1. Raportul Stiintific si Tehnic (RST) in extensocceei.energ.pub.ro/NANOHEALTH/files/etapa_3_...

Cod: PO-04-Ed3-R1-F5

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Anexa 1 - RST

1. Raportul Stiintific si Tehnic (RST) in extenso

1.1 Cuprins

- Obiectivele generale ale proiectului - Obiectivele fazei de execuţie - Rezumatul fazei - Descrierea ştiintifică şi tehnică - Bilbliografie

1.2 Obiective generale

Obiectivul general al proiectului este elaborarea unei tehnologii avansate de diminuare a pulberilor, inclusiv nanoparticule, existente in termocentrale, in vederea reducerii riscului la expuneri profesionale si a imbunatatirii sanatatii si sigurantei la locurile de munca.

1.3 Obiectivele fazei de execuţie

Obiectivul fazei de execuţie este desfăşurarea unor activităţi de cercetare privind evaluarea noxelor profesionale si in special a pulberilor, precum si influenta lor asupra sanatatii personalului.

1.4 Rezumatul etapei In vederea atingerii obiectivelor au fost desfăşurate următoarele activităti: � Evaluarea cantitativa si calitativa a pulberilor, cu estimarea distributiei

dimensionale, a formei si compozitiei chimico-structurale in volumul si la suprafata particulelor; In cadrul acestei etape au fost colectate si analizate cantitativ si calitativ probe de cenuse si carbune concasat sub forma de pulbere, precum si aerosoli de la S.C. Electrocentrale Deva.

� Evaluarea toxicitatii pulberilor pe fractii dimensionale; În cadrul activităţii a fost evaluată in vitro citoxicitatea probelor de cenusa si praf de carbune prin teste comparative cu compusi nanostructurati cu dimensiune similara cu cea a constituientului predominant din probe.

� Evaluarea riscului de sanatate pe un lot reprezentativ selectat din personalul expus la noxe inainte de introducerea tehnologiei de reducere/diminuare a pulberilor; Au fost efectuate analize privind corelatiile intre nivelul PM fine si ateroscleroza, inflamatia sistemica si pulmonara, si alterarea functiei cardiace. Pentru analiza a


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fost selecţionat un lot reprezentativ din personalul care lucrează în medii expuse (ELECTROCENTRALE Deva).

� Coroborarea caracteristicilor chimico-structurale si dimensionale ale

pulberilor cu testele de toxicitate si efectele potentiale asupra sanatatii; În această activitate s-a realizat o legătură între caracteristicile pulberilor (vezi Activitatea 2.2) şi rezultatele obţinute din analizele efectuate pe un grup ţintă din cadrul personalului ELECTROCENTRALE Deva.

� Diseminarea informatiilor pe scara larga privind evaluarea noxelor

profesionale, evaluarea toxicitatii noxelor si coroborarea caracteristicilor chimico-structurale si dimensionale ale pulberilor cu testele de toxicitate si efectele potentiale asupra sanatatii; Au fost elaborate 4 articole după cum urmează: - 1 articol prezentat la o conferinţă organizată în străinătate; - 1 articol prezentat la o conferinţă organizată în România; - 2 articole acceptate spre publicare în două reviste, dintre care una este cotată

ISI � Organizarea si cuantificarea informatiilor obtinute in cadrul etapei si

realizarea planului de management necesar desfasurarii in continuare a proiectului; Se face o analiză a rezultatelor obţinute, în raport cu cele preconizate. Se definesc acţiunile şi termenele aferente etapei nr. 4 a proiectului.

1.5 Descrierea stiintifica si tehnica Activitatea 2.2 – Evaluarea cantitativa si calitativa a pulberilor, cu estimarea distributiei dimensionale, a formei si compozitiei chimico-structurale in volumul si la suprafata particulelor Executanţi: UPB -CCEPM INLPF

In cadrul acestei etape au fost colectate si analizate cantitativ si calitativ probe de cenuse si carbune concasat sub forma de pulbere, precum si aerosoli de la S.C. Electrocentrale Deva.

• Pentru determinarea marimii si distributiei dupa marime a particulelor micronice continute in cenuse si pulberea de carbune s-a folosit un granulometru laser. Am determinat cat la % din totalul volumului de particule e reprezentat de volumul particulelor de o anumita marime.

• Metoda analizei mobilitatii diferentiale a particulelor (DMA). Prin aceasta metoda s-a realizat distributia dupa marimi si concentratia particulelor de cenuse si carbune cu dimensiuni sub 100 nm (nanoparticule).

• Metoda difractiei de raze X- prin care au fost indentificate speciile componente ce sunt in stare cristalina. Totodata s-a facut si o evaluare cantitativa grosiera asupra ponderii fiecarei specii.

• Metoda analizei de dispersie dupa energie (EDAX) – aceasta metoda permite stabilirea compozitiei elementale a probelor.


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• Metoda microscopiei electronice de baleiaj (SEM)- prin care s-au obtinut informatii despre: dimensiune, morfologia si aglomerarea particulelor.

Au fost colectate si analizate 17 probe de cenuse si carbune concasat sub forma de pulbere de la Electrocentrale Deva, prelevate din diferite puncte.

Din diagramele DMA se observa ca in general concentratia de nanoparticule de pulbere de carbune este mare pentru diametrele din domeniul 8-30 nm, cu peakuri de dimensiuni diferite in functie locul unde se efectueaza masuratorile. De asemenea este de notat faptul ca cele mai mari valori ale concentratiei au fost in domeniul de marimi 8 - 18 nm.

Cele mai mari valori ale concentratiei au fost masurate in locul unde carbunele este concasat: sala mori si sala concasoare, concentratia de particule intr-un cm3 ajungand pana la 120.000 respectiv 95.000 de particule/cm3.

Din analiza difractiei de raze X se constata ca dioxidul de siliciu (SiO2) este sub forma cristalina (cuart) si este majoritar cantitativ. De asemenea si silicatul de aluminiu (3Al2O32SiO2 sau 2Al2O3 SiO2) se gaseste sub forma cristalina. Restul compusilor sunt amorfi; totusi pot fi cristaline si alte substante dar cantitatile sunt neglijabile in comparatie cu cei doi compusi mai sus amintiti.

Analiza prin dispersie dupa energie (EDAX) arata ca probele de pulbere din atmosfera contin elemente ca Ti, Ca, Ag, Fe, Al, Si, Pb, K. De asemenea se constata ca peakul cel mai mare este la Si, fapt ce se coreleaza cu masuratorile efectuate cu difractrometrul de raze X, unde proportia cea mai mare a avut-o cuartul si cu analiza compozitiei chimico-oxidice care arata un continut de silice de cca. 50%


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Activitatea 2.3 – Evaluarea toxicitatii pulberilor pe fractii dimensionale

Executanti: INCD Victor Babeş

Probele de cenusa si praf de carbune prelevate din SC ELECTROCENTRALE DEVA contin numeroase metale grele si alti compusi in stare amorfa, in cantitati foarte mici. Silicea (SiO2) este constituentul major, cu peste 50% in probele de cenusa si are o structura cristalina (cuart). Alti constituienti semnificativi sunt Fe2O3 > 7%, Al2O3 >5% si alti oxizi metalici, metale etc in concentratii foarte mici variabile in functie de originea carbunelui.

In compozitia prafului de carbune, silicea este prezenta in stare cristalina in proportie mai mica decat in cenusa (~18%). Alaturi de cuart, alte componente cristaline sunt kaolinitul Al2Si2O5(OH)4 si silicatul de magneziu Mg3Si4O10(OH)2, dar la concentratiii foarte scazute. Silicatul de aluminiu este, de asemenea, cristalin.

Obiectivele etapei au fost evaluarea in vitro a citoxicitatii probelor de cenusa si praf de carbune prin teste comparative cu compusi nanostructurati cu dimensiune similara cu cea a constituientului predominant din probe.

Protocolul de lucru a inclus testarea citotoxicitatii SiO2 ca martor pozitiv si a TiO2 ca martor negativ, la dimensiuni in medie de ~200 nm comparativ cu particule PM* de dimensiuni apropiate obtinute din probele prelevate.

