Development and Validation of an Automated ... - wise-rtd.info

Development and Validation of an 

Automated Instrument for the 

Determination of the Endocrine Disrupter 

17β-Estradiol in the 

Water Cycle 

(Acronym: ESTR-A-LISER) 

Demonstration project in the framework of the support programme 

Life III - Environment: LIFE 00 ENV/D/000346 - ESTR-A-LISER 

Layman-Report

Beneficiary/ Project coordinator 

quo data GmbH 

Siedlerweg 20 

D-01465 Dresden-Langebrück 

Germany 

Contact person 

Kirsten Simon 

Address 

quo data GmbH 

Birkenweiherstr. 9 

D-63505 Langenselbold 

Germany 

Email 

k.simon@quodata.de 

Homepage 

www.estraliser.de 

www.quodata.de

Layman Report . Table of contents 

Table of contents 

1 Objective of the project......................................................................... 1 

2 Realization.............................................................................................. 5 

2.1 Immunoassays ............................................................................................. 5 

2.1.1 ELISA.................................................................................................... 6 

2.2 Statistical analysis methods ......................................................................... 7 

2.3 Pipetting automat ......................................................................................... 7 

3 Results.................................................................................................... 8 

4 Effects on the environment .................................................................. 8 

5 Transferability........................................................................................ 9 

6 Dissemination ........................................................................................ 9 

7 Acknowledgement............................................................................... 11 

quo data GmbH 10.11.2005

Layman Report . Objective of the project 

1 

1 Objective of the project 

First sentence of the EU – Environmental Framework Directive 2000/60/EG: 

„Water is no common article of trade, but an inherited good that needs to be 

protected, defended and handled adequately.“ 

The Environmental Framework Directive of the European Union (EU) 1 is a document 

that has been enacted by the EU member countries. It includes regulations that aim 

at assuring the water quality by active water protection. To achieve these aims, 

measuring systems and instruments that permit an examination and control of the 

water bodies are required. 

A large threat of waters - which can hardly be evaluated so far - is posed by 

hormonal effective substances that are classified as environmentally hazardous, the 

so-called endocrine disrupters (ED). Among them are all substances that influence 

the endocrine system: besides the natural hormones these are the synthetically 

made hormones as well as other industrial chemicals that act like hormones or 

interfere with the hormonal regulation otherwise. 

For fish and amphibians, these hazardous substances and their toxicological effects 

are investigated largely. It is known that a concentration of 9 ng/L of the female 

sexual hormone 

17β-Estradiol in water causes a male roach to produce a female egg yolk protein 

(Vitellogenin) that under ordinary circumstances can only be found in female fish. 

The concentration of the synthetic hormone 17α-Ethinylestradiol in water that causes 

a hormonal reaction is even lower. The male minnow and the male rainbowfish 

produce the female egg yolk protein at a concentration of 1 ng/L resp. at 0,1ng/L. 

For that reason this protein is a sensitive biochemical indicator and detecting this 

protein in fish indicates the pollution of waters with endocrine active substances. 

More signs of hormonal effects of these substances on different animals and in 

different regions can be found: frogs develop deformities and mussels develop 

1 European Environmental Framework Directive 2000/60/EG of the European Parliament and the European 

Council for determining a framework for joint regulation regarding water management policy (October 23 rd , 

2000). 



2 

defective shells. In Florida alligators showed damages of the genitals after a DDTaccident. 

No direct danger for the humans could be detected so far, however several 

phenomena are being attributed to the effects of endocrine active substances. 

A widespread hypothesis by Skakkebeak and Sharpe indicates that the quality 

decrease of spermatozoa of the western males is caused by the influence of 

endocrine disrupters. Additionally an increased risk of breast cancer for females is 

known for the artificially produced 17β-Estradiol. 

A direct effect on humans, e.g. by drinking water that demonstrably contains 

endocrine substances, could not be determinded so far, but the current research 

assumes an endangering of adolescent children and embryos. 

