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304 P.-D. Hansen

with the treated effluent, there is an emission of approximately 2.4 t of biological

treated sewage sludge to the Berlin River Havel System. A volume of approximately

880 t biologically treated sludge is emitted per year and deposited in the river

Havel. The suspended solids of the sludge are carriers of endocrine disrupting

compounds (EDCs) and other organic pollutants.

23.3.2 Xenoestrogenic Effects in the River System of Berlin

The concentrations of EDCs like nonylphenol, different phthalates and lower PCBs

are rather low. For Nonylphenol the safety factor is approximately100 between the

concentration in the effluents and the endocrine effect measured by the enzyme

linked recombinant receptor assay (ELRA). The safety factor for Bisphenol A is

approximately 3,000. There is no safety factor (Hansen 1998) concerning the low-

est effects concentrations (LOEC) of the hormones and the measured concentrations

of the hormones in the effluents. Endocrine effects were measured for 17β-Estradiol

in the range of 0.1–1 ng/l, for Ethinylestradiol [0.2–3.0 ng/l] and for Estrone

[0.5–1.0 ng/l]. These concentrations are already present in the surface water of the

River Havel and the river sediments. Our results regarding endocrine disrupters

(EDCs) are comparable to investigations conducted under the LOES programme of

the Netherlands and cited studies world wide (Vethaak et al. 2002).

Endocrine effects in the sediments of the River Havel, Spree, Dahme and

Teltowkanal are calculated and expressed as 17β-Estradiol equivalents [µg/kg]

in sediment dry weight and for pore water and elutriates of sediments in [µg/l],

see Figs. 23.4 and 23.5. The lower River Havel shows a median concentration

of 8.5 µg/kg—17β-Estradiol equivalents, in the upper River Havel 6.7 µg/kg—

17β-Estradiol equivalents, 34.2 µg/kg—17β-Estradiol equivalents in the River

Spree, 45.7 µg/kg—17β-Estradiol equivalents in the River Dahme and in the

Teltowkanal 14.3 µg/kg—17β-Estradiol equivalents. The results in Fig. 23.4

clearly demonstrate that concentration levels up to 175 µg/l 17β-Estradiol

equivalents in elutriates of sediments are coming from sites with an extreme

suspension rate. In Figs. 23.4 and 23.5 the concentration levels of 17β-Estradiol

equivalents were ranged in seven classes. Endocrine effects measurements

were done by the ELRA-test (ELRA = enzyme linked recombinant assay,

Seifert 1999).

Analysis performed by ELRA (Seifert 1999) and the hER and hAR assay by

McDonalds will be soon part of an intercalibration study for endocrine substances

after the German Institute for Norming (DIN). Sediment loading by xenoestrogens

changes over the year and also because of extreme algal growth (see Fig. 23.1), but

they also contain a substantial amount of phytohormones. Apart from estrogenic

responses there is only a poor evidence of androgens in River Havel sediments.

In Figs. 23.4 and 23.5 the patchiness of the sediments of the River Havel with

endocrine responses in the river bed is shown.

Fig. 23.4 17β-Estradiol equivalents [µg/L] for elutriates of sediments in the Berliner River system

using ELRA in spring 2001. Sediments were sampled at the top 0–5 mm layer of the rivers

Fig. 23.5 17β-Estradiol equivalents [µg/L] for pore water of sediments in the Berliner River system

using ELRA for the year 2001. Sediments were sampled at the top 0–5 mm layer of the rivers

306 P.-D. Hansen

23.3.3 Ecotoxicological Assessment in Relation to the WFD

For an assessment of the endocrine effects reported in Figs. 23.4 and 23.5, a scientific

assessment system is necessary. It was thus decided to use the ecototoxicological

classification system of sediments (Krebs 2005a, 2005b) developed at the German

Federal Institute of Hydrology (BfG). Sediment classification is based on the sci-

entific ecotoxicological approach using so-called pT-values. The method is used to

assess the quality of sediments, dredged materials and toxic effects of waste water

with standardized bioassays. Different bioassays and different test phases (pore

water and elutriate) are considered equal in ranking in this system. The pT-value

depends on the dilution factor and the lowest observed effect concentration, (see

Krebs 2005). The highest dilution level without effects corresponds to the pT-value,

i.e., the dilution step 1:2 represents a pT-value of 1. Toxicity classes are assigned

Roman numerals: it is a seven-level-system based on convention. We combined the

seven-level toxicity classes system using pT-value of the xenoestrogenic effects

measured by the ELRA-test adapted to the five-level ecological classification system

of the EU Water Framework Directive (WFD). Figures 23.6 and 23.7 demonstrate

the risk assessment and ranking using the pT-value as an ecotoxicological classification

concept in correspondence to the WFD.