Evaluarea citotoxicitatii in vitro a utilizat teste clasice de determinare a afectarii viabilitatii si a capacitatii de proliferare celulara (MTS) si a nivelului de eliberare extracelulara a lactate dehidrogenazei (LDH) pe linii celulare de fibroblasti BALB/c 3T3.

In cadrul proiectului au fost efectuate cele doua teste pentru evaluarea efectelor citotoxice in vitro, cel mai frecvent utilizate si considerate adecvate pentru evaluarea preliminara a reactivitatii biologice a particulelor.

Eliberarea LDH, indica o citotoxicitate ireversibila (letala), in general poate fi detectabila la expunerea la concentratii mai mari de toxice comparativ cu MTS, care indica perturbarea statusului vital al celulei si capacitatea ei de proliferare, care poate fi reversibila cand stimulul dispare.

Nivelul ridicat al valorii LDH la dozele mici, poate constitui un indicator al citotoxicitatii crescute a probelor prelevate.


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Modularea capacitatii de proliferare in prezenta particulelor

nanostructurate*

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

25µg/mL 50µg/mL 100µg/mL 150µg/mL 200µg/mL

OD 4

90nm TiO2*

SiO2*

PM *

Eliberare LDH in expunerea la particule nanostructurate*comparativ cenusa si praful de

carbune

0

0,5

1

1,5

2

2,5

3

3,5

25µg/mL 50µg/mL 100µg/mL 150µg/mL 200µg/mL

OD 4

90nm

TiO2*

SiO2*

PM *

cenusa

praf

Din analiza rezultatelor testului MTS in expunerea la probele de cenusa si praf de carbune comparativ cu o componenta nanoparticulata din probe (PM*) si cu SiO2 si TiO2 de dimensiuni similare se poate observa:

- compusii nanostructurati PM*si SiO2 induc efecte apropiate (p>0,05) , dar cu diferente semnificative statistic comparativ cuTiO2 (p**<0,01);

- efectele induse de componentele nanostructurate PM* sunt semnificativ statistic diferite fata de probele de cenusa si praf de carbune din care provin.

Efecte citotoxice ale expunerii fibroblastilor BALB/c 3T3 la cenusa si praful de carbune evaluata prin eliberarea LDH.

Cresterea nivelului extracelular al LDH incepand cu expunerea la dozele mici pana la doze mari de cenusa de carbune – cu diferente semnificative comparativ cu controlul. Probele de praf de carbune induce valori absolute mai mari ale activitatii extracelulare a LDH fata de probele de cenusa si diferite semnificativ comparativ cu controlul


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Activitatea 2.4 – Evaluarea riscului de sanatate pe un lot reprezentativ selectat din personalul expus la noxe inainte de introducerea tehnologiei de reducere/diminuare a pulberilor

Executanti: INCD Victor Babeş

Expunerea pe termen lung la materialele particulate (PM) ambientale au indicat corelatii semnificative intre rata mortalitatii cauzate de cancerul pulmonar si boli cardiopulmonare. S-au gasit corelatii interesante intre nivelul PM fine si ateroscleroza, inflamatia sistemica si pulmonara, si alterarea functiei cardiace. Un impact semnificativ asupra sanatatii umane atribuabile expunerii ambientale si ocupationale au indicat asociatii intre reducerea functiei plamanului, cresterea mortalitatii la pacientii cu boli cardiovasculare si pulmonare pre-existente induse de PM fine si ultrafine. Impactul la nivelul sistemului cardiovascular este complex si nu este sigur daca este dat de semnale ale raspunsului din plaman sau direct de particulele translocate in sistemul cardiovascular.

Persoanele expuse la praf de carbune si silica (SiO2) pot dezvolta diferite patologii pulmonare: pneumoconioza, o afectiune nodulara interstitiala a plamanului care, in cazuri severe poate duce la fibroza masiva progresiva (PMF); de asemenea, expunerea poate induce boala cronica obstructiva (COPD), care include emfizemul si bronsita cronica (Cohen RA et al., 2008).

Citokinele sunt considerate mediatori ai raspunsului la diferiti stresori chimici, noxe din mediul ambiant, stimuli infectiosi si inflamatori. Sunt compusi chimici produsi de diferite tipuri celulare care actioneaza impreuna impotriva agentilor externi nedoriti si a celulelor proprii alterate.

Un obiectiv important al etapei evaluarea riscului de sanatate a personalului expus ocupational la noxe prin determinarea parametrilor serici, hematologici si a comportamentului citokinelor prin tehnologii moderne si utilizand probe biologice noninvasive, pentru evaluarea timpurie a afectiunilor respiratorii la pacientii expusi profesional la noxe in termocentrale

S-au stabilit loturi de persoane expuse la ocupational la noxe in termocentrala de la Deva. S-au alcatuit 2 loturi tinta: persoane expuse in jur de 10 ani ( E10) si persoane expuse in jur de 20 ani ( E20), si respective un lot control cu subiecti sanatosi (nefumatori).

Probele de sange au fost prelevate pentru testari biochimice, hematologice si pentru determinarea citokinelor serice cu consimtamantul fiecarei persoane, conform documentului elaborat de Comisia de etica din Institutul National de Patologie “Victor Babes”.

Parametri biochimici au fost determinati utilizand kituri de reactivi specifici (Roche Diagnostic GmbH, Germania) pe un bioanalizor Hitachi 912 si cei hematologici pe un bioanalizor Horiba ABX. Evaluarea nivelului de citokine s-a efectuat prin tehnologie multiplex xMAP (Luminex 200).

De asemenea au fost determinate interleukinele (IL): IL-1β (interleukina 1 beta), TNF-α (tumor necrosis factor-alpha), IL-6 (interleukin -6), cu kituri de bază pentru determinarea citokinelor umane Human MultiAnalyte Profiling Base Kit A.

Au fost gasite nivele semnificative de TNF-α in fluidele pulmonare ale pacientilor la debutul afectiunilor respiratorii.


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Indicatori biochimici

- parametri biochimici serici uzuali, indicatori ai functionalitatii organelor interne

- proteine serice, bilirubina, creatinina, glicemia, transaminaze (AST, ALT);

- γ-glutamil-transpeptidaza (GGT) ;

- lactic dehidrogenaza (LDH)

- fosfataza alcalina

Valorile parametrilor serici ai persoanelor expuse au fost comparati cu valorile obtinute pe un lot control (martor).

Nivelul seric al IL-6

in functie de timpul de expunere ocupationala

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pg

/mL control

10 ani

20ani

In concluzie, parametri biochimici ai persoanelor expuse au indicat:

- perturbari ale creatininei la grupurile investigate, care poate indica o afectare renala data de expunerea la noxe;

- media indicatorii hepatici pentru loturile investigate nu sunt semnificativ crescute, desi exista indivizi care prezinta valori crescute ale GGT;

- Colesterolul seric, cu componentele HDL si LDL au fost crescute peste limite fiziologice cu variabilitate inter si intra loturi, fiind si insotite de valori crescute si pentru trigliceridele serice; fenomenul este intalnit si la loturi control si nu poate fi atribuit exclusiv mediului de lucru

Rezultatele testelor de citotoxicitate in vitro :

Nivelele apropiate ale efectelor induse de PM* si SiO2 pot indica faptul ca fractiunea PM* alcatuita din particule cu dimensiuni apropiate este alcatuita majoritar din SiO2 si alti compusi cristalini din probele prelevate

Efectele semnificativ diferite ale efectelor componentei nanostructurate PM* fata de cenusa si fata de praful de carbune pot fi atribuite interferentelor celorlalti component citotoxici prezenti in probele prelevate, cunoscute pentru reactivitatea si citotoxicitatea


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Cresterile apreciabile ale IL-1β la loturile expuse la noxe fata de control poate sugera susceptibilitatea cea mai crescuta a acestei citokine la noxe si modularea ei in functie de timpul de expunere.

IL-1β si IL-6, cytokine proinflamatoare, considerate markeri ai instalarii si dezvoltarii afectiunilor mediate de aceste mecanisme.