The biggest endangering of humans by endocrine active substances is the fact that 

these substances imitate human hormones. 

With specific hormones the production of organs in the embryo can be controlled. 

Therefore the presence of an effective hormone in the body at a not intended point of 

time may cause badly damages of the organs. 

The most important industrial chemicals containing endocrine active substances are 

17α-Ethinylestradiol, Bisphenol A, Nonylphenol and Phthalates. 

Between the endocrine disrupters, the so-called birth control pill hormon 17α- 

Ethinylestradiol is considered as being especially active (below 0,1 ng/L). It can be 

found in sewage water due to human excrements and the disposal of pharmaceutical 

products into sewer systems. 

Bisphenol A is employed in the production of plasticisers, resins, lacquers, 

colourings, pesticides and antioxidants. By the elutriation of material the substance 

gets into the environment. 

Nonylphenol often occurs as a tenside in industrial cleaners. Although these are 

voluntarily not used anymore by the industry since 1992, Nonylphenols are still a 

component of import products as adhesives as well rubber and caoutchouc products. 

Phthalates are plasticisers employed in the production of PVC and other synthetics 

that can be elutriated and reach the sewage water where they can develop a strong 

toxicity for aquatic creatures. 

The examples for endocine active substancec can be continued and the number of 

toxic substances is increasing. A current list of endocrine disrupters was introduced 



3 

by Wenzel, Müller und Ternes in 2003 in their study about those disrupters in 

drinking water. 

However not discussed in detail, biocides 2 , pesticides 3 and POP´s (Persistent 

Organic Pollutants 4 ) play an important role that also exhibit a large relevance as 

endocrine active substances. 

The importance of the endocrine active substances increases due to the many 

possibilities of entering the environment. As the main input pathway in the 

environment the water and weather cycle can be considered. Latest knowledge 

demonstrate the connection of input pathways over the whole globe. Within some 

days harmful substances can be carried over the globe and cause pollution in a 

different region. 

An important input pathway as well is the communal and industrial sewage. 

Conventional sewage treatment plants show a very different efficiency in the 

elimination of complex chemical substances. Therefore endocrine disrupters may be 

eliminated only insufficiently. 

These remarks show the significance of endocrine active substances for the whole 

society and therewith the potential danger for humans and animals. Through this the 

necessity to measure these substances precisely with reliable measuring systems 

and instruments becomes clear. 

The measuring of small concentrations is especially required to establish dose-effectrelations 

and to evaluate the efficiency of sewage treatment techniques. 

Present measurement technologies for the recording of water pollution use gas 

chromatography and mass spectrometry as standard analysis. Two approved 

methods that however are extensive, cause high costs, take up a lot of time, require 

qualified personnel as well as a special and expensive instrument. Furthermore 

before the actual measuring, sample enrichment and a clean-up step that is 

supposed to minimise matrix effects (influences on the measurement results due to 

surrounding conditions) have to be carried. Both steps introduce new sources of 

error, lower reliability and reproducibility of results and increase the costs and 

duration of the analysis. 

2 Among them TBT (Tributylzinn) 

3 Among them Isuproturon, Dioron, Endosulfan, Atrazine 

4 Among them DDT, Dioxin, PCB´s, Furane 



4 

An alternative is a method that has been successfully applied in medical science – 

the immunoassays in the form of ELISA-Tests (Enzyme-Linked ImmunoSorbent 

Assay). The transfer of this method to the field of environmental analyses is new and 

has not been ventured yet, since concentrations are in nanogram in this application 

field. 

To get an idea of the dimensions: a concentration of 1 ng/L equals - regarding the 

endocrine disrupters - e.g. 200g 17α-Ethinylestradiol in the Chiemsee, the second 

largest lake in Germany. Measurements in this range of concentration could not be 

accomplished reliably with ELISA-Tests so far. 

