Kim Y.J. (Ed.) Advanced Environmental Monitoring

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22 Monitoring of Dissolved Organic Carbon 293

LIF-signal recorded at the freshwater inlet at the cleaning facility; the grey crosses

in Fig. 22.12 (b) represent the actually applied ozone dosage and the black dots show

the recommended ozone dosage determined by LIF-spectroscopy. After switching

one pumping line to the pumping station ‘Brückerbach,’ there is an enhanced need

for ozone in the first place. The ozone dosage is adjusted, but the demand is too high

resulting in a very low residual ozone concentration. Low residual ozone concentra-

tions are also unfavourable because the disinfection capacity is too low.

At minute 720, there seems to be a short failure at the ozone gas generator,

resulting in a difference between the recommended and the actual ozone dosage

again. In both cases where the actual ozone dosage differs from the recommended

value deduced by LIF spectroscopy, 20 min later a very low residual ozone concen-

tration in the water is recorded. The time delay of 20 min corresponds well to the

retention time for the water in the contact tank (see Fig. 22.6).

These examples document well that the LIF-signal can be used for predicting the

ozone consumption during the oxidation process using different raw waters from

the incoming pumping stations.

22.5 Summary

The investigations have shown that LIF is suitable for the determination of

DOC-values of raw waters. Prerequisite is a calibration to the local conditions of

the raw water. The demonstrated LIF set-up was successfully used for on-line

measurements of DOC at different pumping stations.

Fig. 22.12 (a) Normalized fluorescence, (b) actual ozone dosage (grey cross) and prediction

value (black dot) determined by LIF and (c) residual ozone concentration

294 U. Wachsmuth et al.

Furthermore LIF is a sensitive probe for revealing differences in water quality.

It was possible to distinguish between stagnating water and freshwater in the case

of waterworks in Düsseldorf-Flehe. The investigations at the ‘Grind’ show changes

of LIF from starting water withdrawal to equilibrium.

The investigations demonstrate also a direct correlation between LIF-signal and

ozone consumption. The normalized LIF-signal can be used to determine a

prediction value of the ozone demand for oxidation processes. These measure-

ments have been performed directly at the raw water inlet of the cleaning facility

at the water works ‘Flehe’ of the Stadtwerke Düsseldorf AG. Following this predic-

tion value the ozone dose can be adjusted in real time.

In the future, LIF could be applied to control the input and output of drinking

water plants to get further information about the water cleaning process which is an

important goal in the water safety plans of the suppliers.

References

Baker A. (2002), Spectrophotometric discrimination of river dissolved organic matter. Hydrol.

Process., 3203–3213.

Bünting U., Lewitzka F., and Karlitschek P. (1999), Mathematical model of laser-induced

fluorescence fiber-optic-sensor head for trace detection of pollutants in soil. Appl. Spectrosc.,

53, 49–56.

Esteves V.I., and Duarte A.C. (2001), Differences between Humic substances from riverine,

estuarine, and marine environments observed by fluorescence spectroscopy. Acta Hydrochem.

Et Hydrobiol., 28, 359–363.

Lewitzka F., Niederkrüger M., and Marowsky G. (2004), Application of two-dimensional LIF for

the analysis of aromatic molecules in water. In P. Hering, J.P. Lay and S. Stry (Eds), Laser in

Environmental and Life Sciences, Springer-Verlag: Berlin, pp.141–161.

Marowsky G., Lewitzka F., Bünting U., and Niederkrüger M. (2000), Quantitative analysis of

aromatic compounds by laser induced fluorescence spectroscopy. Proc. SPIE, 42, 218–223.

Niederkrüger M., Wachsmuth U., Konradt N., Rohns H.-P., Nelles T., and Irmscher R. (2004),

Mobiles Laserfluoreszenzspektrometer zum Monitoring auf gelöste organische Verbindungen

bei der Wassergewinnung aus Uferfiltrat. VDI-Berichte, 1863, 17–24.

