**4. Analysis and monitoring of EDCs in environmental samples**

From a historical point of view, the instrumental techniques were first tools to determine trace organic pollutant concentration levels in the environment. With the run of time albo biological methods were introduced into the scientific routine to obtain more comprehensive and reliable information of the pollution levels of given environemtnal compartments. In Figure 7, (A and B below), basic instrumental and biological data together with their short description to present the development of tools in the field of endocrine potency determination with biological methods are presented.

**Figure 7.** (a) Classification of analytical approaches used in order to detect and determine EDCs in the environmental samples and the endocrine potency of different samples. (b) Description of selected bioassays utilized for endocrine potency determination.4.1 BIOLOGICAL METHODS

(b)

#### **4.1. Biological cellular tests used to assess the endocrine potency of the environmental samples**

Cellular biotests are good alternative to traditional analytical procedures, as well as to immunotechniques and methods utilizing living organisms as biomarkers of exposure to EDC [91]. In these types of biotests, the yeast or human cells (e.g. cells of breast or kidney cancer) are used to determine disturbances in the run of hormonal signaling [92]. The cells can be used in unchanged form or altered with proper bioengineering methods to obtain the proper response of cells to the presence of specific chemicals belonging to EDC [93]. For example, the estrogen gene can be introduced to the yeast cells from human, fish, or other species genome. In such case, the term of estrogen equivalent concentration (EEC) finds its application in the form of the formula [42]:

$$EEC\_i = C\_i \cdot EEF\_i \tag{1}$$

where:

**4. Analysis and monitoring of EDCs in environmental samples**

**DETERMINATION OF EDCs IN THE ENVIRONMENT**

methods are presented.

Fig. 7.A)

Determination of endocrine potency

182 Emerging Pollutants in the Environment - Current and Further Implications

sample estrogenic activity.

fiber in the process based on the evanescence phenomena.

Using bioindicating organisms to observe the endocrine potency of sample

controls ensures possibility of determining the estrogenicity potential [1,44‐46].

light will take place (proportional to amount of active chemicals in the sample tested) [1,48,49].

the evanescent response around the glass fiber is obtained and quantitatively measured [35,42].

Tests for estimating the endocrine potency of the samples tested

E‐SCREEN CALUX YES/YAS SCCoR

IR ‐ biomagnification

E‐SCREEN

CALUX

YES/YAS

IR‐ biomagnification

BiocoreTM

EndotectTM

RIANA

RBA

potency determination.4.1 BIOLOGICAL METHODS

From a historical point of view, the instrumental techniques were first tools to determine trace organic pollutant concentration levels in the environment. With the run of time albo biological methods were introduced into the scientific routine to obtain more comprehensive and reliable information of the pollution levels of given environemtnal compartments. In Figure 7, (A and B below), basic instrumental and biological data together with their short description to present the development of tools in the field of endocrine potency determination with biological

> Methods of direct determination of EDCs (non‐cellular test)

Methods of quantitative determination of EDCs

1. sample treatment, isolation, preconcentration, removing interferents

> 3. detection, identification and quantitative determination of analytes 4. spectroscopic

Methods of indirect determination of EDC

> 2. separation of ingredients

techniques (MS)

Biosensors

RIANA

RBA

EndotectTM

BiocoreTM

• Presence of estrogen induces response of cells proliferation rate being proportional to estrogen concentration in the sample studied. The cells line being used are estrogen‐defendant ones, e.g. cell lines of human breast cancer MCF‐7. These cells are being incubated in the presence of sample testes while in the control sample either the 17 β‐estradiol is present (positive control) or not (negative control). Comparison of cells proliferation rates in samples tested and

• Cells show sensitivity to given types of chemicals, these may be estrogenic, androgenic or xenoestrogenic substances (e.g. dioxin‐like chemicals). Similarly to YES/YAS assay the main receptor gene is hyphenated with the reporting gene. If the sample tested poses the activity to proper group of cells the emission of

• The human estrogen receptor gene is introduced to the *Saccharomyces cerevisiae* (in case of YES test, for YAS it is androgenic gene) hyphenated to LacZ reporter gene [43,47]. Such cells are becoming estrogen activity controls. In case of substance's estrogenic potential it binds the estrogen receptor and is signaling presence of estrogenic chemical initiating the reporting gene activity. The reporting gene is coding synthesis of β‐galactosidase which afterwards takes part in process of transforming the dye present in the sample solution from yellow to red one [1,48]. The intensity of red color is directly related to the