On this account the quo data GmbH and the project partners, the University of Bonn 

(Germany) and the University of Aveiro (Portugal) as well as the Federal 

Environment Agency GmbH Vienna (Austria), applied a demonstration project within 

the framework of the support programme LIFE III – Environment which aimed at 

developing ELISA-Tests for a special substance for the first time and optimising them 

by statistical analysis methods in order to obtain valid results. 

Together with the project partners, the quo data GmbH met this challenge and 

developed an automated analytical system for the determination of endocrine active 

substances (examplarily for the estrogen 17ß-Estradiol) within the limits of the project 

duration. 


Layman Report . Realization 

5 

2 Realization 

2.1 Immunoassays 

Immunoassays are chemical tests used to detect or quantify a specific substance, 

the analyte, in a blood or body fluid sample, using an immunological reaction. 

Immunoassays are highly sensitive and specific. Their high specificity results from 

the use of antibodies and purified antigens as reagents. An antibody is a protein 

(immunoglobulin) produced by B-lymphocytes (immune cells) in response to 

stimulation by an antigen. Immunoassays measure the formation of antibody-antigen 

complexes and detect them via an indicator reaction. 

The purpose of an immunoassay is to measure an analyte. Common uses include 

measurement of drugs, hormones, specific proteins, tumor markers, and markers of 

cardiac injury. Radioimmunoassay (RIA) is a method employing radioactive 

isotopes to label either the antigen or antibody. The major advantages of RIA, 

compared with other immunoassays, are higher sensitivity, easy signal detection, 

and well-established, rapid assays. The major disadvantages are the health and 

safety risks posed by the use of radiation and the time and expense associated with 

maintaining a licensed radiation safety and disposal program. 

Enzyme immunoassay (EIA) as developed as an alternative to radioimmunoassay 

(RIA). These methods use an enzyme to label either the antibody or antigen. The 

sensitivity of EIA approaches that for RIA, without the danger posed by radioactive 

isotopes. One of the most widely used EIA methods is the enzyme-linked 

immunosorbent assay (ELISA). 

For a long time immunoassays belong to the routine methods in biochemistry and 

medical diagnosis. However in the last 10-15 years their importance in different areas 

like the environmental analysis and food inspection increased intensely. They are 

used for the determination of environmental contaminates (aromatic hydrocarbons, 

pesticides or explosives in soil or water), food additive and pharmaceuticals. 

For the determination of substances with small sizes of molecules, immunoassays 

compete with chromatographic methods that are mainly applied today. 

Immunoassays have several advantages compared to other analytical methods: they 

are fast and inexpensive and the analysis does not require long-winded clean-up 



6 

steps for the samples. Thereby a high number of samples can be analysed in a short 

time and the needed sample volume is low. Additionally this method is suited for an 

automation. 

2.1.1 ELISA 

The first step of an ELISA is coating a microtiter plate with antibodies that can bind to 

the analyte. For this purpose a small amount (e.g. 5 µL) of the antibody serum is 

diluted (e.g. 1:50.000) in a special buffer solution. This buffer solution corresponds to 

the characteristics (pH value, ionic strength) of the serum fluid in blood from where 

the antibodies were being isolated. From this solution a defined volume (200 µL) is 

being pipetted in the wells of the microtiter plate. The plate is shaken over a defined 

time and the antibodies adsorb on the surface. Afterwards excrescent antibodies are 

edulcorated by a special washing buffer. Now the sample that contains the analyte 

(i.e. the substance to be determined) is added. The next step consists of a short 

waiting time (pre incubation). While this time, the molecules of the analyte can bind to 

the antibodies that adhere to the surface. Following the so-called enzyme tracer is 

added. An enzymer tracer is an analyte molecule that is bound to an enzyme that 

catalyses a specific reaction, at the end the product of this reaction can be 

measured. The enzyme tracer is also diluted with a specific buffer solution. During a 

second incubation time, the enzyme tracer competes with the analyte over the 

binding sites of the antibody due to the structural similarity. The unbound analyte and 

enzyme tracer are being washed out afterwards, while a part of the added enzyme 

tracer is remaining bound on the surface. The amount of the bound enzyme tracer 

can be made observable by adding a special substrate solution. This solution 

contains two substances and their reaction is catalysed by the bound enzyme. 