Evolutia moderata a TNF-α in expunerea la noxe poate fi atribuita faptului ca aceasta citokina este crescuta de timpuriu ca raspuns la agenti toxici si se stabilizeaza la un nivel crescut, stimuland alte procese implicate in afectiunile inflamatoare

Activitatea 2.5 – Coroborarea caracteristicilor chimico-structurale si dimensionale ale pulberilor cu testele de toxicitate si efectele potentiale asupra sanatatii

Executanti: UPB – CCEPM ICEMENERG INCD Victor Babeş

Efectul pulberilor, inclusiv al nanoparticulelor asupra sanatatii, prin cuantificarea atat a numarului de particule si a distributiei dimensionale a acestora, cat si a formei si compozitiei chimice sunt in stransa corelare cu gradul lor de toxicitate pe fiecare tip de particule. Expertizarea locurilor de munca din cadrul ELECTROCENTRALE DEVA are ca scop stabilirea nivelului noxelor care face parte integranta In literatura de specialitate este unanim acceptat [Åke Thidell, Peter Arnfalk- Nanoparticles: A Closer Look at the Risks to Human Health and the Environment/2004] faptul ca nanoparticulele produc un efect inflamator mult mai pronuntat atunci cand sunt inhalate si depozitate in plamani, decat particulele mai mari ale aceluiasi material, ca urmare a cresterii suprafetei lor specifice odata cu scaderea dimensiunii particulelor.

Prin coroborarea rezultatelor masuratorilor si analizelor efectuate cu cercetarile de influenta asupra starii sanatatii salariatilor, se pot trage urmatoarele concluzii:

1. Din masuratorile si cercetarile de laborator efectuate in cadrul proiectului s-a evidentiat faptul ca pulberile din atmosfera locurilor de munca din cadrul SC ELECTROCENTRALE DEVA au un continut ridicat de silice cristalina, de 30-50%.

2. Din diagramele DMA se observa ca in general concentratia de nanoparticule de pulbere de carbune este mare pentru diametrele din domeniul 8-30 nm, cu peakuri de dimensiuni diferite in functie locul unde se efectueaza masuratorile. De asemenea este de notat faptul ca cele mai mari valori ale concentratiei au fost in domeniul de marimi 8 - 18 nm. Cele mai mari valori ale concentratiei au fost masurate in locul unde carbunele este concasat: sala mori si sala concasoare, concentratia de particule intr-un cm3 ajungand pana la 120.000 respectiv 95.000 de particule/cm3.

3. Prezenta compusilor cristalini a fost stabilita prin procedeul de difractie de raze X

Din diagramele difractiilor de raze X se observa ca dioxidul de siliciu (SiO2) prezent in pulberi este sub forma cristalina(cuart) si majoritar cantitativ.

4. Din masuratorile efectuate prin Spectroscopia de Dispersie de Raze X dupa Energie (EDAX) s-a constatat ca probele de pulbere din atmosfera locurilor de munca din ELECTROCENTRALE DEVA contin elemente ca Ti, Ca, Ag, Fe, Al, Si, Pb, K. De asemenea se observa ca peakul cel mai mare este la Si.


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5. Studiu toxicologic in vitro s-a efectuat prin testul de proliferare celulara (test TMS). In rezultatele experimentarilor testul MTS a indicat o citotoxicitate crescuta chiar incepand cu concentratiile mici, exprimata printr-o crestere semnificativa intre concentratia cea mai mica (25µg/mL) si proba martor.

6. Testarea efectelor citotoxice (eliberarea LDH). Scaderea LDH intracelular si eliberarea lui in mediu de cultura este un indicator de moarte celulara ireversibila data de afectarea membranei celulare. Testul eliberarii LDH indica efecte intracelulare primare date de expunerea la noxe. Eliberarea LDH indica o citotoxicitate ireversibila (letala) si este detectabila la concentratii mai mari de toxice comparativ cu MTS care indica perturbarea (ce poate fi reversibila cand stimulul dispare) a statusului vital al celulei si capacitatea ei de proliferare.

MTS indica efecte intracelulare primare date de expunerea la noxe, anterioare aparitiei afectarii permanente (ruperii) membranei celulare care duce la pierderea LDH intracelular si eliberarea lui in medium de cultura ca indicator al mortii celulare ireversibile

Din analiza datelor comparative se pot mentiona faptul ca toate testele arata afectarea starii de sanatate comparativ cu probele martor, dar sunt diferente de efecte intre probe:

7. Pentru monitorizarea starii de sanatate a personalului din cadrul SC ELECTROCENTRALE DEVA S-au stabilit loturi de persoane expuse la noxe in termocentrala Deva. S-au alcatuit 2 loturi tinta: persoane expuse in jur de 10ani ( E10) si persoane expuse in jur de 20ani ( E20), si respective un lot control cu subiecti sanatosi (nefumatori). Probele de sange au fost prelevate pentru testari biochimice, hematologice si pentru determinarea citokinelor serice. Au fost analizati urmatorii indicatori biochimici:

- parametri biochimici serici uzuali, indicatori ai functionalitatii organelor interne

- proteine serice, bilirubina, creatinina, glicemia, transaminaze (AST, ALT);

- γ-glutamil-transpeptidaza (GGT) ;

- lactic dehidrogenaza (LDH)

- fosfataza alcalina

Din comparatia nivelului citokinelor serice IL-1β, TNF-α , IL-6 din loturile tinta cu lotul control, reies diferente apreciabile E10 fata de C, si diferente semnificative statistic E20 fata de C. S-au observat cresteri 5,20x ale IL-1β la E10 si de 20x la E20 fata de control. Nivelul IL-1β este, de asemenea de 3,84x la lotul E20 de fata de E10.

Variatia nivelului IL-6 in functie de timpul de expunere: E10 creste de 2,37x si E20 de 8,86x fata de control, iar E20 creste de 3,73x fata de E10.

Analiza indicatorilor biochimici sugereaza afectarea functiei renale (nivelul creatininei) si sistemului cardiovascular (nivelul colesterului si trigliceridelor)

Cresterile apreciabile ale IL-1β la loturile expuse la noxe fata de control poate sugera susceptibilitatea cea mai crescuta a acestei citokine la noxe si modularea ei in functie de timpul de expunere.

IL-1β si IL-6, cytokine proinflamatoare considerate markeri ai instalarii si dezvoltarii afectiunilor mediate de aceste mecanisme au prezentat o crestere semnificativa fata de control, dar si o crestere semnificativa a grupului cu expunere prelungita E20 fata de grupul E10.


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Evolutia moderata a TNF-α in expunerea la noxe poate fi atribuita faptului ca aceasta citokina este crescuta de timpuriu ca raspuns la agenti toxici si se stabilizeaza la un nivel crescut, stimuland alte procese implicate in afectiunile inflamatoare.

Nivelul citokinelor indica aparitia patologiilor inflamatoare pulmonare si probabil a altor organe la persoanele expuse la noxe, cu amplificarea patologiei in expunere cronica.

Activitatea 2.6 – Diseminarea informatiilor pe scara larga privind evaluarea noxelor profesionale, evaluarea toxicitatii noxelor si coroborarea caracteristicilor chimico-structurale si dimensionale ale pulberilor cu testele de toxicitate si efectele potentiale asupra sanatatii

Executanti: UPB – CCEPM ICEMENERG

În cadrul acestei activităţi au fost elaborate 4 articole, după cum urmează:

1. MONITORIZATION IMPACT OF ELECTRICAL EQUIPMENTS ON HUMAN

HEALTH – PRIORITY OF CNTEE TRANSELECTRICA IN POLICY OF SECURITY

AND HEALTH, autori: Alice Raducanu, Marin Stefan, dr.Cornelia Marcolt, Camelia

Suvergel, lucrare prezentată la FOREN 2010, Neptun, iunie 2010

2. Structural characterization and in vitro cytotoxic potential of coal dust and

ash in a romanian power plant, autori: Alice Raducanu, Aurica Suvergel,

George Darie, Ileana Rau, Constantin Grigoriu, Cristian Viespe, Traian Vasiu,

Ionela Daniela Popescu, Eleonora Codorean, articol acceptat spre publicare în

revista Environmental Engineering and Management Journal, 2010

3. Characterization of ultrafine particles generated at “Deva-Mintia” power

plant, autori Alice Raducanu, Angela Stanca, George Darie, Ileana Rau, Constantin

Grigoriu, Cristian Viespe, Traian Vasiu, articol acceptat spre publicare în revista

METALURGIA INTERNATIONAL (cotată ISI).