• There are numerous mammalian cell lines utilizable in the SCCoR (*Single Cell Coactivator Recruitment*). Genetically introduced ability of indicator to

• It is the only cell test not created with genetic engineering methods engagement or elaborating cells 'proliferation potency. Application of this test is based on fact that changes in cells functioning can be assessed due to changes in IR radiation changes caused by the cell organelles. The IR microscopy is used for this purpose. The IR radiation in the mid‐IR range is characterized with sufficiently low energy not destroying the cell organelles [51]. The light

• Utilizes the plasmon resonance phenomena) in order to assess reactivity between chemicals and estrogenic receptor at the detector surface being the golden plate. The microflow system enables of transporting the sample stream at the surface of golden plate and the system of optical detection enables measurement of the plasmon surface resonance. Numerical value of this parameter is directly proportional to the concentration of xenoestrogen tested. It is also possible to use other physical phenomena to detect the estrogenic activity e.g. the piezoelectric effect or chelation of the nickel atoms

• Based on activity of human estrogenic receptors (hER) which are bind to promoters enabling fluorescence measurable with new type of detector called evanescence‐type detector*.* For this reason the hER and promoter are bound on the glass fiber and the total fluorescent response is being measured along the

• Used to determine the estrogenic properties of selected analytes. Its usability has been tested for assessing presence of xenobiotics (atrazine, isoproturone, estrone) in the water samples [57]. The method utilizes laser to fluorescently induce the antibodies that is specifically bound to analyte. Similarly to EndotectTM

• Based on competition between radiolabelled estradiol and the tested EDC to bind to the estrogenic receptor active site [58]. The purified protein of human estrogenic receptor is being added to the sample of standards mixture – estradiol (of known concentration and labeled with tritium) and ligand being determined (with increasing concentration) and incubated. During this period the receptor‐ligand complex settles on the surface of hydroxyapatite followed by

(a)

fluorescence enables unique possibility of distinguishing whether or not the analyte is agonist or antagonist of the estrogen receptor in the cell [50]. SCCoR

dispersion is being measured with set of 128 sensors and the response is calibrated against naturally functioning cell.

Immunoanalytical techniques

(b)

**Figure 7.** (a) Classification of analytical approaches used in order to detect and determine EDCs in the environmental samples and the endocrine potency of different samples. (b) Description of selected bioassays utilized for endocrine

flushing out the unbound ligand from the surface. The product bound to the surface is radiolabelled thus can be measured.

*Ci* – concentration of particular EDC in the sample studied*EEFi* – numerical value of the en‐ docrine equivalent factor

Numerical values of this factor determines in the relative way the endocrine character of given the chemical in relation to the endocrine potency of the reference chemical, most often estradiol or 17β-estradiol.

In this way, the endocrine potency can be described using the equation [32]:

$$
\Sigma EEC\_i = \Sigma EEC\_i \tag{2}
$$

In Table 5, there are given numerical values of EFF of selected chemicals belonging to EDC.


**Table 5.** Numerical values of EFF of selected chemicals belonging to EDC

In Table 6, the data concerning the analysis of various samples with bioassays is given.



**Table 6.** Concentrations of selected EDCs determined in environmental samples using biological and instrumental methods.

#### **4.2. Analytical and instrumental methods used for detecting EDCs**

Detection, identification, and quantitative determination of EDC-like chemicals are currently achieved mostly with chromatographic techniques. Prior to chromatographic separation and detection (mostly with mass spectrometry or time of flight detection), complex and time- and labor-consuming sample treatment are necessary as presented in Figure 8 (as exmple on the basis of data revision [67,68]).

**Figure 8.** The schematic presentation of selected estrogen determination in sewage samples with LC-MS.

Figure 8. The schematic presentation of selected estrogens determination in the sewage samples with LC-MS.

