SUMMARY

The development of analytical tools to diagnose situations of stress is key in the evaluation of the environmental deterioration. The study of the presence of the pollutants must go joined to the biological answers. These studies are specially important when they are considered to be emergent pollutants, since they are the generic medicaments, which reach the aquatic systems as consequence of not arranging of filter systems efficiently for these pollutants again type. The adjustment of analytical suitable and efficient methodologies for these pollutants must go joined to the development of technologies 'ómicas', which can play an essential role for a major knowledge of the biological answers.

The zone of study centres on marshes and aquatic ecosystems of the cross-border region of Spain and Portugal. The Bay of Cadiz is considered to be especially, it Odiel River in Huelva, the Natural Place Marimas del Odiel, the Tideland Domingo Rubio, Isla Cristina's Marshes, the River Guadiamar (in the opposite Spain Portugal) and River Formosa (Portugal). Since bioindicadores there were used flat Scrobicularia, Chamalaea hen and Nerea diversicolor, though the study does not exclude the study of any 'ictícola' species or seafood shops of interest comecial or food.

There will use analytical methodologies of last generation like the couplings HPLC-MS, HPLC-ICP-MS, procedures 'proteómicos' based in 2 - D with MALDI-TOF-MS, and techniques 'metalómicas' on multidimensional chromatography, ICP-MS, and spectrometry of masses in tandem.

PURPOSE OF THE PROJECT

Precedents

Between the numerous pollutants that concern the aquatic ecosystems there must be included today the medicaments used for the treatment of human diseases and of the animals. These substances are considered to be emergent pollutants neither for that there does not exist an analytical established methodology and the levels are not known in the aquatic, terrestrial and marine systems, nor the metabolitos that they generate. These compounds are not totally degraded in the filter systems and have a toxicological important action. The potential actions of these compounds are very diverse, different biological mechanisms of defense being able to be activated after an exhibition prolonged to the same ones. These substances can concern numerous functions of the terrestrial or marine organisms, which alter the viability of the populations, and the possibility of use of these organisms as food. As consequence of it, hisinfluence on the conservation of the ecosystems is very critical. Some of these medicaments can concern all kinds of alive organisms. Between these compounds the analgesics can be mentioned, antinflamatórios, antibiotic and antidepressing.

In addition, the increasing production of species is prominent ictícolas and seafood for the human consumption in aquicultural facilities, which is constituting an additional source of pollution, in view of the use of antibiotics for the preservation of diseases. These substances can concern finally the species of the fishing capture. It is necessary to think that though the medicines are not in the habit of being persistent compounds, hisconstant contribution turns them into chronic pollutants of the aquatic organisms.

Purpose

One tries to develop analytical procedures for the study, the characterization and diagnostics of the presence of medicaments of generic character in marshes with ecological importance of the south border of Spain - Portugal, in the zone The Algarve - Huelva-Cádiz.

AIMS OF THE PROJECT

General Aim

1. To develop analytical procedures for the study, the characterization and diagnostics of the presence of medicaments of generic character in samples of marshes with ecological importance of the south border of Spain - Portugal, in the zone The Algarve - Huelva

Specific Aims

1. To design analytical methodologies for the analysis of endocrine disruptors in the ecosystems in study.
2. To apply analytical methodologies in ecosystems of the southwest of Spain - Portugal (region Algarve-Huelva).
3. To study biological molecular signal based on the characterization of enzymes antioxidative, enzymes biotransformadoras, oxidative damages in biomolecule, and levels of 'metalotioneinas' and 'acetilcolinoesterasa'.
4. To study the effects caused by medicaments in diverse bioindicadores of the zone of study: (i) S. page (hepatopancreas and gills) and Nereis diversicolo.
5. To integrate the results and analysis of information.

METHODOLOGY AND PLAN OF WORK

There would be realized a sampling of sediment and organisms(organizations) on stations selected in each of the zones of study in order:

• A. Adjustment of methodology for analysis of medicaments
  
  

  The medicaments will be extracted using a mixing with acetonitrilo-waters down (3:7) in phosphoric way and probe of ultrasounds. The extract purified by means of extraction in solid phase (C-18), it is analyzed by means of liquid chromatography by detector of fluorescence and ultraviolet, using as mobile phase an acetonitrilo-water mixing with tampon phosphate and laurilsulfato sodium.
  
  The penicilinas will determine by means of HPLC of phase reverse and detector of UV. The extraction of the compounds will be carried out in watery phase by means of the rainfall of organic components by a mixing of H2SO4 and wolframato sodium. The cleanliness of these extracts will be realized by means of SPE by an adsorbent 'polimérico' (divinilbenceno-co-N-vinilpirrolidona), it completing by means of extraction liquid - liquid with dietileter. The extracts are worked with anhydride benzoico and I reactivate 1,2,4-Hg the (II)nd-triazol.
  