În Anexa sunt prezentate în extenso aceste articole.

Activitatea 2.8 – Organizarea si cuantificarea informatiilor obtinute in cadrul etapei si realizarea planului de management necesar desfasurarii in continuare a proiectului

Executanti: UPB – CCEPM Prezenta lucrare reprezinta etapa nr. III a proiectului PNCDI II: “Tehnologie avansata de diminuare a pulberilor (inclusive nanoparticule) existente in termocentrale in vederea reducerii riscului la expuneri profesionale si a imbunatatirii sanatatii si sigurantei la locurile de munca”, contract nr. 22-090/2008, intitulata “Cercetari privind evaluarea noxelor profesionale si in special a pulberilor, precum si influenta lor asupra sanatatii personalului”.


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Obiectivele urmarite in acest proiect vor fi identificarea solutiilor tehnice moderne de diminuare sau eliminare, a noxelor profesionale (in special pulberi), elaborarea de masuri de reducere a expunerii la noxe profesionale, a personalului din sistemul energetic si constuirea de instalatii pilot moderne de reducere a noxelor profesionale.

Pentru diminuarea riscului asupra sanatatii produs de aceste pulberi la locurile de munca din incinta termocentralelor, proiectul propune o solutie bazata pe o conceptie stiintifica novatoare de realizare a unui echipament de filtrare in mai multe trepte, ce include in final reducerea dimensionala prin precipitare electrostatica, prin preluarea impulsurilor de la descarcarea corona.

In cadrul proiectului au fost realizate pana in prezent 3 etape :

Etapa 1

Stadiul actual al cunoasterii privind noxele profesionale specifice termocentralelor, influenta pulberilor, inclusiv a nanoparticulelor asupra sanatatii personalului la locul de munca si posibilitati de reducere a noxelor profesionale

Etapa 2 Cercetari preliminare privind evaluarea noxelor profesionale

Etapa 3. Cercetari privind evaluarea noxelor profesionale si in special a pulberilor, precum si influenta lor asupra sanatatii personalului

In cadrul acestor etape s-au realizat urmatoarele activitati principale:

� Cercetari privind stadiul actual al cunoasterii privind noxele profesionale specifice unitatilor energetice pe baza de combustibil fosil, cu accent pe termocentralele care functioneaza cu combustibil solid

� Studiu privind influenta pulberilor, inclusiv a nanoparticulelor asupra sanatatii personalului la locul de munca din termocentrale

� Studiul privind posibilitatile de reducere a noxelor profesionale, in special a pulberilor din termocentrale, in vederea imbunatatirii sanatatii si sigurantei la locul de munca

� Masuratori de noxe profesionale in cadrul termocentralei DEVA

� Evaluarea cantitativa si calitativa a pulberilor, cu estimarea distributiei dimensionale, a formei si compozitiei chimico-structurale in volumul si la suprafata particulelor

� Evaluarea toxicitatii pulberilor pe fractii dimensionale

� Evaluarea riscului de sanatate pe un lot reprezentativ selectat din personalul expus la noxe inainte de introducerea tehnologiei de reducere/diminuare a pulberilor

� Coroborarea caracteristicilor chimico-structurale si dimensionale ale pulberilor cu testele de toxicitate si efectele potentiale asupra sanatatii

� Diseminarea informatiilor pe scara larga privind evaluarea noxelor profesionale, evaluarea toxicitatii noxelor si coroborarea caracteristicilor chimico-structurale si dimensionale ale pulberilor cu testele de toxicitate si efectele potentiale asupra sanatatii


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Etapa urmatoare a proiectului, etapa nr. 4, intitulata “Elaborare solutie noua si realizarea modelului experimental de reducere a pulberilor existente la locurile de munca din termocentrale, prevede realizarea urmatoarelor activitati:

� Studiu de solutie privind diminuarea concentratiei pulberilor in vederea imbunatatirii performantelor tehnologiei propuse

� Elaborarea si demonstrarea functionalitatii modelului experimental de retinere a pulberilor din termocentrale

� Evaluarea riscului de sanatate apersonalului prin metodologii complexe in vitro si ex-vivo; studiu intermediar

� Diseminarea informatiilor pe scara larga privind elaborarea studiului de solutie privind privind diminuarea pulberilor (nanoparticulelor) in vederea imbunatatirii sanatatii si sigurantei la locurile de munca

In vederea realizarii activitatilor propuse in proiect, managementul proiectului coordoneaza partenerii in realizarea activitatilor specifice fiecarui partener si planifica si urmareste realizarea obiectivelor si activitatilor propuse, astfel:

Planul de management pentru desfăşurarea în continuare a proiectului (etapa 4):

Nr.crt. Activitate Responsabil Termen Rezultate 1. Sedinta de consortiu etapa

nr.4. Stabilire obiective Manager de proiect

15.12.2010 Planificare activitati cu partenerii, obiective;necesitati

2 Studiu de solutie privind diminuarea concentratiei pulberilor in vederea imbunatatirii performantelor tehnologiei propuse

Coordonator; Partener nr.1 Partener nr. 5

28.02.2011 Selectare si stabilire solutie tehnica de reducere a pulberilor de la locurile de munca

3 Elaborarea modelului experimental de retinere a pulberilor din termocentrale

Partener nr.1 partener nr.2

15.03.2011 Elaborare model experimental pentru o tehnologie de reducere a emisiilor de pulberi in termocentrale

4 Demonstrarea functionalitatii modelului experimental de retinere a pulberilor din termocentrale

Partener nr.1 partener nr.2

15.04.2011 Experimentari in instalatiile ELECTROCENTRALE DEVA pentru demonstrarea functionalitatii modelului experimental de echipament

5 3.3 Evaluarea riscului de sanatate a personalului prin metodologii complexe in vitro si ex-vivo; studiu intermediar

Coordonator; Partener nr.4

30.04.2011 Studiu si analize privind evaluarea riscului de sanatate a personalului expus la noxe

6 Diseminarea informatiilor pe scara larga privind elaborarea studiului de solutie privind privind diminuarea pulberilor (nanoparticulelor) in vederea imbunatatirii

Toti partenerii 10.05.2011 Comunicari stiintifice, articole, pagina web, CD-ROM


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sanatatii si sigurantei la locurile de munca

7 Sedinta de consortiu etapa nr.4. Analiza rezultate

Manager de proiect

15.05.2011 - Raport de cercetare in extenso

- Raport financiar etapa

- Raport de audit intern

- Organizarea si cuantificarea informatiilor obtinute in cadrul etapei si realizarea planului de management necesar desfasurarii in continuare a proiectului

Îndeplinirea în condiţii optime a Planului de management prezentat mai sus depinde puternic de modul de finanţare a proiectului pe parcursul anului 2011.

1.6 Bibliografie 1. G. Oberdorster, Pulmonary effects of inhaled ultrafine partilcles, Int. Arch. Occ. Env.

Hea. 2001, 74 (1), 1-8 2. D. M. Brown, V. Stone, P. Findlay, W. Macnee, K. Donaldson, Increased inflamation

and intracelular calcium caused by ultrafine carbon black is independent of transition metals or other soluble components, Occup. Enviroment, Medicine 57, 685-691,2000.

3. K. Donaldson, X.Y. Li, W. Macnee, Ultrafine (nanometre) particle mediated lung injury, J. Aerosol Sci. 29, 553-560, 1998

4. K. Donaldson, V. Stone, A. Clouter, L. Renwick, W. Macnee, Ultrafine particles. Occup environ. medicine 587, 553-560, 2001

5. A. Nemmar, P.H. Hoet, B. Vanqiuckenborne, D. Dinsdala, M. Thomeer, M.F. Hoylaerts, Passage of inhaled particles into the blood circulation in humans, Circulation, 105 (4): 411-414, 2002

6. A. Nemmar, M.F. Hoylaerts, P.H. Hoet, D. Dinsdale, T. Smith, H. Xu, Ultrafine particles affect experimental thrombosis in an in vivo hamster model, Am. J. respir. Crit. care. Med. 166 (7):998-1004, 2002

7. A. Nemmar, M.F. Hoylaerts, H.P. Hoet, J. Vermylen, B. Nemery, Size effect of intratracheally instilled particles on pulmonary inflammation and vasular thrombosis, Toxical. Apll. Pharmacol, 186(1):38-45, 2003

8. S. Hasegawa,M. Hirabayashi, S. Koboyashi, Y. Moriguchi, Y. Kondo, K. Tanabe, S. Wakamatsu, Size Distribution and Characterization of Ultrafine Particles in Roadside Atmosphere, J. of Environmental Sceince and Health, part. A, Vol. A39, no 10, pp 2671-2690, 2004.