#### **•** Pre-treatment

**No. ANALYTES**

1 Bisphenol A

Estradiol and estrone

Estriol

Alkylphenol ethoxylates

1,3,4,6,7,8 hexahydro-4,6,6,7,8,8 hexamethylcyclopenta[*g*] -2-benzopyran (HHCB)

Testosterone

Estrone, estradiol

Estradiol

6 Estradiol

<sup>5</sup> Estrone Sewage

17 α-ethinilestradiol Surface and

2

3

4

7

**SAMPLE TYPE**

184 Emerging Pollutants in the Environment - Current and Further Implications

Bottled water samples

River water

Wastewate r

**EXTRACTIO N**

Extraction on C-18 columns

Filtration with a glassfiber filter, SPE (HLB)

Norethindrone 15 ng/dm3

Levonorgestrel 15 ng/dm3

River water SPE (C18)

Cleaned wastewater s

> sewage waters

Testosterone 0.3 ng/dm3 >0.4 ng/dm3

Ethinylestradiol 0.1 ng/dm3 >0.2 ng/dm3

Bisphenol A 5–500 *μ*g/dm3 0.08–1.55 *μ*g/dm3 17*β*-estradiol 0.05–1 *μ*g/dm3 0.57–1.73 *μ*g/dm3 17α-ethinylestradiol 0.12 ng/cm3 0.5–1000 ng/cm3

Musk xylene 0.5 ng/g <LOQ–13.0 ng/g

Nonylphenol 1.3 ng/dm3 <LOD–118

Ethinylestradiol - 1.4–19.4 ng/dm3

**DETECTION TECHNIQUE**

Radioimmunoass ay

ELISA

LC-MS/MS ELISA

Radioimmunoass

ELISA

ELISA

Estradiol - 0.085±0.010

effluents SPE (C18) MCF-7 - 70 ng/dm3 [63]

SPE (C18) YES - 1.1–11.1 ng/dm3 [64]

**LOD**

20–1000 *μ*g/dm3

ELISA 0.05 ng/cm3 0.01–1.33 ng/cm3 [59]

0.3 ng/dm3 1.2–9.4 ng/dm3

0.1 ng/dm3 >0.5 ng/dm3

1.3 ng/g <LOQ–62.1 ng/g


ng/dm3

YES - 0.8–35.5 ng/dm3 [62]



µg/dm3

ay - 3.2–4.3 ng/dm3

**Concentration determined**

> 0.724–78.15 *μ*g/dm3

**References**

[60]

[61]

[65]

Water and sewage samples collected for determination of endocrine disrupting compounds contain other various impurities. At this moment of sample treatment, the majority of samples is subjected to filtration to remove solid impurities. Pre-existing coagulation facilitates the filtration. Then the sulfuric acid, hydrochloric acid, methanol, or formaldehyde can be added to the samples to obtain the appropriate pH. The addition of one of these compounds also prevents the degradation of the assayed analytes. Samples are stored in darkness and low temperature, usually <4o C, in bottles made of amber glass to avoid photodegradation of analytes [69, 70].

#### **• Extraction**

The usual method of trace organic pollutants extraction is solid-phase extraction (SPE). The first step is filling the column-conditioning of the sorbent. After conditioning, the column is percolated with test sample. The target analytes and other compounds absorb in the sorbent. The next step is the elution of interfering compounds from the column. At the end, the target compounds are eluted with proper solvent mixtures. Solid phase extraction can be either: online where the extraction is directly integrated into the system of the quantitative analysis; or it may be off-line where the extraction column is not connected in any way with the gas or liquid chromatograph. In on-line SPE, full automation of the process occurs and the method is characterized with ease of application of samples and a large throughput. However, despite the higher costs off-line SPE, it is often used because when combined with GC, water must be removed totally prior to eluting analytes [70].

When choosing the appropriate sorbent for the SPE column, one has to take into account the chemical and physical properties of assayed compounds. One of the most frequently used cartridge packing is Oasis HLB. It allows to obtain high recovery of both the acidic, basic, and neutral compounds. Recovery exceeds 70%. This sorbent can be used for the large range of pH of the samples, ranging from 2–7. Lichrolut ENV+ cartridges are used when the sample has a low pH and contains polar organic compounds or when sample contains neutral drugs and its pH is neutral. Columns packed with C-18 are suitable for non-polar or moderately polar compounds. The extraction process must then be optimized: sample volume, the volume of sorbent cartridge, percolation rate, type of eluent and its volume. The elution solvent is selected depending on the properties of the compounds eluted and its elution strength.

A less frequently used method is the Solid Phase Microextraction (SPME). It depends on the distribution of the analyzed chemical compounds between the sample and the sorbent. This method is fast, moderately new, and an easy method of extraction. SPME coupled with GC content allows the study of semivolatile, volatile, and non-polar analytes. More difficult is the combination of this technique with liquid chromatography. Non-volatile compounds are not totally desorbed during the thermal desorption. SPME has many advantages thus it is more attractive than the SPE method, but has a more restricted choice of sorbent and too little sorption capacity. Therefore, the parameters of this technique are still optimized for wider application and greater sensitivity. Samples after SPE or SPME are concentrated using evaporation under a gentle nitrogen stream [70].