Peuravuori J., Koivikko R., and Pihlaja K. (2002), Characterization, differentiation and classi-

fication of aquatic humic matter separated with different sorbents: synchronous scanning

fluorescence spectroscopy. Water Res., 36, 4552–4562.

Section 4

Biosensors, Bioanalytical

and Biomonitoring Systems

Chapter 23

Biosensors for Environmental

and Human Health

Peter-D. Hansen

Abstract Sensors and biosensors as well as biochemical responses (biomarkers) in

ecosystems owing to environmental stress provide us with signals (environmental

signalling) of a potential damage in the environment. These responses are per-

ceived in this early stage, but in ecosystems, the eventual damage can be prevented.

Once ecosystem damage has occurred, the remedial action processes for recovery

could be expensive and pose certain logistical problems. Prevention of ecosystem

deterioration is always better than curing. Ideally, “early warning signals” in eco-

systems using sensing systems and biochemical responses (biomarkers) would not

only tell us the initial levels of damage, but these signals will provide us as well

with answers to develop control strategies and precautionary measures with respect

to the water framework directive. To understand the complexity of the structure

of populations and processes behind the health of populations, communities and

ecosystems, we have to direct our efforts to promote rapid and cost-effective new

emerging parameters of ecological health. New emerging parameters are bio-

chemical effect (biomarker) related parameters in the field of immunotoxicity and

endocrine disruption. Environmental effects such as genotoxicity and clastogenic-

ity were detected in organisms from various “hot spots”. Vital fluorescence tests

are one means that allow us to unmask adverse events (i.e., genetic alterations in

field-collected animals or in situ-exposed organisms) by a caging technique. New

emerging ecosystem health parameters are closely linked to biomarkers of organ-

isms measured in monitored areas. One problem is always to find the relevant

interpretation and risk assessment tools for the environment.

Keywords: Biosensor, effect assessment, ecotoxicological classification in sedi-

ment, endocrine effects, assessment of “good ecological status”, drug exposure.

Technische Universität Berlin, Faculty VI, Department of Ecotoxicology,

Franklin Strasse 29 (OE4), D-10587 Berlin, Germany

(Tel: +493031421463, Fax: +49308318113)

297

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

297–311.

298 P.-D. Hansen

23.1 Introduction

For risk assessment and risk assessment tools new recommendations are described

in the Technical Guidance Document of EU – Edition 2, in the new EU Chemicals

Legislation REACH and in the status report for toxicological methods of the

European Centre for the Validation of Alternative Methods. In the EU water frame-

work directive (2000) a general requirement for ecological protection, and a gen-

eral minimum chemical standard was introduced to cover all surface waters. For the

description of “good ecological status” and “good chemical status”, effects moni-

toring tools are needed for the description of the ecological status of river basin

systems. In some cases, biomarkers were very helpful to promote an environmen-

tally sensitive and sustainable use in studies with coastal zone samples (Baumard

et al. 1999, Hansen et al. 1985, 1990, 1995, 2006). A very promising tool is the

scale classification based on toxicity studies and environmental monitoring to clas-

sify sediments: i.e., “Ecotoxicological Classification of Sediments by Bioassays”

(Krebs 2005a, 2005b). The currently available biomarkers or biochemical responses

are used as environmental monitoring tools to assess information on early responses

of living organisms to environmental stressors, and to deliver signals of ecosystem

damage and pathology owing to both man-made and natural pollutions. Biomarkers

are already being applied to the water matrix and to benthic organisms that include

new emerging biomarkers such as endocrine effects and immunotoxicity. Usefulness

for new emerging effect parameters such as immunotoxicity (phagocytosis) in the

context of biotoxins is demonstrated by mussels exposed to sediments in coastal

areas and exposure of mussels under controlled conditions to the water matrix.