9. C. Asbach, H. Kaminski, H. Fissan, C. Monz, D. Dahmann, S. Mulhopt, H. R. Paur, H. J. Kiesling, F. Herrman, M. Voetz, T. A. J. Kuhlbusch, Comparision of four mobility particle sizer with different time resolution for stationary exposure measurements, J. Napoparts Res (2009)

10. B. Davison, D. Whyatt, C. Boardman, Aerosol eolution from a busy road in North-west Engleand, Metoorologische Zeits


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11. Albulescu R., Codorean E., Albulescu L., Caraene G., Vulturescu V., Tanase C., In vitro biocompatibility testing of implantable biomaterials, Romanian Biotechnological Letters. 2008, 13, 3863-387

12. Allermann L, and Melchior Poulsen O, Interleukin-8 Secretion from Monocytic Cell Lines for Evaluation of the Inflammatory Potential of Organic Dust, Environmental Research, 2002, 88, 188-198

13. Allermann L and Melchior Poulsen O, Inflammatory Potential of Dust from Waste Handling Facilities Measured as IL-8 Secretion from Lung Epithelial Cells In Vitro Ann. occup. Hyg., 2000, 44, 259-269

14. Bakand S., Winder C., Khalil C. and Hayes A., A novel in vitro exposure technique for toxicity testing of selected volatile organic compounds, J. Environ. Monit., 2006, 8, 100-105

15. Barbarin V, Nihoul A, Misson P, Arras M, Delos M, Leclercq I, Lison D and Huaux F, The role of pro- and anti-inflammatory responses in silica-induced lung fibrosis, Respiratory Research 2005, 6, 112

16. Borm P.J., Toxicity and occupational health hazards of coal fly ash (cfa). A review of data and comparison to coal mine dust, Aim. accup. Hyg., 1997, 41, 659-676

17. Borm P.J., Particle toxicology: from coal mining to nanotechnology, Inhal.Toxicol, 2002, 14, 311-324

18. Borm P.J., Klaessig F. C., Landry T. D., Moudgil B., Pauluhn J., Thomas K., Trottier R. and Wood S., Research Strategies for Safety Evaluation of Nanomaterials, Part V: Role of Dissolution in Biological Fate and Effects of Nanoscale Particles, Toxicological Sciences, 2006, 90,23-32

19. Codorean E., Tanase C., Iosif C., Popescu I.D., Raducan E., Modulation of chemical toxicity by alternative mediators of apoptosis – Toxicology and Applied Pharmacology, 2004, 197, 3, 373

20. Codorean E., Tanase M., Albulescu L., Popescu I.D., Mihai S., Murariu A., Tanase C., Novel developmental immunotoxicology monitoring risk assessment for human populations from environmental pollution; alternative methods in vitro, In: „Environmental Health Risk V”, Brebbia CA (Ed), WIT press, United Kingdom, 2009, 233-246

21. Codorean E., Albulescu L., Raducan E., Popescu ID, Albulescu R, Nichita C, Tanase C., Multiplexed immunoassay for monitoring cytokine profile in immunopharmacological studies in vitro, Toxicology Letters, 2009, 189, S266

22. Codorean E, Nichita C., Albulescu L, Raducan E, Popescu ID, Ionita AC, Albulescu R, Correlation of xMAP and ELISA cytokine profiles ; development and validation for immunotoxicological studies in vitro ,Romanian Archives of Microbiology and Immunology, 2010,vol 68 nr. 1, p 13-19

23. Cohen RA, Patel A, Green FH, Lung disease caused by exposure to coal mine and silica dust. Semin Respir Crit Care Med. 2008, 29, 651-61

24. Deaton P. R., Mckellar C. T., Culbreth R., Veal C. F., Cooper J. A. D. Jr , Hyperoxia stimulates interleukin-8 release from alveolar macrophages and U937 cells : attenuation by dexamethasone, American journal of physiology. Lung cellular and molecular physiology, 1994, 2, L187-L192

25. Donaldson K, Stone V, Clouter A, Renwick L, Macnee W, Ultrafine particles. Occup environ. medicine 2001, 587, 553-560

26. Fotakis G., Timbrell J. A., In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride, Toxicology Letters, 2006, 160, 171–177

27. Gosset P, Lassalle P, Vanhée D, et al. Production of tumour necrosis factor-alpha and interleukin-6 by human alveolar macrophages exposed to in vitro coal mine dust.Am J Respir Cell Mol Biol 1991; 5: 431–436


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28. Lambroussis C. G., Soares B. D., Perez S., Gaipa D., Elie A.L., Ament C. M., Rouf F.T., Bassa L. M. and DiLorenzo A. M., Indications of Potential Toxic/Mutagenic Effects of World Trade Center Dust on Human Lung Cell Cultures, Journal of Biological Science, 2009, 9, 81-85

29. Ling S H, van Eeden S F, Particulate matter air pollution exposure: role in the development and exacerbation of chronic obstructive pulmonary disease, International journal of chronic obstructive pulmonary disease 2009, 4, 233-43

30. Kennedy I M., The health effects of combustion-generated aerosols Proceedings of the Combustion Institute 2007,31, 2757-2770

31. Meghea A., Murariu A., Tanase C. and Codorean E., Heavy metals contamination of commercial fish foodstuff – potential health risks on human consumers, Environm Engin and Manag J, 2009, 8, 233-236

32. Muramatsu D., Sasaki K., Kuroda S., Hayashi K., Tanaka N. and Sakai A., Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB/c 3T3 cell transformation assay, Mutation Res. Academic J. 2009, 675, 66-70

33. Oberdörster G., Ferin J. and Lehnert B. E., Correlation between Particle Size, In Vivo Particle Persistence, and Lung Injury, Environ Health Perspect, 1994, 102,173-179

34. Oberdörster G., Pulmonary effects of inhaled ultrafine particles, Int.Arch.Occup. Environ Health, 2001, 74, 1-8

35. Oberdörster G., Maynard A., Donaldson K., Castranova V., Fitzpatrick J., Ausman K., Carter J., Karn B., Kreyling W., Lai D., Olin S., Monteiro-Riviere N., Warheit D., Yang H., Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy, Particle and Fibre Toxicology, 2, 1-35

36. Oberdörster G., Stone V., Donaldson K., (2007), Toxicology of nanoparticles: A historical perspective , Nanotoxicology, 2005, 1, 2-25

37. Rom WN, Relationship of inflammatory cell cytokines to disease severity in individuals with occupational inorganic dust exposure. Am J Ind Med. 1991, 19, 15-27.

38. Sakai A., BALB/c 3T3 cell transformation assays for the assessment of chemical carcinogenicity, AATEX Academic J., 2008, 14 , 367-373

39. Stone V, Johnston H, Clift MJ.Air pollution, ultrafine and nanoparticle toxicology: cellular and molecular interactions. IEEE Trans Nanobioscience. 2007, 6, 331-40

40. Stone V., Johnston H., Schins R. P. F., Development of in vitro systems for nanotoxicology: methodological considerations, Critical Reviews in Toxicology, 2009, 39, 613-626

41. Unfried K., Albrecht C. , Klotz L.-O.,von Mikecz A. , Grether-Beck S. and Schins R.P.F., Cellular responses to nanoparticles: Target structures and mechanisms, Nanotoxicology, 2007, 1, 52-71

42. van Eeden SF. Kitagawa Y. Klut ME. Lawrence E. Hogg JC. Polymorphonuclear leukocytes released from the bone marrow preferentially sequester in lung microvessels. Microcirculation. 4(3):369-80, 1997

43. Vanhée D, Gosset P, Marquette CH, Wallaert B, Lafitte JJ, Gosselin B, Voisin C, Tonnel AB. Secretion and mRNA expression of TNF alpha and IL-6 in the lungs of pneumoconiosis patients. Am J Respir Crit Care Med. 1995 , 152, 298-306

44. Zhai R, Liu G, Ge X, Bao W, Wu C, Yang C, Liang D. Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and their soluble receptors in coal workers' pneumoconiosis, Respir Med. 2002, 96, 829-34


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2. Indicatorii generali si specifici de activitate

Indicatori generali:

Indicatori de rezultat

Denumirea indicatorilor UM 1. Număr de produse şi tehnologii rezultate din activitatea de cercetare, bazate pe brevete, omologări sau inovaţii proprii.