23.4 Discussion

A relevant question meriting discussion is the following: can biosensors give

additional information? For on-line measurements, there are some doubts that

“state of the art” biosensors will give additional information and will replace

chemical analytes or even bioassays. The case study emphasized the need to

develop biosensors which do not only measure “conventional” contaminants but

also new emerging parameters like endocrine effects which are needed for compliance

with the drinking water directive as well as the water frame work directive (WFD)

and which are currently used in monitoring programs. The classification of the

contamination of river sediments by xenoestrogens after the pT-values and

expressed as 17β-estradiolequivalents contributes to the evaluation by receptor

assay—sensors for the ecological risk assessment (ERA), risk communication and

risk management. To exploit the principal advantage of biosensors, standardisation

and harmonisation is needed for governmental decision making in collaboration

with industry and governmental authorities. A promising sign is that ISO

(ISO=International Standardization Organisation) already has a working group for

Biosensors (ISO TC 147/SC2) and a basic working document by ISO on biosensors

is already available. Very promising also is the additional information available

especially in the field of bio-effect related sensors like whole cell sensors and receptor

sensors. Progress is being made in the development of receptor assays-sensors (Hansen

2003; Hansen et al. 2006). The format of the receptor assays has changed meanwhile

to nanotechnology levels, already well established in water and food analysis. For

Fig. 23.6 pT-values and classification of River Sediments (Elutriates) in the Berliner River

System for the year 2001 as per the WFD classification system (Krebs, 2005a, 2005b). For

xenoestrogenic effects, see Fig. 23.4 (Huschek and Hansen 2006)

Fig. 23.7 pT-values and classification of pore water of sediments in the Berliner River system for

the year 2001 as per the WFD classification system (Krebs, 2005a, 2005b). For xenoestrogenic

effects, see Fig. 23.4 (Huscheck and Hansen 2006)

308 P.-D. Hansen

assessment of surface water, the WFD has requested the establishment of “good

ecological status” and “good chemical status”. The challenge in this study was to

develop a strategy to include effect related data of river sediments into WFD concepts.

Finally, we proposed a convention to combine the seven level toxicity classes system

using pT-value of the xenoestrogenic effects measured by the ELRA-test and

adapted these to the five-level ecological classification system of the EU WFD.

Bringing in to the WFD the effect related approaches concerning bioassays and

biomarkers is only relevant in the context of the “quality norms (QN)” of environmental

relevant substances and the “good chemical status”. The QN developed after

the WFD in correspondence to “good chemical status” has to be enlarged for

endocrine disrupting compounds, as it was shown in this study for estrogens

(i.e., 17β-Estradiol) and Xenoestrogens (i.e., Nonylphenol and Bisphenol A).

Other classes of concern and their potentially hazardous environmental effects

should also be investigated.

Besides loading aquatic systems with endocrine disrupting compounds, there

are several medicinal products used in large quantities for humans and livestock

in industrialised countries (see Table 23.3) that have been frequently found in

sewage treatment plants (STPs), surface waters (Huschek et al. 2004), sediments,

pore water and groundwater. Because of the low current and loading by treated

secondary effluents, there are several hot spots. Most of the beaches of the River

Havel are protected areas linked to the drinking water directive. Because of the

very active beach filtration (see Fig. 23.3) along the River Havel for the produc-

tion of drinking water, there is intensive monitoring of sediments and groundwater

mainly by instrumental analysis (LC-ESI-MS/MS). The results clearly show that

several polar drugs are detected in groundwater and beach filtration. Several

compounds are effectively removed by beach filtration but others are still present

at higher concentrations (see Table 23.3). In total there are approx. 50–70

substances present.

Apart from drug residues (Huschek et al. 2004) several polyaromatic substances are

present in River Havel sediments. In the River Havel sediments, Tributyl-Sn (TBT)

concentrations were detected in the range of 1.600 ng/g dry weight. Exposure experi-

ments with mussels showed feminisation and an increase of vitellogenin like proteins

(Blaise et al. 2003) and exposed fish displayed an increase of vitellogenin as well as

intersex determined by microscopic examination of the gonads (Arab et al. 2006). The

results were a clear indication of ecosystem health status (see Table 23.2).

