

Wilk and Kolmogorov-Smirov tests) and homogeinity of

variance (Levene’s test). The BaPMO activities were trans-

formed using the logarithmic function f(x) 5 log(1 1 x).The Ames test results (number of revertants) were trans-

formed using the square root function f(x) 5 Hx. Thesetransformations resulted in homogenous variance and nor-

mal distribution of data enabling the usage of parametric

statistical methods. For comparison of groups treated with

different equivalent of water, single-way analysis followed

by Dunnet’s or Tukey’s posttest was used. Kruskal-Wallis

followed by Dunn’s posttest and Spearman rank correlation

yielded similar results as parametric methods.

148 BRITVIC ET AL.

Environmental Toxicology DOI 10.1002/tox

RESULTS

To investigate the potential usage of Blue Cotton extraction

method for measurement of MFO induction and bioactiva-

tion potential in river monitoring research several experi-

ments were conducted.

BaPMO Activity in Experimental Carp

Results of the MFO inducting experiment showed that the

xenobiotics extracted from the Sava River induced BaPMO

activity in experimental carp in a dose-dependent manner,

according to the river water volume equivalent [Fig. 1(a)].

Namely, the amount of xenobiotics in 1 L, 3 L, and 6 L of

extract equivalents from the Sava River caused 3.9, 5.3,

and 9.3-fold increase of the BaPMO activity (12.4 6 2.3,

16.8 6 4.1, and 29.9 6 1.8 pmol BaPOHmgprot21min21,

respectively) when compared with the basal BaPMO activ-

ity (3.2 6 0.3 pmol BaPOHmgprot21 min21), i.e., negative con-

trol. Those values expressed as 3-MC toxicity equivalents

would correspond to activities induced with i.p. injection of

19.3 mg/kg, 26.2 mg/kg, and 46.6 mg/kg 3-MC, respectively.

The increase obtained with 6 L river water equivalent was

close to maximum induction obtained with 50 mg kg21 of

3-MC (32.1 6 3.8 pmol BaPOHmgprot21 min21), i.e., positive

control. In contrast, Blue Cotton water extracts from Mreznica

River, as expected, did not significantly induce BaPMO activ-

ity in experimental carp. The highest induction of only

1.2-fold over control (4.5 6 0.3 pmol BaPOHmgprot21 min21)

was obtained with 6 L river water volume equivalent. The

other two water volume equivalents did not show statistically

significant difference between themselves or with the control

(P\0.05).

In the experiment with ANF, the induction of BaPMO

with extracts from Sava River decreased significantly [Fig.

1(b)]. Obviously, highest difference, when compared with

the previous experiment, was with the 6 L extract of both

rivers showing that when larger volumes of water are

extracted the quantity of MFO inducers increased. Even

though, no statistically significant difference was observed

between 3 and 6 L extract of Sava River and positive con-

trol, and with all three extract quantities of Mreznica River

and negative control. Six liter water extract from the Sava

River (9.3 6 0.8 pmol BaPOHmgprot21min21) elicited

stronger BaPMO induction than the positive control (8.3 62.2 pmol BaPOHmgprot

21 min21).

BaPMO Activity in Native Fish

Apart from laboratory, a field experiment with native fish

populations from both rivers was conducted. In all fish spe-

cies native to polluted Sava River, when compared to

untreated juvenile carp used as a negative control, an

increase in BaPMO activity was measured (from 1.3

to 11.3-fold) (Fig. 2). Simultaneously, of all fish species

native to the Mreznica River only nose carp showed a slight

increase in BaPMO activity when compared to control (1.8-

fold). Interestingly, this species showed the highest induction

of BaPMO activity in both rivers (11.3 and 1.8-fold) (Fig. 3).

