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400

500

Minho Lima Douro

EROD Activity

(% of reference value)

100

150

200

Minho Lima Douro

GST Activity

(% of reference value)

100

150

Minho Lima Douro

BaP-type Metabolites

(% of reference value)

100

150

200

250

Minho Lima Douro

BaP-type Metabolites

(% of reference value)

Fig. 30.1 Liver EROD activity (A), liver GST activity (B), and BaP-type metabolites in the bile

Douro (contaminated sites). Results are presented as the percentage of reference site mean levels

(EROD: 39.3 ± 11.1 pmol/min/mg protein; GST: 112.5 ± 7.8 nmol/min/mg protein; BaP-type

metabolites in the bile: 74.3 ± 14.1 ng BaP/mg protein; BaP-type metabolites in the liver: 21.0 ±

1.6 ng BaP/mg protein) with corresponding S.E.M. bars. Different characters indicate statistical

significant differences among sampling sites as indicated by Tukey honestly significant difference

multiple-comparison test (p < 0.05) for each parameter

100

150

Minho LimaD ouro

GSH/GSSG

100

150

Minho Lima Douro

Oxidized Glutathione

(% of reference value)

100

150

200

Minho Lima Douro

GSH

(% of reference value)

100

150

200

Minho Lima Douro

Total Glutathione

(% of reference value)

Fig. 30.2 Liver TG (A), GSH (B), GSSG (C) and GSH/GSSG ratio (D) determined in eels from

the estuaries of rivers Minho (reference site), Lima and Douro (contaminated sites). Results are

presented as the percentage of reference site mean levels (TG: 0.88 ± 0.04 nmol/min/mg protein;

GSH: 0.34 ± 0.03 nmol/min/mg protein; GSSG: 0.25 ± 0.01 nmol/min/mg protein; GSH/GSSG:

1.53 ± 0.18) with corresponding S.E.M. bars. Different characters indicate statistical significant

differences among sampling sites as indicated by Tukey honestly significant difference multiple-

comparison test (p < 0.05) for each parameter

30 Biomonitoring Studies Performed with European Eel Populations 395

100

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Minho Lima Douro

GR Activity

(% of reference value)

100

150

200

Minho Lima Douro

CAT Activity

(of reference value)

100

150

200

Minho Lima Douro

SOD Activity

(% of reference value)

a,b

100

150

200

Minho Lima Douro

GPx Activity

(% of reference value)

Fig. 30.3 Liver SOD (A), CAT (B), GPx (C) and GR (D) activities determined in eels from the

estuaries of rivers Minho (reference site), Lima and Douro (contaminated sites). Results are

presented as the percentage of reference site mean levels (SOD: 1.1 ± 0.1 U/mg protein; CAT:

16.8 ± 0.8 µmol/min/mg protein; GPx: 1.8 ± 0.2 nmol/min/mg protein; GR: 3.8 ± 0.3 nmol/min/mg

protein) with corresponding S.E.M. bars. Different characters indicate statistical significant

differences among sampling sites as indicated by Tukey honestly significant difference multiple-

comparison test (p < 0.05) for each parameter

SOD activity was significantly higher (~32%) in the liver of eels collected in

the Lima estuary than in eels from the estuary of Minho River (Fig. 30.3A). Liver

CAT (~32%) and GPx (~33%) activities were significantly higher in eels from

the Douro estuary than in eels collected in the estuaries of Lima and Minho

Rivers (Fig. 30.3B, C). Although not significant, the eels from Douro estuary

showed a slightly higher GR activity (~12%) than eels from Lima and Minho

estuaries (Fig. 30.3D).

The oxidative damage, assessed as LPO, was significantly higher in the liver

of eels from the estuary of Lima River than in the liver of eels collected in the

estuaries of Minho and Douro Rivers (Fig. 30.4). Moreover, LPO was significantly

lower in the liver of eels from the estuary of the Douro River than in eels from

the estuary of Minho River (Fig. 30.4).

30.4 Discussion

In the present study, induced EROD activity was found in eels from the estuary

of Douro River that is polluted with xenobiotics both from urban and industrial

sources (Ferreira et al. 2004, 2006), therefore, in good agreement with previous

studies reporting an induced liver EROD activity in mullets from this estuary

396 C. Gravato et al.

(Ferreira et al. 2004, 2006). EROD activity increase was previously observed in sev-

eral fish species after exposure to organic pollutants, such as PAHs, causing very

strong increases in CYP1A catalytic activities (Van der Oost et al. 2003). The present

study demonstrated not only a significant liver EROD induction in eels from the

Douro estuary, but also a significant increase in liver GST activity. This phase II

biotransformation induction in eels was associated with low BaP–type metabolites in

bile and liver of eels from Douro suggesting that chemicals other than PAHs are

responsible for the induction found in eels from this estuary.