A comprehensive strategy regarding the demands of WFD is necessary and this

study is a contribution to the chemistry quality component, but morphology and

biology are also of importance. The uniqueness of these three quality components

of the WFD has to be discussed in further studies and in relation to ecosystem

health. The environmental aspects have to be strongly assessed in the context of

public health. Effects in ecological systems are closely linked to health therefore

strongly suggesting that biosensors, biomarkers and bioassays should be included

in ecosystem health assessment in-line with the WFD. The most successful strategy

would be to include bioassays and biosensors standardised by ISO and CEN into

the approaches to define a “good chemical status” in the river basin systems. For

23 Biosensors for Environmental and Human Health 309

Table 23.3 Concentration ranges of active drug substances in µg/L in effluents of two STP’s in

Berlin (Huschek G and Hansen P.-D. 2006)

Active drug substances

Sales (kg) of active

substance in 2001

Effluents of two different

in Berlin STP [µg/L]

during 2001–2002

Ibuprofen 344884.6 0.054–0.35

Carbamazepine 87604.9 0.9–1.2

Diclofenac 85800.7 1.47–2.1

Metoprolol 68364.4 0.99–1.18

Bezafibrate 33475,6 0.10–1.75

Propyphenazone 28140,2 0.11

Phenazone 24843,2 0.09

Sotalol 26649,2 0.57–0.68

Erythromycin 19199,0 0.15–0.43

Atenolol 13594,4 0.19–0.27

Trimethoprim 11426,6 0.16–0.42

Clarithromycin 7159,1 0.071

Propranolol 6519,0 0.056–0.09

Gemfibrozil 5243,7 0.28

Roxithromycin 9554,5 0.11–0.52

Naproxen 5060,1 0.1

Indomethacine 3720,6 0.14

Bisoprolol 2956,8 0.13

Ethinylestradiol 47,5 n.d. - 0.0003

n.d. = not detectable

the understanding of the status of the environment, a more realistic effect related

real time sensor system is required.

The case study on a receptor assay–sensor showed clearly that biosensors are

complementary to chemical methods since they can signal the presence of toxic

(i.e., Xenoestrogens, Biotoxins) compounds that require further effect related

chemical analytical studies. In addition this will allow the correlation of toxicity

biosensor data with individual chemical compounds. Because of this cross activity,

biosensors can detect chemical compounds which were not target analytes but they

are detectable by the cross active biosensors or by the bioeffect related sensor but

not by the chemical instrumental system (Alcock et al. 2003).

In summary, biosensors are early recognition systems that indicate the presence

of unknown compounds which are responsible for the signals they detect. It is well

known and accepted that in addition to chemical analysis effect related parameters

are necessary. Sometimes chemical toxicity and related information can not be

obtained by chemical instrumental measurements.

For diagnosis concerning human health, new technologies like proteomics are

presently under developmental stages. These tools will eventually help to validate

effects in humans and in the environment and this could be one relevant direction

for the future. Many techniques have been developed to meet the demands of the

310 P.-D. Hansen

lucrative biomedical markets and await adaptation for environmental applications,

while the demands of continuous on-line monitoring are still hampered with prob-

lems that will require unique solutions.

Acknowledgements The author thanks for funding by Senate Department of Urban Development

of the City of Berlin—Fischereiamt (Berlin Fishery Board, BFB), the European Commission

(SANDRINE: ENV4-CT98–0801 and CITY FISH: ENK1-CT 1999–00009 projects) and the

BMBF (MARS 1, BMBF- Az 03F0200A).

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lyl phthalate and tertebromodiphenyl ether. Aquat. Toxicol., 78, Suppl 1, 86–92.

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Chapter 24

Biological Toxicity Testing of Heavy

Metals and Environmental Samples Using

Fluorescence-Based Oxygen Sensing

and Respirometry

Alice Zitova

, Fiach C. O’Mahony

, Maud Cross

, John Davenport

and Dmitri B. Papkovsky

1,3

Abstract A new methodology for simple, rapid, high throughput biological testing

of potentially hazardous chemical and environmental samples has been developed,

which is based on measurement of oxygen consumption of aquatic test organ-

isms using phosphorescent oxygen-sensitive probes and detection on a fluorescent

plate reader. Test organisms are exposed to potential toxicants and then allowed to

respire in a sealed measurement compartment in the presence of soluble oxygen

probe added to the sample. The resultant depletion of the dissolved oxygen causes

an increase in sample fluorescence over time, thus reflecting the organism respira-

tion rate and its alterations. Dedicated low-volume sealable 96-well plates provide

improved sensitivity, convenience and miniaturization in such respirometric assays.