  The tetracyclines will be extracted by a mixing of acid metafosfórico/metanol (8:2) to 0.5 % in water followed of purification with cassettes C-18. The analysis carries out by means of HPLC with phase reverse and detector UV, using as mobile phase MeCN/NaH2PO4 0.05 M.
  
  The results obtained in the previous procedures will be confirmed analyzing the samples simultaneously by means of HPLC-MS/MS.
  
  Antiinflammatory and analgesics will extract to themselves with a mixing methanol - water (1:1) and probe of ultrasounds. The watery extracts will purify by means of cassettes EXtrelut NT. The analyses will carry out by means of HPLC of phase reverse and elution with gradient using the following mobile phases: A) 20 mm of acetate ammonium 1 acid acetic M; B) methanol; and C) waters down. The detection will be realized by means of spectrometry of masses.

• B. Global evaluation of the presence of medicaments in the ecosystems
  
  There will apply to themselves the analytical methodologies developed previously to samples of sediments and biosigns that will be obtained in a campaign of sampling.
  
  The biosigns selected depending on the experience acquired in the previous studies of NETWORK CONTAMAR and PROTEOBIOMAR, are: the bivalves flat Scrobicularia and Chamelaea hen and the poliqueto Nereis colored.

• C. Study of biomarcadores conventional
  
  The levels will be evaluated of molecular biosign based on the characterization of enzymes antioxidatatives, enzymes bioconverter, oxidatives damages in biomolecules, and levels of 'metalotioneinas' and 'acetilcolinoesterasa' in the biosigns and zones selected.

• D. Studies ómicos ('proteómicos' and 'metalómicos').
  
  The effects of the medicaments will be evaluated using new technologies of massive information about the evolution of the biomolecules (processes of on expression or inhibition) in the biosigns of the ecosystems in study.

  I study them of effect of the medicaments it will be realized using S. page (hepatopancreas and gills) and Nereis colored. Technologies will be in use proteomics (2-D GE with analysis of the critical spots with spectrometry of masses) to verify the changes of multifaceted expression caused by the presence of medicaments; and metalomic technologies  (multidimensional chromatography with detection ICP-MS) to identify the action of the medicaments on the presence of metalobiomolecules.

• E. Coordination and integration of results.
  
  The groups of the consortium will be in permanent contact, thanks to the current telematic resources, for the coordination of his works. In addition workshops will be organized every 6 months of alternative form in Huelva and Faro for exchange of experiences and information, harmonization of results and transfer of methodological knowledge.
  
  With the information that is generated it will be possible establish recommendations on marker actions to avoid the effect of the emergent pollutants considered in the coastal area of the southwest of Spain - Portugal.

TEAMS PARTICIPANTS AND RECORD OF THE GROUP OF RESEARCH

Teams participants

1. Mr. JOSE LUIS GÓMEZ ARIZA. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
2. Ms. MARIA JOAO BEBIANO. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
3. Mr. JULIAN BLASCO. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
4. Ms. TAMARA GARCÍA BARRERA. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
5. Ms. RUT FERNANDEZ TORRES. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
6. Ms. MACARENA GONZÁLEZ FERNÁNDEZ. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
7. Mr. FERNANDO MORENO ROLDÁN. Email: fernando.moreno@ dqcm.uhu.es
8. Mr. JOSE MANUEL MANSO SAYAGO. Email: jose.manso@ dqcm.uhu.es
9. Ms.ROCÍO JARA BIEDMA. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
10. Mr. MANUEL CONTRERAS ACUÑA. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
11. Ms.AMANDA GAGO TINOCO. Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Record of the group of research

The group of research " Environmental Analysis and Bioanálisis " belonging to the University of Huelva (FQM141) and the groups of research of the Teachers M. Bebiano (University of the Algarve - Faro, Portugal) and J. Blasco (Institute of Marine Sciences - Cadiz) that take part in the present project, have developed a very intense and fruitful collaboration in projects and previous studies and there has made possible the present offer in which really innovative methodologies are approached in the field of the Analytical Chemistry and of the 'ómicas', looking for an integration that allows to evaluate the biological response to the effects of the pollutants in the environmental systems.

The group FQM141 of the Andalusian Plan of Research (PAI) goes more than 25 years developing new procedures of analysis, specially in the field of the metallic especiación and methodologies related to the environment and the pollution. The experience of the group in this field has led him to taking part in diverse projects like 1FD97-0610, REN2002-04366 and CTM2006-08960, NETWORK CONTAMAR and PROTEOBIOMAR that constitute clear precedents of the present offer.

The group FQM-141 is pioneering in Spain in the field of the chemical especiación, the metalómica and the environmental studies related to differential and emergent pollutants, having taken part in 11 national projects and 6 Europeans, who have generated approximately 180 publications in the last 15 years. In addition the group has been consulted as expert in environmental topics by diverse organizations and public entities by means of agreements or contracts of research.