Here, results of immunotoxic effects in mussels and the endocrine load of the sedi-

ments are demonstrated. Immunotoxic response of the blue mussel was quantified

by phagocytosis activity (Phagocytosis Index) of its hemocytes. It appears that both

immunosuppressive and immunostimulative effects are likely to occur at specific

sites and that responses will be influenced by the type and intensity of contaminants

present. Immune system function in bivalves can be adversely affected by long-

term exposure to environmental contaminants. Investigating alterations in immu-

nity can therefore yield relevant information about the relationship between

exposure to environmental contaminants and susceptibility to infectious diseases.

23.2 Biochemical Responses and Biosensors for Effects

Monitoring in the Environment

Use of biomarkers (biochemical responses) in multi-arrays for environmental

monitoring is complementary to chemical analysis since they can alert for presence

of toxic compounds that require further instrumental analysis or “bioresponse-

linked instrumental analysis”. Biosensors are by definition analytical devices

incorporating a biological component like micro-organisms, organelles, cell receptors,

23 Biosensors for Environmental and Human Health 299

enzymes, antibodies, nucleic acids and a physicochemical transducer system:

optical, electrochemical, thermometric, piezoelectric or magnetic. An enzyme

linked recombinant receptor assay like the ELRA is a biosensor according to this

definition: the biological component is the enzyme and the transducer is the optical

component. Biosensor and biochemical responses for the assessment of environ-

mental health are listed in Table 23.1. It is rather difficult to transfer the monitored

biochemical responses or the sensor responses into an operational effect related

standard (EQN = environmental quality norm) for environmental monitoring.

Sensing systems based on the induction and inhibition of a functional system relat-

ing to function, interference and effect related endpoints are listed in Table 23.1 and

depicted in Fig. 23.1.

Table 23.1 Environmental monitoring of effect related biochemical responses (biomarkers) and

potential sensing systems with their endpoints (“effects at the level of” neurotoxicity etc.)

(Modified after Bilitewski et al. 2000.)

Targets Example Interference by compounds Endpoints

Proteins Acetylcholine esterase Organophosphorus and Neurotoxicity

Enzymes carbamic compounds

Protein phosphatase Microcystins Hepatotoxicity

1 and 2A

Ion channels Na+ channel (voltage- Saxitoxin, tetrodotoxin, Neurotoxicity

gated channel) procaine

Transport SHBG, CBG, TBG Endocrine disruptors Growth,

protein reproduction

Receptors Estrogen receptor Endocrine disruptors (e.g. Growth,

o,p-DDT, nonylphenol) reproduction

Nicotinic acetylcholine Anatoxins neurotoxicity

(ACh) receptor

(ligand-gated

channel)

Electron QB protein Photosynthesis II herbicides Photosynthesis

carriers (e.g. s-triazines,

phenylureas), phytotoxins

Nucleic acids DNA double strands PAHs, Pesticides, PCBs, Genotoxicity

DNA EDCs, intercalating

polycyclic aromates

(ethidium, acridine, caffeine);

DNA adducts (metabolites

of chloracetamide

herbicides)

Cytoskeleton Tubulin Colchicin, taxol; anti-tubulin Cytotoxicity

herbicides (e.g. trifluralin,

oryzalin)

Ribosomes rRNA (ricin) Ribotoxins (ricin, abrin, cytotoxicity

Shga toxin)

300 P.-D. Hansen

Biosensors together with effect related parameters or biochemical responses for

environmental monitoring are very complex but they will give a clear picture of the

health status of the investigated system. In Fig. 23.1 the biochemical responses are

demonstrated by three phases of reaction by the organisms. The enzymes and

oxyradicals are helpful tools for a final biosensor. Enzyme biosensors are sensors

based on enzymatic bio-recognition elements. Therefore the definition of “biosensors”

can be widened: “Biosensors are analytical devices incorporating a biologically

derived and/or bio-mimicking material (i.e., cell receptors, enzymes etc.), associated

with or integrated within a physicochemical transducer or transducing microsystem,

which may be optical, electrochemical, thermometric, piezoelectric or magnetic.