Nr. -

2. Număr de cereri de brevete depuse în urma proiectelor din care: a) Naţionale b) EPO (Europa) c) USPTO (SUA) d) Triadice (Europa, SUA, Japonia)

Nr. - - - - -

3. Număr de cereri de brevete acordate (în urma proiectelor) din care: a) Naţionale b) EPO c) USPTO d) Triadice

Nr. - - - - -

4. Număr de articole publicate în urma proiectelor, din care: a) în reviste indexate ISI b) în reviste indexate în alte baze de date internaţionale recunoscute

Nr.: 1 - 1

5. Număr de articole acceptate spre publicare în urma proiectelor, din care: a) în reviste indexate ISI b) în reviste indexate în alte baze de date internaţionale recunoscute

Nr.: 2

1 1

6. Număr de produse transferabile - 7. Număr de studii de necesitate public din care: a) de interes naţional b) de interes regional c) de interes local

Nr.: - - -

8. Număr de IMM participante - 9. Ponderea contribuţiei financiare private pe proiecte din care contribuţie financiară directă

0 %

10. Numărul mediu de poziţii echivalente cu normă întreagă pe proiect, din care: a) doctoranzi b) postdoctorat

0,90

0,1 -

11. Mobilităţi, din care internaţionale

0,5 om/lună -

12. Valoarea investiţiilor în echipamente pentru proiecte

0 RON

13. Rata de succes în depunerile de proiecte - 14. Număr reţele de cercetare susţinute -

Notă: � Pentru indicatorii 4 şi 5 (articole) datele de identificare sunt prezentate în

Secţiunea 1 a RST.


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Indicatori specifici fiecarei directii de cercetare:

Directia de cercetare

Denumirea indicatorului Numarul Informatii despre indicator

DC 1 Tehnologiile societăţii informaţionale

Nr. de tehnologii IT performante Nr. tehnologii suport pentru comunicatii; Nr. metode/sisteme de inteligenta artificiala; Nr. produse nanoelectronice si fotonice; Nr.nano- si microsisteme

DC 2: Energie

Nr.concepte de utilizare de noi surse energetice Nr. de tehnologii de reducere a pretului in domeniul energetic Nr. de tehnologii/produse in domeniul securitatii energetice

-

- 1

Tehnologie avansate de diminuare a pulberilor, inclusiv nanoparticule, existente in termocentrale, in vederea reducerii riscului la expuneri profesionale si a imbunatatirii sanatatii si sigurantei la locurile de munca.

DC 3: Mediu

Nr. de sisteme şi tehnologii energetice durabile Nr. de tehnologii curate de produs si proces pentru reducerea poluării mediului (green chemistry) Din care: in transporturi Nr.de tehnologii eco-eficiente de valorificare a deseurilor; Nr.concepte si tehnologii de consolidare a diversitatii biologice si ecologice; Nr. de metode si solutii tehnice in domeniul amenajarii teritoriului

DC 4:Sănătate

Nr.concepte/studii ale mecanismelor de adaptare ale organismului; Nr. metode pe baze moderne de investigatie in medicina; Nr. terapii moderne; Nr. de metode de preventie si interventionale la nivel naţional, arondate la spaţiul european de operare

DC 5: Agricultura, securitatea şi siguranţa alimentară

Nr. de produse corespunzătoare principiilor dezvoltării durabile şi securităţii alimentare, inclusiv alimente funcţionale; Nr. de metodologii de detectare a reziduurilor şi contaminanţilor din


18

întreg lanţul alimentar DC 6: Biotehnologii

Nr.de medicamente noi; Nr.protocoale de diagnostic şi tratamente medicale; Nr.de tehnologii pentru producţia de alimente cu siguranţă maximă asupra sănătăţii umane; Nr.de tehnologii avansate in domeniul produselor farmaceutice; grupurilor biocatalitice; noi enzime şi microorganisme Nr. de sisteme bioinformatice

DC 7: Materiale, procese şi produse inovative

Nr. de materiale avansate Nr.de tehnologii de reciclare a materialelor avansate Nr. de tehnologii avansate de conducere a proceselor industriale Nr. de tehnologii şi produse mecanice de înaltă precizie şi sisteme mecatronice Nr. de tehnologii nucleare Nr. de produse şi tehnologii inovative destinate transporturilor şi producţiei de automobile

DC 8:Spaţiu şi securitate

Nr. de aplicaţii spaţiale integrate Nr. de tehnici aeronautice Nr. de tehnologii aerospaţiale Nr. de tehnici pentru securitate

DC 9:Cercetări socio-economice şi umaniste

Nr. de noi metode manageriale, de marketing şi dezvoltare antreprenorială; Nr. de studii referitoare la calitatea educatiei si a ocuparii; Nr. de studii referitoare la capitalul uman, cultural şi social; Nr.de tehnici de conservare a patrimoniului

Nota: La completarea acestor indicatori se va tine seama de directia de cercetare si de obiectiectivele proiectului. Acesti indicatori se vor completa acolo unde este cazul.


19

3. Procesele verbale de avizare si receptie a lucrarilor


20

Nr. Inreg.: ..........................

SE APROBA, AVIZAT, REPREZENTANT AUTORIZAT, DIRECTOR ECONOMIC,

Rector Prof. Dr. Ing. Ecaterina Andronescu

Ec. Dorina Adamescu

PROCES VERBAL DE AVIZARE A LUCRARILOR

DE CERCETARE-DEZVOLTARE (P V A) Comisia de avizare constituita de executantul proiectului Tehnologie avansata de diminuare a pulberilor (inclusive nanoparticule) existente in termocentrale in vederea reducerii riscului la expuneri profesionale si a imbunatatirii sanatatii si sigurantei la locurile de munca, prin decizia nr 1623/20.10.2008 in cadrul etapei nr. 3 care fac obiectul contractului nr. 22-090/2008 incheiat cu Centrul National de Management Programe, a constatat urmatoarele:

a) Lucrarile executate corespund clauzelor contractuale;

b) Toate documentele necesare efectuarii platii exista si sunt corect intocmite;

c) Concluziile lucrarii, principalele rezultate obtinute si datele privind efectuarea cheltuielilor sunt prezentate in Raportul intermediar de activitate si in documentele sale insotitoare;

d) Planificarea activitatilor si resurselor aferente realizarii etapei curente in derulare a proiectului, prezentata in Raportul intermediar de activitate, este corespunzatoare realizarii obiectivului propus si in concordanta cu prevederile contractului;

Comisia avizeaza FAVORABIL lucrarile si documentele si considera ca pot fi prezentate pentru evaluare la CNMP – Programul 4 “Parteneriate in domeniile prioritare” – Domeniul Energie

COMISIA DE AVIZARE FUNCTIA IN COMISIE NUME SI PRENUME SEMNATURA

PRESEDINTE Prof. Dr. Ing. Ilie Prisecaru MEMBRI (cel putin trei specialisti)

Prof. Dr. Ing. Gh. Lazaroiu Prof. Dr. Ing. Gh. Comanescu Conf. dr. ing. Sorina Costinas

SECRETAR prof. dr. ing. George Darie


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4. Articole realizate pe baza rezultatelor obţinute în cadrul proiectului