Fig. 1. BaPMO activities in carp i.p. induced with Blue Cot-ton river water volume extracts from Sava River ( ) 1 L, ( )3 L, and ( ) 6 L and Mreznica River ( ) 1 L, ( ) 3 L, and ( ) 6L, and BaPMO activities for positive control (i.p. inductionwith 3-MC) ( ) and negative control (only corn oil) ( ), (A)without addition of ANF and (B) with addition of ANF. Statis-tically significant differences are compared to negative con-trol: P\0.05 (*), P\0.01 (**) and P\0.001 (***).

Fig. 2. BaPMO activities in native fish specimens caught inSava ( ) and Mreznica ( ) River.

149BLUE COTTON COUPLED WITH MFO INDUCTION AND AMES TEST

Environmental Toxicology DOI 10.1002/tox

From the fish species caught in the Sava River lowest

BaPMO activity was recorded in Danubian roach (4.1 6 0.4

pmol BaPOHmgprot21 min21). From the Mreznica River, except

of nose carp, all tested fish species had similar BaPMO activ-

ities (from 2 6 0.3 pmol BaPOHmgprot21 min21 to 2.7 6 0.6

pmol BaPOHmgprot21 min21) and they were all lower than the

negative control (3.26 0.3 pmol BaPOHmgprot21 min21).

Ames Test

The increase of the potential for bioactivation of BaP to S.typhimurium TA100 mutagens was proportional with the

increase of river water volume equivalent, i.e. the increase

was dose dependent, for both rivers (Table I). The only two

samples that were not statistically significant (P \ 0.05)

when compared with control were 1 L and 3 L extracts

from Mreznica River, as the mean number of revertants

was 137 6 3 and 138 6 7, respectively. Postmitochondrial

fractions (S9) of carp liver pretreated with volume extracts

(1, 3, and 6 L) from Sava River increased the bioactivation

potential of BaP to TA100 mutagens and calculated muta-

genicity ratio (MR) was 3.0, 5.0, and 9.0, respectively. At

the same time, S9 from carps induced with Blue Cotton

extracts from Mreznica River did not induce liver potential

to bioactivate BaP to bacterial mutagens using TA100

strain, the highest MR (1.5) was calculated for the 6 L

equivalent (Table I). Postmitochondrial fractions (S9) from

native fish species generated similar bioactivation as S9

from Blue Cotton induced carp (Table II). Namely, none of

the fish species from Mreznica River increased the potential

for bioactivation of BaP to S. typhimurium TA100 muta-

gens, and the number of revertants after test was more or

less the same as the spontaneous number of revertants. On

the other hand, S9 from most of the Sava River fish species

increased the bioactivation potential of BaP to S. typhimu-rium TA100 mutagens. Only Danubian roach had calcu-

lated MR below 2. The MR of other tested species ranged

from 2.8 to 7.4.

TABLE I. Results of the Ames test using Salmonella TA100 strain for the carp liver S9 previously induced with differentBlue Cotton river water volume equivalents from Sava and Mreznica rivers

Mean Number of Revertants/Plate, Standard Deviation (SD), and Mutagenic Ratio (MR)

DoseSava Mreznica 1 Con 2 Con MMS (2S9) BaP (1S9)

RWE/l Mean SD MR Mean SD MR Mean SD Mean SD Mean SD Mean SD

1 381 9 3.0 137 3 1.1 1085 23 125 6 2000 16 1025 14

3 631 12 5.0 138 7 1.1

6 1125 26 9.0 186 4 1.5

Fig. 3. Correlation between number of revertants in Ames test and BaPMO activity innative fish species from (A) Sava and (B) Mreznica River.

150 BRITVIC ET AL.

Environmental Toxicology DOI 10.1002/tox

DISCUSSION

In the past few decades, many researches have been con-

ducted to assess chemical pollution and consequent poten-

tial ecological hazard of surface freshwaters such as rivers

and lakes. The need for an appropriate and efficient bioas-

say or a set of tests for evaluation of surface waters on

potential hazard to human and the water environment was

the main actuator of this researches. Although, many mod-

ern sophisticated techniques has been developed the need

for a cost-effective methods should not be neglected. In this

work we investigated further potential and efficacy of two

well known tests, MFO inducting experiment and Ames

test, in combination with Blue Cotton extraction technique

to assess the possible potential mutagenicity of two rivers.