Thereby the colourless input product becomes a coloured reaction product that is 

optically ascertainable. In the last step the interruption of the catalysed reaction is 

caused by adding sulphuric acid. Again a change of colour is taking place. 

Since enzyme tracer and analyte competed over the available binding sites, from the 

intensity of the colouration the amount of the bound analyte can be concluded. Since 

only the enzyme tracer possesses the property to catalyse, an intensive colouration 

indicates a big amount of bound tracer, but small amount of bound analyte while a 

faint colouration indicates a small amount of bound tracer and a big amount of bound 



7 

analyte. The intensity of the colouration (optical density) is mesaured by a microtiter 

plate photometer. 

2.2 Statistical analysis methods 

Besides the immunochemical standardisation of the ELISA-Tests, statistical methods 

for the design of experiments and the analysis of the samples as well as the 

measurement results are of utmost importance. 

The randomised pipetting of the microtiter plate minimises measurement errors by a 

defective microtiter plater. In addition to this possibility to arrange the samples, also a 

calibration on the basis of standards can be carried out by using a regression, the 

maximum-likelihood-estimator. Following with the help of the calibration curve that 

depends on the standards or with the help of the robust mean value of the samples a 

trend can be calculated. With this trend analysis the estimation of the concentrations 

by including all samples is carried out. 

For very precise results that are unassailable in front of the court, the analysis 

software offers the possibility to specify the measurement uncertainty and the limit of 

detection by the Bootstrap method. 

2.3 Pipetting automat 

The ESTR-A-LISER has been designed on the basis of specifications from the 

optimisation of ELISA-Tests as well as from the statistical preliminary analyses. 

The essential part of the instrument is a pipetting automat that is controlled by a 

newly developed software which ensures a narrow control of the whole measurement 

procedure. 

The automat enables a time and amount exact pipetting of samples, standards and 

reagents. The software also controls the tempering device that keeps the automat on 

a stable temperature during the procedure. The temperature can be determined 

within the range of 18°C and 40°C. 

Before starting the measurements, all single steps of the ELISA are defined in the 

control software, including the volumes and waiting times. Furthermore the order in 

which the samples and standards are finalised is defined. Afterwards all necessary 

reagents (samples, standards, immunochemical reagents) as well as the microtiter 

plate are placed into the automat. This is the only manual step during the test. 

Thereby effects by different users are almost completely eliminated. The necessary 


Layman Report . Results . Effects on the environment 

8 

buffer solution that is needed for the washing step is provided from a container 

outside of the pipetting system. During the test, the pipetting needle is cleaned in the 

wash station between every single pipetting step to avoid the displacing of reagents. 

The absorption reader for the determination of the optical density at the end of the 

procedure is integrated in the automat and forwards the measurement result to the 

control software automatically. The integrated analysis software provides a detailed 

report of the results. Due to the almost complete automation the instrument is easy to 

operate. 

3 Results 

By means of highly sensitive immunoassays and standardised ELISA-Tests very 

small limits of detection were received without the complex clean-up steps. 

The statistical methods ensure reliable measurement values, since the determined 

concentrations are specified together with e.g. the extended measurement 

uncertainty. 

Within the project, the prototype of the ESTR-A-LISER was employed successfully in 

several validation and field studies for surface water and sewage treatment plants, 

thereby limits of detection of 2 ng/L were obtained. In the routine operation between 

10 and 20 samples per day can be analysed by the automat. 