Another common method of extraction is liquid-liquid extraction (LLE). LLE relies on shaking the sample with an organic solvent for a specified period of time. One can perform this operation several times. The organic phase is separated from the water, and mixture of all the extracts is obtained. The resulting solution is dried, for example, using anhydrous sodium sulfite. When the sample volume is sufficiently small, determination of analytes can be started [71].

## **• Determination of concentration levels of target analytes**

filtration. Then the sulfuric acid, hydrochloric acid, methanol, or formaldehyde can be added to the samples to obtain the appropriate pH. The addition of one of these compounds also prevents the degradation of the assayed analytes. Samples are stored in darkness and low

The usual method of trace organic pollutants extraction is solid-phase extraction (SPE). The first step is filling the column-conditioning of the sorbent. After conditioning, the column is percolated with test sample. The target analytes and other compounds absorb in the sorbent. The next step is the elution of interfering compounds from the column. At the end, the target compounds are eluted with proper solvent mixtures. Solid phase extraction can be either: online where the extraction is directly integrated into the system of the quantitative analysis; or it may be off-line where the extraction column is not connected in any way with the gas or liquid chromatograph. In on-line SPE, full automation of the process occurs and the method is characterized with ease of application of samples and a large throughput. However, despite the higher costs off-line SPE, it is often used because when combined with GC, water must be

When choosing the appropriate sorbent for the SPE column, one has to take into account the chemical and physical properties of assayed compounds. One of the most frequently used cartridge packing is Oasis HLB. It allows to obtain high recovery of both the acidic, basic, and neutral compounds. Recovery exceeds 70%. This sorbent can be used for the large range of pH of the samples, ranging from 2–7. Lichrolut ENV+ cartridges are used when the sample has a low pH and contains polar organic compounds or when sample contains neutral drugs and its pH is neutral. Columns packed with C-18 are suitable for non-polar or moderately polar compounds. The extraction process must then be optimized: sample volume, the volume of sorbent cartridge, percolation rate, type of eluent and its volume. The elution solvent is selected

A less frequently used method is the Solid Phase Microextraction (SPME). It depends on the distribution of the analyzed chemical compounds between the sample and the sorbent. This method is fast, moderately new, and an easy method of extraction. SPME coupled with GC content allows the study of semivolatile, volatile, and non-polar analytes. More difficult is the combination of this technique with liquid chromatography. Non-volatile compounds are not totally desorbed during the thermal desorption. SPME has many advantages thus it is more attractive than the SPE method, but has a more restricted choice of sorbent and too little sorption capacity. Therefore, the parameters of this technique are still optimized for wider application and greater sensitivity. Samples after SPE or SPME are concentrated using

Another common method of extraction is liquid-liquid extraction (LLE). LLE relies on shaking the sample with an organic solvent for a specified period of time. One can perform this operation several times. The organic phase is separated from the water, and mixture of all the extracts is obtained. The resulting solution is dried, for example, using anhydrous sodium

depending on the properties of the compounds eluted and its elution strength.

C, in bottles made of amber glass to avoid photodegradation of

temperature, usually <4o

removed totally prior to eluting analytes [70].

186 Emerging Pollutants in the Environment - Current and Further Implications

evaporation under a gentle nitrogen stream [70].

analytes [69, 70].

**• Extraction**

Liquid chromatography combined with tandem mass spectrometer is the most widely used analytical technique because it allows ion fragmentation that is needed for accurate and precise determination of the analytes. LC-MS/MS determines the compounds that have identical molecular weight but disparate product ions. Using MS/MS increases the selectivity and sensitivity of the method. Atmospheric pressure chemical and electrospray ionization (APCI and ESI) are modes of ionization interfaces that are the most widely used with LC-MS/MS. Low or medium polar compounds are determined by APCI, and the analysis of polar analytes is conducted using ESI. The main use of liquid chromatography is to determine non-volatile, polar, or degradable under high temperature substances. For example, beta-blockers and antibiotics can by analyzed using only LC-MS/MS [70].