Similarly to carp exposed to Blue Cotton extracts, exposure

of carp to a flow of polluted Sava river water for 29 days

resulted in rapid 9.5-fold increase in the BaPMO activity

when compared to untreated carp (Britvic et al., 1993).

Simultaneously, BaPMO in Blue Cotton extract induced

liver was measured with the addition of ANF, a specific in-

hibitor for fish CYP1A (Stegeman and Hahn, 1994). ANF

is known to inhibit CYP1A activity in rainbow trout (Taka-

hashi et al., 1995; Hodson et al., 2007) and in cultured fish

hepatocytes (Manik et al., 2000). In this work, ANF has

decreased measured BaPMO activity in a dose-dependent

manner. The stronger BaPMO induction with 6 L water

extract from the Sava River than the induction elicited with

3-MC in positive control could be explained with the sur-

plus of PAHs causing the induction kinetics to override the

ANF inhibitory effect, or possibly the AhR agonists present

in the complex mixture of river water may have caused syn-

ergism (Basu et al., 2001; Grung et al., 2007). The inducing

potency of a complex mixture of 16 PAHs can be about 2-

fold higher than predicted from their individual induction

equivalency factors (Till et al., 1999). The induction of

BaPMO activity from natural fish specimens from both riv-

ers showed the correlation with the induction of carp

exposed to Blue Cotton extracts. Namely, highest induction

in fish from Sava River was 11.3-fold, and from Mreznica

River 1.8-fold when compared to control. While an

enhanced BaPMO activity in fish from Sava River may

indicate the presence of unknown xenobiotics causing pro-

found pathophysiological and molecular responses, values

from Mreznica River fish could as well be the normal val-

ues of MFO activity in all native fish species living in

unpolluted waters (Britvic et al., 1993) and are suggesting

the absence of xenobiotics with the BaPMO-inducing prop-

erty. Indeed, previous study of BaPMO activity in the popu-

lation of fish living in the polluted Sava river and fish living

in two less polluted Kupa and Krka rivers, showed high

correlation between the level of pollution by communal and

industrial waste and the level of BaPMO induction (Kezic

et al., 1983).

Furthermore, various fish species revealed different lev-

els of BaPMO induction. This species differences in the

magnitude of the response may be influenced by both bio-

logical variables (sex, age, spawning status, size, and

genetic strain) and habitat variables (temperature, pH, and

water flow) (Stegeman and Chevion, 1980; Koivusaari

et al., 1981; Luxon et al., 1987; Lindstrom-Seppa, 1985;

Pedersen et al., 1976; Jimenez and Burtis, 1989). Addition-

ally, large species differences in the induction measured

during field studies may be due to variations in exposure,

accumulation or sensitivity to inducers (Fuchsman et al.,

1999). In this study none of the investigated fish species

where exclusive carnivores or herbivores, their calculated

trophic level ranged from 2.0 6 0.00 (nose carp) to 3.03 60.46 (chub) (Froese and Pauly, 2000). Hence, no unambigu-

ous conclusions could be made from the MFO induction

and species feeding habits or contact with sediments. All

aforementioned leads to large variations in induction capa-

bilities of MFO enzymes in various fish species using very

similar protocols and inducing agents (Kloepper-Sams and

Benton, 1994; Focardi et al., 1995; Smith and Gagnon,

1998). A nose carp (Chondrostoma nasus) had the highest

level of BaPMO activity in both Sava (36.2 6 4.2

BaPOHmgprot21 min21) and Mreznica (5.8 6 0.4

BaPOHmgprot21 min21) rivers (Fig. 2). Previous studies on

this species from Sava River also showed high BaPMO

activities at different sampling sites; with the highest being

69.7 6 9.8 BaPOHmgprot21 min21 measured in liver S9 from

nose carp (Britvic and Kurelec, 1999). The largest differ-

ence between rivers was observed in chub as BaPMO activ-

ity was even 15.5 times higher in the Sava River. However,

in the study of Krca et al. (2007), where a set of biomarkers

TABLE II. Results of the Ames test using Salmonella TA100 strain for the native fish species S9 from Sava and Mrezn-ica rivers