4 Effects on the environment 

With the examples of 17β-Estradiol and 17α-Ethinylestradiol the successful 

demonstration project showed that by means of ELISA-Tests with an adequate 

standardisation and automation in combination with statistical methods small limits of 

detections can be obtained validly. This technology permits the production of 

inexpensive automats that easily can be adapted to further ELISA-Tests for the 

determination of other substances. 

Since by the EU – Environmental Framework Directive a multitude of substances is 

classified as potentially hazardous to water, a measuring system that can be applied 


Layman Report . Transferability . Dissemination 

9 

flexibly to a great number of substances could be created by these technologies. 

Thus an important instrument for the environmental analysis could be the result in the 

near future. 

5 Transferability 

The transferability of the used methodology and the immunochemical procedure 

depends on the properties of the substance to be measured. The already mentioned 

water hazardous substances of the EU-Directive possess relatively different chemical 

structures and properties. The characterisations and prerequisites that underlie the 

immunoassays require specific binding properties of a substance in order that a 

measurement with ELISA-Test is possible. For every substance it has to be verified if 

the requirements are met. 

6 Dissemination 

On many occasions the quo data GmbH presented the results of the demonstration 

project ESTR-A-LISER in front of laymen and experts. 

Likewise many PR-activities can be named where the quo data GmbH informed 

about the research project in form of press releases for the daily newspapers and 

professional articles in scientific journals. 

Additionally the quo data GmbH and the project partners are taking part in scientific 

symposiums that deal with endocrine active substances. At this scientists present 

particular aspects of the research and development project in form of papers, posters 

and lectures to the interested expert audience 5 . The participation of quo data 

employees in research networks about endocrine active substances also plays an 

important role. Considering the Water Management Initiative of the North Rhine- 

5 Example: Endo-Workshop: ESTR-A-LISER: automated immunoassays for the determination of the endocrine 

active substances 17β-Estradiol and 17α-Ethinylestradiol in surface and sewage water. Dresden 2005. 


Layman Report . Dissemination 

10 

Westphalia as an example, the positive effect of research networks that enables an 

active exchange and information transfer between experts becomes clear. 

An especially good platform for the dissemination of the results offered the final event 

of the ESTR-A-LISER project in the BioZentrum at the University of Bonn, which was 

organised by the quo data GmbH and the project partner on April 8 th 2005. 

A multitude of experts from the fields of analytical chemistry and environmental 

analysis as well as from institutes of water management was invited to this final 

event. 

The daily and scientific press was also invited and many articles about the event 

were released. 

The different activities of the research partner created a wide interest in the project. 

Many requests by practitioners from economy and research regarding the status quo 

or the functionality of the ESTR-A-LISER reached the quo data GmbH. 

Another important component for the communication of the research activities and 

results is the homepage www.estraliser.de which has been constantly updated with 

new information by quo data employees and thereby led to a widespread public 

notice of the research project. 

The permanent contact of the quo data GmbH and the project partners to other 

scientists results in the positive evaluation of the demonstration project which is 

continued as a new project PHARM-A-LISER. With the expansion of the 

measurement system to substances that are endocrine and pharmaceutical active, 

shows their further importance in the future as well as the necessity of precise 

measuring and determination of toxicological substances in the environment. 


Layman Report . Acknowledgement 

11 

7 Acknowledgement 

The quo data GmbH as project coordinator would like to thank: 

European Commission within the framework of the financial support programme LIFE 

III – Environment 

Co-financed by the Development Bank of Saxony (SAB) 

Project partner: 

• University of Bonn . Institute of Agricultural Chemistry 

Karlrobert-Kreiten Straße 13 

53115 Bonn, Germany 

Contact person: PD Dr. R. Schneider 

• Federal Environment Agency GmbH . Analytical Chemistry 

Spittelauer Lände 5 

1090 Wien, Austria 

Contact person: DI P. Hohenblum 

• University of Aveiro . Chemistry Department 

Analytical Chemistry Group 

3810-193 Aveiro, Portugal 

Contact person: Prof. Dr. Esteves

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