Biosensors are distinct from bioassays in that the transducer is not an integral part of

the analytical system. Figure 23.1 shows an example of biochemical responses for

diagnosis of the health status of environmental systems (Hansen 2003).

The effect related parameters or biochemical responses are complex but they will

give a clear picture of the health status of the investigated system. “Ecosystem

Health” is synonymous with “environmental integrity”, from which it follows that

the scope of ecosystem health (EH) research encompasses all the tools and

approaches which are efficacious in increasing the cognitive, curative and preventive

knowledge as the goal to the preservation of environmental integrity. Ecosystem

health research thus directs its attention to the prediction of reversible and irreversible

insults which human or other activities could potentially inflict on the environment.

For the assessment of ecosystem health, very promising biomarker approaches are

centred on quantifying biochemical effects in organisms and populations. One

important tool for the acceptance of biomarkers in science, technology and govern-

mental legislature are the so-called inter-laboratory comparison studies of measurements

Fig. 23.1 Environmental signalling and diagnosis by biosensors and biochemical responses

(biosensors endpoints) In figure: Genotoxic effects; Oxyradicals

23 Biosensors for Environmental and Human Health 301

of biomarkers. Laboratory studies have established a strong causal link between

exposure of fish to PAHs and co-planar PCBs and the expression of cytochrome

450

1A1 and its associated 7-ethoxyresorufin-O-deethylase (EROD) activity. The

induction of EROD activity in fish liver has particularly been used as a biomarker

for the effects of these organic contaminants in several inter-calibration exercises

(see Stagg and Addison 1995). EROD induction is a classical biomarker and well

established for MFOs (mixed function oxigenases) and biotransformation in

ecotoxicology. So far, however, only phase I of the MFOs is investigated and not

phase II with conjugation and the real detoxification process. Additionally, another

often used biomarker is cholinesterase inhibition. The basic concept is that organo-

phosphorus pesticides and carbamates inhibit cholinesterase at different levels

(Sturm and Hansen 1999; Sturm et al. 1999). For the quantification of neurotoxicity

there are two well known cholinesterases (acetyl cholinesterase and butryl

cholinesterase) and the methodology in principle is standardised after DIN (German

Institute for Norming: DIN 38415-T1 1995). There is an application of validated

biomarkers available with endpoints including new emerging biomarkers for endocrine

effects and immunotoxicity in addition to genotoxicity. There is a high potential of

biochemical responses and the development of fast and reliable biochemical tools

(biosensors) for on-site screening in environmental and human health analysis. The

principles associated with the different scales of biochemical processes relating to

ecosystem and human health are shown in Table 23.2.

23.3 The River System of Berlin and the “Good Ecological

Status” Classified by a Biosensor System

In considering the impact of either natural stress or man-made stress we always

encounter detoxification, disease defence, regulation and adaptation processes. This

situation makes the assessment approach by biomarkers rather complicated. However,

in symptoms analysis including functional (behaviour, activity and metabolism) and

structural changes in organisms (cells, tissues and organs), biomarkers do have a

significant ecological assessment potential. For landscape planning and environmental

management it is necessary to get significant data from biochemical responses for

relevant and sustainable actions. As an example of transfer of new substances from

sediments to groundwater – the River Havel case study in Germany has demonstrated

close aspects between environmental monitoring and ecosystem health aspects.