Fish Species

Sava Mreznica MMS (2S9) BaP (1S9)

Mean SD MR Mean SD MR Mean SD Mean SD

Danubian roach 126 4 1 116 2 0.9 2000 16 2000 16

Roach 356 12 2.8 118 2 0.9

Bleak 468 8 3.7 122 4 1

Chub 868 6 6.9 120 6 1

Nose carp 926 16 7.4 124 8 1

151BLUE COTTON COUPLED WITH MFO INDUCTION AND AMES TEST

Environmental Toxicology DOI 10.1002/tox

were measured in chub, this species was found to be a valu-

able indicator of environmental pollution. A correlation

was found for the results of Ames test and BaPMO mea-

surement in native fish specimens from Sava River (R 50.96) (Fig. 3). When liver S9 from native fish specimens

was used in the Ames test, only Danubian roach from Sava

River increased its potential to bioactivate BaP less than a

twofold. And this species also had the lowest BaPMO activ-

ity induction in the MFO inducting experiment. On the

other hand, no correlation was found for the results from

Mreznica River (R 5 0.62) (Fig. 3). All tested fish species

from the Mreznica River had MR around one, having

almost the same number of revertants as the number of

spontaneous revertants. These results are in accordance

with previously conducted experiments with native fish

specimens from Sava and Korana Rivers (Britvic et al.,

1993). In the experiment with carps the number of revertants

increased proportionally with the volume of river water in

the Blue Cotton extract from Sava River, and from Mreznica

River the number of revertants increased only with 6 L

extract. The liver potential to bioactivate BaP using the TA

100 strain was increased fourfold in the experiment with

carp exposed to Sava River water for 29 days (Britvic et al.,

1993). All three calculated MR’s for Sava River were higher

than two (3, 5, and 9, respectively). Even though the MR

from two separate experiments testing Sava River water

were greater than two, suggesting the positive mutagenic

activity (Fujimoto et al., 2007; Umbuzeiro et al., 2007), other

experiments with 32P-postlabeling analyses of DNA adducts

in chub, bream and barbel failed to show any pollution-

related DNA adducts (Kurelec et al., 1989).

CONCLUSIONS

The results of this investigation demonstrate that the inte-

gration of used bioassays with Blue Cotton extraction tech-

nique may provide an effective water monitoring tool. Even

though this experiment does not represent environmentally

realistic exposure scenario, measured biological effects

show that the extracted substance that caused the effect

would certainly caused it in such a scenario. This combina-

tion of extraction and biological endpoints can affect which

analytical techniques will be used for chemical analysis

and, consequently, the cost of preparation of fraction for

testing. The investigations and existence of cost-effective

methods for determination of wastewater mutagenic poten-

tial are important particularly for the developing countries.

Additionally, as in ecotoxicological studies it is essential

to assess the toxic response of indigenous fauna as indicator

species or sentinels of environmental contamination this

experiment demonstrated that the MFO-sensitivity, i.e.,

inducibility, is species-dependent.

From the results of this experiment it is evident that the

inducibility gradation was equal in the same species from

differently polluted sites (Fig. 3). Consequently, it implies

the possibility of freshwater monitoring of mutagenic com-

pounds in the sense of MFO induction comparison between

tested and reference location.

Finally, since the drinking water supply in the Sava ba-

sin relies almost exclusively on the rich resources of high-

quality groundwater, which are under the direct influence

of the Sava River (Kallqvist et al., 2008) it is important to

have easily conductible, reliable and interpretable biomoni-

toring methods.

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