One of the biggest difficulties with LC-MS/MS is interference in the matrix effects. This effect causes the strengthening or suppression of the analyte signal, thus producing erroneous results. When contaminated environmental samples are analyzed, for example wastewater, it is necessary to perform efficient clean up of samples. The process of optimizing the analytical methods, such as of liquid chromatography, involves making a series of studies to determine the parameters that give the best results for all determined substances. MS parameters are also optimized for each analyte by conducting the flow injection analysis (FIA). To obtain credible results, it is needed to optimize the separation of compounds by liquid chromatography and mass spectrometry parameters.

In case of GC-MS analysis, the matrix effects occur less frequently than during the analysis of LC-MS/MS. The disadvantage is that it is a more time-consuming technique and requires complex preparation of the sample in case of derivatization step.

As a result of derivatization of polar components, their analogs are less polar and more thermostable. It increases the sensitivity of analysis but also increases the loss of sample by performing additional operations. A negative aspect of derivatization is the use of carcinogenic and toxic reagents. Derivatization reaction should allow the detection of analytes that have polar functional groups. It is effective when the reaction occurs in a given time with 90% efficiency.

In literature there can be found many applications of GC-MS for the analysis of drugs, PAHs, PCBs, and other pollutants in water and wastewater samples [70].

In Table 7, there are presented examples of determining the EDCs in environmental samples, mainly in samples of river, drinking, surface, and sewage water. They are also determined in samples of food, air, in the tissues of the Chinese sturgeon, in house dust, and in human serum.

It can be stated that LC-MS/MS and GC-MS are the most often used techniques in determining EDCs. Other methods such as high performance liquid chromatography with fluorescence or diode array detector are less frequently used in the analysis of EDCs. The best results are given by the combination of LC-MS/MS with GC-MS.

On the basis of data collected in Table 1, it can be concluded that people and other living organisms are exposed to EDCs throughout their entire life. Even low concentrations levels of EDCs can have a significant impact on animal and human health and the existence/health state of entire populations. Many people do not realize that even these small amounts can be significantly harmful after long exposure. The concentration of some compounds from the EDC groups has decreased because their application was banned. Unfortunately, they have long half-lives, so trace amounts are present in the samples assayed decades after the release of specific chemicals.

Determination of EDCs poses many challenges and problems. Newer and more accurate analytical methods are required and need to be used by the scientific community. There are more and more articles/books about detection of EDCs in environmental samples and their harmfulness. Application of EDC should be reduced as far as possible because contamination of these compounds poses a huge risk to the environment.



On the basis of data collected in Table 1, it can be concluded that people and other living organisms are exposed to EDCs throughout their entire life. Even low concentrations levels of EDCs can have a significant impact on animal and human health and the existence/health state of entire populations. Many people do not realize that even these small amounts can be significantly harmful after long exposure. The concentration of some compounds from the EDC groups has decreased because their application was banned. Unfortunately, they have long half-lives, so trace amounts are present in the samples assayed decades after the release

Determination of EDCs poses many challenges and problems. Newer and more accurate analytical methods are required and need to be used by the scientific community. There are more and more articles/books about detection of EDCs in environmental samples and their harmfulness. Application of EDC should be reduced as far as possible because contamination

> **DETECTION TECHNIQUE**

> HPLC-MS/MS

Carbon nanotubetyrosinase based amperometric enzymatic biosensors

LC–ESI-MS

Estrone 2.5 ng/dm [75] <sup>3</sup> <LOD–0,022

**LOD**

**Measured concentratio n**

ng/cm3

ng/dm3

ng/dm3

ng/dm3

0.02 µM 0.5 µg/dm3 [74]

µg/dm3

µg/dm3

µg/dm3

6.3 ng/dm3 <LOD–0,007

LC–APCI-MS 1.61 ng/dm3 0.002–0.003


11.6 ng/dm3 1.49–29.9

**REFERENCE S**

[72]

[73]

of these compounds poses a huge risk to the environment.

**EXTRACTION**

LLE (diethyl ether)

River water SPE LC-MS/MS

LiChrolut RP-18 SPE

RP-18 SPE

hydroxyprogesterone - 0.1 ng/cm3

estradiol - 0.1–50.0

acid 2.3 ng/dm3 3.24–9.35

Erythromycin 13 ng/dm3 3.08–134.5

Androstenedione - 0.1 ng/cm3

**SAMPLE TYPE**

188 Emerging Pollutants in the Environment - Current and Further Implications

Human serum

Drinking and surface water

> Natural water

Desethylatrazine LiChrolut

of specific chemicals.