23.3.1 Description of the River System of Berlin

and the Biosensor (Receptor Assay–Sensor) Application

The River Havel is an intensively monitored waterway because of its multiple use

for beach filtration and finally as a source to produce drinking water. The River

302 P.-D. Hansen

Havel is a water stretch of 30 km in length with a surface of more than 20 km

. It is

a slow flowing river with several conflicting uses such as (1) receiving waterway

for secondary effluent and rain water, (2) active beach filtration and drinking

water production, (3) sport- and professional fisheries, (4) leisure and recreation

concerning water sports and the EU water directive for swimming areas with

beaches along the river and (5) waterway for leisure boats and commercial cargo

vessels. With an extended surface, a low water depth (mean water depth 7 m) and

a steady input of nutrients, the river Havel has a potential for algal blooms.

Blooms begin in February (Fig. 23.2) and continue until May. During May and

June the algae growing are reduced and a second bloom of blue greens with

microcystins start at the end of June. There is a combination of increasing

temperature (up to 30°C) and decreasing flow of water (approx. 30 m

/s), which

trigger increases in biomass and in the amount of suspended solids. Finally, the

latter are eventually deposited as sediments.

Table 23.2 Selected biochemical responses for assessment of environmental health and the

biomarker methods used for their examination (Modified after Bresler et al. 1999)

Method Characteristic of health

Measurement of blue and green fluorescence

of NADH and FAD in living tissues

Metabolic state of mitochondria, cells or

tissues respiration and glycolysis

Quantitative fluorescent cytochemistry DNA, RNA, proteins and lipids content

Using permeable fluorogenic substrates of

enzymes, specific inhibitors, and kinetic

analysis

Enzyme activity in living cells in situ:

a. Non-specific esterases b. Detoxifying

enzymes c. Marker enzymes

Using special fluorescent anionic markers Alterations of permeability of plasma

membranes, epithelial layers and histo-

hematic barriers

Using specific fluorescent transport substrates,

inhibitors and kinetic analysis

State of carrier-mediated transport system

for xenobiotics elimination

Using fluorescent xenobiotics or fluorescent

analogue of xenobiotics

Xenobiotics distribution, extra- and intrac-

ellular accumulation and storage

Using special fluorescent xenobiotics or

fluorescent analogues of xenobiotics

State and function of xenobiotic-binding

proteins

Vital tests with Acridine Orange or

Neutral Red

State of lysosomes and cell viability

Metachromatic fluorescence of intercalated

or bound Acridine Orange, 590/530 nm

Microfluorometry

Functional rate of nuclear chromatin, DNA

denaturation

Complete cyto- and histopathological

examination

Early pathological alterations and signs of

environmental pathology

Electron microscopy Cell structures and organoids

Cytogenetic examinations Detection of environmental genotoxicity

and clastogenicity

Mass Spectrometry (MALDI/TOF/MS;

ESI-TOF-MS/MS)

Identification and detection of membrane

proteins, epitope-binding areas of

proteins

23 Biosensors for Environmental and Human Health 303

Besides algal blooms, there is a second source for suspended solids in the river

because of effluent loading by sewage treatment plant emissions. The river consists of

10%, 20%, 30% and 40% treated effluents (tertiary treatment and sometimes membrane

filtration). In winter (high water flow = approx. 100 m

/s) loading corresponds to

10% effluent to 20%–30% in spring and autumn (medium water flow = approx.

30 m

/s) and to 40% of treated effluent in summer (low water flow = approx. 5 m

/s).

The second source of suspended solids in the receiving river Havel on top of

extreme eutrophication by algae (see Fig. 23.2) is the outlet of the Berlin-Ruhleben

sewage treatment plant. The effluent contains 10 mg/l suspended solids in treated

sewage sludge. The sewage plant close to the river Havel is one of the main sewage

plants in Berlin with an output of 240,000 m

of treated effluent per day. Together

Fig. 23.2 Algal blooms in the Berlin waterways River Havel, Spree, Dahme and the Teltowkanal

Fig. 23.3 Schematic hydro geologic section of the transect at the River Havel (Lieper Bucht),

location of monitoring wells (shallow and deep) and of the raw-water supply well for drinking

water (Modified after Heberer and Mechlindki 2003)