Previous studies showed that the hepatic cytochrome P450 1A activity was

inversely related to egg viability, fertilization success and to the successful

development from fertilization through hatching, demonstrating that this biotrans-

formation activity interfere with the ability to regulate sex steroids (Spies et al.

1988). Therefore, it may be hypothesized that EROD induction found in eels from

Douro River estuary may have negative effects on sexual maturation of the indi-

viduals. In addition, eels from this estuary showed high CAT, GPx, TG, GSH and

GSSG levels that were associated with low LPO and B(a)P-type metabolites either

in the bile or in the liver. High liver biotransformation and antioxidant capacity in

eels from the estuary of the Douro River might be important to their adaptation to

the presence of contaminants in this estuary. It has been proposed that animals

inhabiting polluted environments can develop some adaptations or compensatory

mechanisms (Regoli and Principato 1995). Chronically intoxicated fish can, for

example, reduce the inflow of toxicants by building up morphological barriers and

they may activate mechanisms of detoxification (Lindström-Seppä and Pesonen

1986; Andersson et al. 1988), which make them more resistant to toxicants (Bucher

and Hofer 1990). However, the mechanisms used to face chemical stress require

100

150

Minho Lima Douro

LPO

(% of reference value)

Fig. 30.4 Liver LPO determined in eels from the estuaries of rivers Minho (reference site), Lima

and Douro (contaminated sites). Results are presented as the percentage of reference site mean

levels (LPO: 92.4 ± 3.0 nmol/g wt) with corresponding S.E.M. bars. Different characters indicate

statistical significant differences among sampling sites as indicated by Tukey honestly significant

difference multiple-comparison test (p < 0.05) for each parameter

30 Biomonitoring Studies Performed with European Eel Populations 397

energy that could have been invested in growth and weight increase, factors that are

determinant for the success of the long migration until the reproduction area

(Sargasso Sea).

Eels from the estuary of Lima River showed low liver TG, GSH, GSH/GSSG

and LPO levels associated with high B(a)P-type metabolites liver concentrations.

These results suggest an impairment of their detoxification and anti-oxidative

defenses and the occurrence of lipid peroxidation. The low levels of GSH found

may be related with the low EROD activity found in these animals. The consumption

of GSH due to the scavenging of oxyradicals or as a cofactor (GPx and GST

activities) represents a drain on intracellular reducing equivalents (Winston and Di

Giulio 1991). The tissue thiol status modulates CYP 1A gene expression and

catalytic activity (Otto et al. 1997), suggesting that the low liver EROD activity

observed in eels from the estuary of Lima river can be a consequence of GSH

depletion. Overall, these results indicate that eels from Lima River estuary are

under oxidative stress probably due to long-term PAHs exposure. Fish vitellogenesis

can be affected by pollutants with know affinity for the estrogenic receptor, such as

PAHs (Nicolas 1998). Since, eels from Lima estuary are exposed to PAHs as indi-

cated by the high levels of liver bile metabolites found, negative effects on sexual

development may be expected. In addition, oxidative stress may also interfere with

the health condition of the individuals and, thus, to decrease their probability of

survive during the long migration to reproduce. In fact, previous studies

demonstrated clear relationships between biochemical alterations induced by

environmental pollution (PAHs) and fish diseases, such as preneoplastic hepatic

lesions, hepatic neoplasms and skin diseases (Myers et al. 1994; Vethaak et al.

1996). Winston and Di Giulio (1991) observed elevated rates of idiopathic lesions

and neoplasia among fish inhabiting polluted environments that were related to the

increased oxidative stress associated with pollutant exposure.

In conclusion, the findings of this study suggest that the exposure of eels to pollu-

tion during their development in the estuaries of Lima and Douro Rivers is inducing

changes on biomarkers involved in physiological functions determinant for the

survival and performance of the eels, namely, biotransformation enzymes and anti-

oxidative stress defences. Studies from several authors suggest that these alterations

may have negative effects on sexual development. In addition, mechanisms used to

face chemical stress need energy that is probably allocated from other functions such

as tissue repair, growth and weight increase which are determinant for a successful

migration until the reproduction area. Therefore, exposure to pollution due to their

continental phase may reduce the contribution of Lima and Douro eel populations for

the species evolution. More studies are required to relate the alterations found in

biomarkers with possible effects on reproductive parameters and health condition of

the animals.

Acknowledgements The authors express their gratitude to Dr. C. Antunes, A. Oliveira and to I.