**No. ANALYTES**

Testosterone

17-

Cortisone and

DEET

2,4-dichlorobenzoic

3 Bisphenol A

Bisphenol A

1

2

5



**No. ANALYTES**

1,1,1-trichloro-2,2 bis(*p*-chlorophenyl) ethane (DDT)

Estrone

Sulfadiazine

Ethinylestradiol

Testosterone

Bis(2-ethylhexyl)

Progesterone

14

15

17

**SAMPLE TYPE**

190 Emerging Pollutants in the Environment - Current and Further Implications

stomach, intestines, adipose, gill, pancreas, kidney, gallbladder, and roe from 13 female Chinese sturgeons

> Waste water

Drinking water

17α-ethynylestradiol The CLLE

<sup>16</sup> Atrazine Wastewater SPE (Oasis

**EXTRACTION**

methanol mixture solution)

Wastewater SPE (Oasis) GC-MS

SPE (Oasis HLB)

HLB)

extracts derivatization

17β-estradiol 11.2 ng/dm3 10.9–224

Bisphenol-A 17.4 ng/dm3 15–890

4-tert-Octylphenol 8.5 ng/dm3 29–710

17α- Ethynylestradiol 10 ng/dm3 <487 ng/dm3

Estriol 5 ng/dm3 4648–22633

SPE (HLB) LC-MS-ESI

phthalate 7–20 µg/dm3

HPLC-MS/MS

GC-MS

**DETECTION TECHNIQUE**

**LOD**

**Measured concentratio n**

0.2 ng/g <LOQ–480

5.6 ng/dm3 21–128.5

ng/g

ng/dm3

ng/dm3

ng/dm3

ng/dm3

ng/dm3

5.7–30.8 ng/dm3

0.116–0.214 µg/dm3

0.073–0.831 µg/dm3

0.11–0.199 µg/dm3

1 ng/dm3 6–50 ng/dm3

LC-MS/MS 1118 ng/dm3 [83]

**REFERENCE S**

[81]

[82]

[84]


**Table 7.** Concentrations of selected EDCs determined in environmental samples using instrumental methods.

### **5. Summary**

The poor state of knowledge about the mechanisms of action and effects of EDC chemicals has forced the interdisciplinary scientific teams to intensify their work in the subject. Nowadays, many institutes are carrying out research focused on exploring the properties and metabolic pathways of EDCs and their mixtures in the environment. Good knowledge about the environmental fate, endocrine potential, and distant toxic effects of ecoestrogens will allow to estimate the levels of the pollution and minimal exposure on certain compounds. Moreover, this knowledge can be applied for upgrading the common tools used to detect and perform quantitative determination of EDCs, and can be the basis for the development of new techni‐ ques that will provide information about the composition of the sample and about its endocrine potential [94,95].

The discovery of micropollutants occurring in the environment resulted in new methodologies being put into the analytical practice. These methodologies are developed in two different directions. The first is based on methodological solutions designed to detect, identify, and determine xenobiotics that occur in various environmental samples. For this purpose, instru‐ mental methods such as gas and liquid chromatography with mass spectrometry detection are usually used. These techniques provide reliable information about the presence, quantity, and influence of EDCs.

The second approach is to put into the analytical practice new bioanalytical methodologies. These methodologies allow estimation of the sample endocrine potential, but they do not provide information on which of the sample ingredient is responsible for causing the toxic effect. The results of the analysis of this biological response are valuable source of information for chemists and ecotoxicologists. These results can be the basis for estimating the endocrine potential of the environment exhibited by certain species. Moreover, bioanalytical techniques may be supplementary to the techniques of quantitative and qualitative determination of endocrine disrupting chemicals. It is not possible to estimate the environmental risk of EDC presence based only on the information about the sample composition. It is necessary to determine both the magnitude and how in particular the endocrine homeostasis may be impacted by xenobiotics. These tasks can be realized only by using a well-chosen bioassays battery. In the recent years there has been a significant increase in the importance of the biological methodologies in environmental research because of their numerous advantages. It is reflected in the research literature and in the increase in the number of scientific publications on this subject.

In this chapter the information about some of the estrogenic compounds, their environmental fate, and biological influence can be found. Special attention was given paid to the review of the analytical approaches used at the stage of detection and determination of EDCs in the environmental samples. Also a brief characterization of both the cellular and non-cellular bioassays is presented, as well as the information regarding the changes occurring in the bioindicators as results of being exposed to a specific ecotoxins.