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MONITORIZATION IMPACT OF ELECTRICAL EQUIPMENTS ON HUMAN HEALTH – PRIORITY OF CNTEE TRANSELECTRICA IN POLICY OF SECURITY AND HEALTH

ing.pr.spec - Alice Raducanu – CNTEE Transelectrica SA

insp. sef - Marin Stefan – CNTEE Transelectrica SA

dr.Cornelia Marcolt - CNTEE Transelectrica SA

cercet. st. – Camelia Suvergel – ICEMENERG

Abstract: The complex character of the issues the scientific community is confronted with at present includes, besides chemical pollution, exposure to electromagnetic fields of different intensities and frequencies. If chemical pollution has made the object of numerous studies with actual results, the effects of exposure to electromagnetic fields are not as well-known. The present study aims at investigating the effects of the electromagnetic fields on the health of the personnel exposed to this type of noxe. In order to evaluate the health state of the personnel exposed to the possible influences of the electromagnetic fields generated within these stations on the homeostasy of the human body, a medical investigation protocol, including clinical, neuropsychological, functional investigations, laboratory exams of the biological products (blood, urine), has been devised for establishing the hematological and biochemical constants. The present study also determines the levels of the melatonin neurohormone synthesis and secretion through the ELISA method. This has also represented a major difficulty in establishing a harvesting protocol enabling us to register the variable, physiological concentrations of the hormone, its metabolism, respectively, as accurately as possible. The harvesting protocol is new and unique in this field. As it is difficult to be carried out at the site, the harvesting protocol has been applied in the clinic, with the subjects admitted to the clinic. The protocol presupposes the harvesting of a urine spot for each urination over 48 hours. This procedure has allowed us to notice the circadian variations and the possible influence of the electromagnetic fileds due to the fact that some of the subjects that have been admitted during the two day period of time have worked at least one shift. The results obtained and presented in this study are partially confirmed by other similar studies. In the course of time, the initial functional disorders of the melatonin circadian secretion rhythm have been followed by circulating concentration diminishing, and, after this, by the imbalance in the human body mechanisms protecting it against the free radicals. This effect could be compensated by increasing the intake of natural antioxidants such as food (vegetal) rich in carotenoids.Considering the experimental results obtained and corroborating them with the results at the international level, the research could be continued over a new period of time on the same subjects, in the same substations.

Key-Word: electric and magnetic field, melatonin neurohormone, circadian secretion rhythm

1. Introduction The complex character of the issues the scientific community is confronted with at present includes, besides chemical pollution, exposure to electromagnetic fields of different intensities and frequencies. If chemical pollution has made the object of numerous studies with actual results, the effects of exposure to electromagnetic fields are not as well-known. The present


23

study aims at investigating the effects of the electromagnetic fields on the health of the personnel exposed to this type of noxe. In previous studies, complementary aspects relating to the health of the operating personnel from the electric substations that may have been affected by problems that could be included in the electromagnetic compatibility category have been examined. As a result of these studies, remedies to the major problems in this field could also be identified. Most of the damaging effects that have been encountered in the course of time have been related to the voltages and currents induced in the metallic structures or objects that are not properly grounded. In the case of the electric substations and overhead high voltage lines, the corona discharge also generates noise in the acoustic or electromagnetic spectrum, affecting the public activity in the nearby areas.

For a number of years the concern of the public relating to the possible negative effects of the low level and low frequency electric and magnetic fields, both on the health of the operating or residential personnel in the nearby of the sources of such fields, has increased.

In principle, the electric and magnetic fields whose frequency varies between 0 Hz (continuous voltage installations) and 30 kHz, with intensities in the air of up to 20 kV/m, 50 mT, respectively, are considered.

As regards the high frequency electromagnetic radiation (300 kHz ÷ 300 GHz), this has captured the interest due to the spectacular evolution of the technical microwave applications, in particular due to radiotelephony. The studies on this phenomenon are included in the larger field of bio electromagnetism and are carried out in the field of epidemiology, of the laboratory studies and dissymmetry. At low levels of exposure and in the absence of the thermal effects, some effects have been noticed in the laboratory. At present, the research activities focus on the independent. reproduction of these first conclusions. Until now no risks to health have been identified in cases where exposure has not led to thermal effects in the radiated organism, too. In the high frequency field, research has concentrated on the mobile phones, on the effect of the field they radiate in the close vicinity of the human brain. Special attention has been given to the low frequency electromagnetic field generated by the high voltage transmission and distribution grids, internal distribution networks, low voltage electric equipment (apparatus) and video terminals. Numerous independent organizations such as US Environmental Protection Agency (USEPA), World Health Organisation (WHO), National Radiological Protection Board (NRPB) and International Radiological Protection Association (IRPA) have developed a large number of reports in this field. Concern about the possible negative effects on the health of the personnel working in areas of the electric and magnetic fields generated by the electric installations (even by the low voltage ones) has been registered. The main aspects from this point of view are related to the ions and ozone resulting from the corona discharge. Some reports on the involved electric phenomena and on their possible consequences have further increased the concern of the electric substation personnel and of the specialists who, unlike the former, could reproduce the claimed effects by systematic studies. Every time, they have led to the conclusion that there was no clear-cut indication of the influence of the low voltage electromagnetic field exposure on the incidence of certain affections (cancer, especially). 2. Theoretical aspects relating to the electric and magnetic fields In spite of the numerous studies carried out so far, epidemiologists are still uncertain whether individual exposure to extremely low frequency electromagnetic radiations can be associated with certain effects on human health.


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One of the bio-effects that have been frequently noticed is the change in the production, secretion or utilization of a hormone secreted by the pineal gland located in the brain, a hormone that is commonly known under the name of melatonin. Melatonin (5- methoxy –N- acetyltryptamine) is a chemical mediator playing an important role in the human body and that is why the alteration of its level can be associated with a wide range of pathological conditions, including cancer. Due to this potential association when melatonin was found in low concentrations in the laboratory animals exposed to the electromagnetic fields it was assumed that this reduction could explain the higher than normal incidence of cancer in the people living in the nearby of the high voltage electric lines. The synthesis and secretion of the melatonin follows a circadian pattern, reaching the maximum production during nighttimes. The electromagnetic radiations, light, determine the normal amount of melatonin produced within 24 hours. Light has two main (primary) effects on the production of melatonin by the pineal gland: • It synchronizes the circadian rhythm, thus limiting the production of melatonin when it is dark; • It marks the reduction in the melatonin production when people and lab animals are exposed to light during nighttimes. Melatonin behaves like an antioxidant in the body. Many studies point out that exposure to the electromagnetic fields generates a higher rate of different cancer types than the normal one. Melatonin administration to the lab animals reduces the increase in the initial or transplanted tumours, while pinealtomia reduces the level of melatonin and accelerates the tumour increase. Such conclusions point out that an increase in the level of melatonin during nighttimes can hinder the increase in the already existing tumours. In contrast, any factor, for example exposure to light or electromagnetic radiations, reduces melatonin production during nighttimes, enabling acceleration of the tumour cell increase as the oncostatic agent has been lost. In the human body, the reduction in the melatonin production rhythm has been often associated with the tumour increase. In the women who have breast cancer and the men with prostate cancer it was found that the nocturnal melatonin peak was attenuated, the total melatonin being less than in the investigated individuals without tumours. As there is no clear evidence, these individuals are supposed to have developed cancer due to a lack of an adequate reserve of this natural oncostatic hormone. Anyway, experimental studies have pointed out that high melatonin levels diminish the tumour increase while low levels accelerate the tumour cell proliferation. Other experimental data indicate, through the exposure to the electromagnetic field, the melatonin production and cancer initiation. In 1993, melatonin was reported to be the most efficient scavenger discovered for the hydroxyl radical. The free radicals of oxygen, including hydroxyl, can be very toxic and affect the DNA, thus favouring cancer occurrence. At the same time, it has been noticed that melatonin is a potential scavenger for the peroxi radical. The peroxi radicals can initiate secondary cancer. Another activated form of oxygen, the singlet oxygen, seems to be neutralized by melatonin. The singlet oxygen is toxic enough so that it can partially affect the DNA base. Finally, melatonin has been indicated as stimulating an important antioxidant enzyme, glutathione peroxidase. Considering these antioxidant effects of the pineal melatonin, as well as the role of the free radicals in cancer initiation, any factors, such as the electromagnetic fields, or others, that reduce the endogen levels of melatonin, could be expected to increase cancer probability. The protective effect of melatonin against the oxidative effects of DNA is also supported by the observations that melatonin is to be found in great concentrations in the cell nuclei. These theories explain the reports in which the increased cancer risk is associated with the exposure to power electromagnetic fields. These theories underline the importance of reducing the level of melatonin produced and secreted by the pineal gland. Melatonin has become well - known for its anticancer action in the experimental studies. Thus, melatonin can prevent cancer initiation by reducing the frequency with which the DNA