Cunha for their help during the capture of the eels. This study was funded by “Fundação para a

Cíência e a Tecnologia” of Portugal (FCT) (project EELEANORA, POCTI/BSE/47918/2002;

Post-Doc grant to C. Gravato, POCTI/BPD/21070/2004) and by EU FEDER funds.

398 C. Gravato et al.

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Chapter 31

In Vitro Testing of Inhalable Fly Ash

at the Air Liquid Interface

Sonja Mülhopt

, Hanns-Rudolf Paur

, Silvia Diabaté

, and Harald F. Krug

Abstract The aim of this study is to analyse the toxicological potential of fine and

ultrafine particles from industrial combustion processes using a biotest. This biotest

is performed by near-realistic exposure of cultivated lung cells at the air-liquid

interface and analysing the biological responses. Important steps in this work are

to develop the exposure system for the use at industrial particle sources, to provide

reproducible deposition conditions for submicron particles and to validate the

exposure protocol for the bioassay. The presented technique maintains the viability

of the cells but is sensitive for inflammatory effects. Exposure experiments with

the ultrafine fraction of fly ash from a municipal waste incinerator have shown an

increased release of IL-8 as a function of exposure time and dose. The presented

exposure method and the lung specific bioassay seem to be an appropriate model

to simulate the inhalation of particulate air pollution and to screen the biological

effects of particulate emissions from different sources.

Keywords: Aerosol, bioassay, PM10, toxicity, ultrafine particles

31.1 Introduction

Particle emissions from industry and traffic are important sources of anthropogenic

airborne particles in the environment. In recent years epidemiological studies

have shown that there is an association between the concentration of fine parti-

cles (PM

, PM

2.5

) in the atmosphere and the rate of mortality or morbidity due to

respiratory and cardiovascular diseases (Peters et al., 1997; Wichmann et al.,

Forschungszentrum Karlsruhe, Institute for Technical Chemistry, Thermal Waste Treatment

Division, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein – Leopoldshafen, Germany;

Tel: + 49 7247 82 3807, Fax: + 49 7247 82 4332

Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, Hermann-von-

Helmholtz-Platz 1, 76344 Eggenstein – Leopoldshafen, Germany

402

Y.J. Kim and U. Platt (eds.), Advanced Environmental Monitoring,

402–414.

31 In Vitro Testing of Inhalable Fly Ash at the Air Liquid Interface 403

2000a). The causes of the toxicological effects of fine particles to the human

organism are largely unknown. Besides their chemical composition, the particle

number and the surface of the particles seem to be of particular importance for

the effects. So not only particle mass but more the number concentration seems

to be a measure for the toxic potential of an inhaled aerosol (Wichmann et al.,

2000b). For the quantitative assessment of the toxicity of industrial submicron

particles the dose-response relationship is essential. The long term objective of

this project is to use the toxicological results on relevant particle parameters for

the developments of emission control strategies which will reduce the aerosol

components responsible for the health effects in the population. Fly ash from a

municipal waste incinerator plant was selected to study in vitro its responses in

target cells of the lung.

Inhalation experiments with animals (in vivo studies) provide the most

comprehensive data on the toxicological potential of an aerosol; however, they are

very complex and expensive. In vitro exposure of cultivated lung cells allows a

series of experiments in short time periods under reproducible conditions (Diabaté

et al., 2002; Voelkel et al., 2003; Wottrich et al., 2004).

In vitro exposure of cells usually takes place in culture medium in which the

particles are resuspended. However resuspension in a liquid changes the properties

of the particles, moreover the dose is not applied directly to the cells. The amount

of particles which finally reach the cell culture remains unknown unless radioactive

labelled particles are used and quantified after the exposure. Therefore a newly

developed technique to study the biological effects of an aerosol was applied, in

which the particles directly contact the air-liquid interface of the cells (Aufderheide

et al., 2005). An exposure at the air-liquid interface is technically challenging,

however it resembles the situation in the lung during inhalation of atmospheric

particulate matter.

The aim of this study is to analyse the toxicological potential of fine and

ultrafine particles from industrial combustion processes using a biotest. This biotest

is performed by near-realistic exposure of cultivated lung cells at the air-liquid

interface and analysing the biological responses. Important steps in this work were

to develop the exposure system for the use at industrial particle sources, to repro-

ducibly generate a test aerosol, to determine the deposition efficiency of the aerosol

and to provide an exposure protocol for the bioassay.

31.2 Experimental Procedures

31.2.1 Aerosol Generation

Particles are generated in the flow reactor AEOLA (Fig. 31.1). AEOLA (AErOsol

LAboratory facility) is a pilot plant for the generation of dry aerosols in a wide

range of concentrations. Ambient air or flue gas from a light-oil burner is used as

carrier gas. Relevant concentrations of gaseous trace substances (SO

, NO

, dioxin,