These assays are carried out using standard laboratory tools and fluorescence plate

readers, with multiple samples processed in parallel in 96-well microtitter plates.

Oxygen consumption rate is a universal biomarker of general viability and metabolic

responses of aerobic organisms, hence, this methodology is applicable to various

organisms, including those currently used in toxicity testing. In this study, the new

respirometric platform has been demonstrated with different organisms including

prokaryotes (E.coli), eukaryotes (Jurkat T-cells), invertebrates (Artemia salina),

and validated with toxicity testing of environmentally relevant chemicals. A panel

of water samples discharged from wastewater treatment plants was also analyzed

with this panel of test organisms. Organisms were exposed to the samples for a

period of time (0–24 h depending on the organism chosen), and then assessed for

their respiration rates (1 h assay). Toxicity was recorded as EC

, i.e., the concen-

tration of the toxicant which caused a 50% decrease in the respiration compared

to untreated organisms. Responses of various organisms to certain chemicals can

Biochemistry Department & ABCRF, University College Cork, Cavanagh Pharmacy Building,

Cork, Ireland

Zoology Ecology and Plants Science Department, University College Cork, Distillery Fields,

North Mall, Cork, Ireland

Luxcel Biosciences Ltd., Suite 332, BioTransfer Unit, BioInnovation Centre, UCC, Cork, Ireland

312

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

312–324.

24 Biological Toxicity Testing of Heavy Metals 313

be cross-compared and correlated to established toxicity tests (based on LD

). In

terms of sample throughput, sensitivity, speed, flexibility and convenience, the new

screening platform is seen as being superior to the existing toxicity tests currently

used. It provides adequate assessment of biological hazards of complex chemical and

environmental samples, allows for the monitoring of sub-lethal effects and provides

information-rich data reflecting the mode of toxicity. It is therefore highly suitable for

environmental monitoring and screening of potentially hazardous samples, including

large scale programs such as EU Water Framework Directive and REACH.

Keywords: Toxicity testing, animal-based testing, oxygen consumption assay,

fluorescence-based oxygen sensing, optical oxygen respirometry

Glossary of acronyms: EC

(median effective concentration), LD

(median

lethal concentration), PAH (polyaromatic hydrocarbons), PBDEs (polbrominated

diphenylethers), RST (respirometric screening technology), Swift (Screening

methods for Water data InFormaTion), WWT(wastewater treatment), WFD (water

framework directive).

24.1 Introduction

In recent years the European Commission has published two new EU initiatives of

particular concern to those involved in environmental monitoring. The first was a

regulatory system for chemicals called REACH (EU, 2001b) which stands for

Registration, Evaluation and Authorization of Chemicals. Under REACH, each

producer and importer of chemicals in volume of 1 tonne or more per year and per

producer/importer—around 30,000 substances—will have to register them with a

new EU Chemicals Agency, submitting information on properties, uses and safe

ways of handling them. Through evaluation each member state or agency is

required to look in more detail at registration dossiers and at substances of concern,

limiting animal testing to the absolute minimum in addition to prescribing the use

of alternative methods wherever possible. Chemicals of particular concern include

those which are known to cause cancer, mutations, toxicity to reproduction, includ-

ing those which are persistent, bio-accumulative and toxic, or very persistent and

very bio-accumulative. Based on such studies the Commission can decide to ban

certain uses of chemicals or ban the chemicals in question altogether. The second

initiative called the Water Framework Directive (EU, 2000) and the associated

Swift-WFD (EU, 2003) is concerned with the expansion of the scope of water

protection to include all waters, to set clear objectives in order that a “good status”

be achieved by 2015 and that water use be sustainable throughout Europe. The

directive initially identified 33 substances (EU, 2001a) which had been shown to be

of major concern for European Waters. These include existing chemicals, plant

protection products, biocides, metals and other groups like polyaromatic hydrocar-

bons (PAHs) and polbrominated diphenylethers (PBDEs) that are used as flame