25

is attacked by the free radicals. Melatonin reduces the increase in the cancer cells in human cultures. The breast cancer cells, as well as other types of human tumours, are inhibited by the presence of melatonin. A variety of tumours, both hormones dependent or independent, have been associated with the electromagnetic exposure. The decrease in melatonin reduces the total antioxidant potential of the human body because it plays a scavenger role on the free radicals of the oxygen. This fact alone could determine a small increase in the initiation of cancer. In the last years a number of researchers have suggested that the free radicals, that are paradigmatic molecules, can be influenced by the applied external electromagnetic fields. The halving time of the free radicals, in particular, may increase in the presence of the magnetic fields. It was supposed that the recombination of the free radicals can be delayed by the magnetic fields this way prolonging the halving time. Thus, the probability of their interaction with the DNA increases and the number of the affected DNA molecules will be greater. Therefore, the antioxidant protection mechanism is less efficient during the exposure to the magnetic fields due to the melatonin level decrease and, if this is coupled with the increase in the free radicals halving time, normally produced as a result of the oxygen metabolism in cells, the frequency with which all the cells are affected, the DNA ones, especially, could increase, too. This association may favour an increase in the risk of cancer. Another factor can be also added to this scheme. A variety of processes (for example, the ionizing radiations from the environment) and toxins (carcinogenous, herbicides) generate free radicals in the cells. In the individuals whose protection system is compromised by the exposure to the electromagnetic fields and the free radicals have a halving time greater than normal, generation of supplementary radicals can create an important detriment. The researchers have agreed on the fact that the electromagnetic fields cannot cause hemolytic scissions (breakage) of the covalent bonds that can generate the formation of the free toxic radicals, but electromagnetic fields can increase the risks of exposure in the processes or substances that initiate, in fact, the generation of free radicals. The briefly presented theories are not exclusive. All the processes, for example the reduction in melatonin, increase in prolactine and estrogens and the increase in the halving time of the free radicals can contribute to the increase in the breast cancer incidence by initiating certain DNA affections and by promoting the already existing tumours. These interactions are currently tested on lab animals and are simultaneously clearer defined by the epidemiological studies that are trying to determine whether there are effects associated with the human exposure to electromagnetic fields. The epidemiological studies and most of the experimental investigations that have been carried out on the exposure to electromagnetic radiations have led to the identification of a potential risk of cancer and, if the free radicals area component of this risk, then a series of other effects occur. A great number of affections and ageing can occur at the same time with the generation of the active oxygen species, such as the free radicals of oxygen. No clear conclusions could be drawn as regards the effects of exposure to the electromagnetic fields due to this ambiguity, the efforts will intensify and there is hope that in the future they will lead to conclusive answers. If the melatonin reduction in the human body is one of the consequences of exposure it is easy to rectify this deficiency; melatonin can be found in a number of plants used for food; more than that, melatonin is not toxic and is quickly absorbed. Thus, the melatonin supplement could represent a treatment for the individuals with less melatonin than the normal level due to the exposure to the electromagnetic fields or other causes. Further, a synthesis of the results of seven years of investigations tackling the main aspects, as well as the synthesis results will be presented. The methodology that has led to these results and the values obtained are further presented.


26

3. Application overview (synthetically presentation) At present, the focus is on the magnetic field generated by the electric installations, although both the electric and the electromagnetic fields are present at the same time in the area where these installations are located. By examining the experimental results registered în cele 3 electric substations of CN TRANSELECTRICA S.A company, maximum values have been identified at the level of the individual substations presented in Table 1.

Table 1

The maximum values of the electric and electromagnetic fields in the electric substations of S.C. TRANSELECTRICA S. A. (Monitored betweeni 2001 and 2087)

Substation no.

Name of the electric

substation

Voltages E kV/m

B µT

Obs.

01 Dârste 400 kV/ 110 kV

19,41 (36.5)

11.39 MOP At the level of the lid

With the lid open and

With the cell energized

02 Lacu Sărat 400 kV /220 kV

/110kV

20.83 (36.3)

20.1

03 Bradu 400 kV /220kV/ 110 kV

18.74 (36.7)

19.73

• The values Epresent in all the 3 substations, that are higher than 10 kV/m, have been registered as maximum values in the measurement points located around the drives of the circuit breakers of the MOP type. Due to the measurements carried out in other operating conditions and the possibility to carry out measurements in working areas that have been de-energized, the determinations of the electric field have focused both on certain points of interest from the 400 kV electric substation and on the identification of the factors of risk to the personnel during the work hours (e.g. the measurement of the electric field intensity at the MOP equipment, from the front, with the lid open, at the lid top, (where maximum values surpassing 30 kV/m have been registered). Very low values of the electric field intensity have been registered in the three objectives where the measurements have been carried out, including the high work platform in the AT-s. On the bases of these determinations and on the basis of those carried out in the electric substations in similar conditions, it has been established that the four working points, namely: • The autotransformer revision works (at the soil level, within the working area) • The disconnecting switch transfer coupling • Transfer coupling MOP, with the lid open (at 220 kV) pose no biological risks, regardless of the period of time spent there, the values being under the 10 kV/m limit. Preliminary results concerning the health state of the personnel from the DARSTE-BRASOV, LACU SARAT-BRAILA, BRADU-ARGES electric substations In order to evaluate the health state of the personnel exposed to the possible influences of the electromagnetic fileds generated within these stations on the homeostasy of the human body, a medical investigation protocol, including clinical, neuropsychological, functional investigations, laboratory exams of the biological products (blood, urine), has been devised for establishing the hematological and biochemical constants.


27

The present study also determies the levels of the melatonin neurohormone synthesis and secretion through the ELISA method. The focus on this hormone has been determined by the previous findings suggesting modifications of metabolism as a result of the electromagnetic field influence. The effect of the decrease in the melatonin secretion level could prove extremely important not only because this hormone plays a major role in regulating the sleep-wake relationship, but also because it seems that it acts as anti-cancer protection, too. Physiologically, melatonin is synthetized and secreted in an ondulatory way, according to the circadian rhythm, characterized by day and night, by minimum and maximum values, respectively. This has also represented a major difficulty in establishing a harvesting protocol enabling us to register the variable, physiological concentrations of the hormone, its metabolism, respectively, as accurately as possible. The harvesting protocol is new and unique in this field. As it is difficult to be carried out at the site, the harvesting protocol has been apllied in the clinic, with the subjects admitted to the clinic. The protocol presupposes the harvesting of a urine spot for each urination over 48 hours. This procedure has allowed us to notice the circadian variations and the possible influence of the electromagnetic fileds due to the fact that some of the subjects that have been admitted during the two day perio of time have worked at least one shift. 4. Methodology The health condition of the working personnel from the investigated high voltage substations has been evaluated considering the specific activity they carry out and their occupational exposure. The clinical examination has been carried out on 47 persons (approximately 15 persons by each investigated electric substation), representing the operating personnel in the respective substations and has been completed by their neuropsychological exam and biological product harvesting for lab analyses. The hematological determinations that have been performed referred to the study of the peripheral blood picture: number of red cells, hematocrit, average eritrocit volume, hemoglobin concentration, number of white cells and the leucocite formula. These determinations have been carried out by means of the MINITRON equipment, manufactured by the Diatron company; the blood sample harvesting has been carried out on an anticoagulant (EDTA). 5. Clinical and psychomedical examinations 47 subjects have been examined three times consecutively over a 8 – year period of time in the three electric substations under investigation. The results of these examinations have been registered in their medical sheets. On this occasion, associated pathology personal anamnestic data have been taken over, too. A questionnaire on previous occupational anamnesis (uninterrupted activity in a workplace, work places, occupational exposure, or no exposure, to different noxes) has been filled in as all this information (data) is useful for eliminating the confusing factors from the analysis, as well as for establishing, as homogenously as possible, the groups for the endocrine determinations. To this goal (namely identification of the possible confusing factors), data referring to random exposures to other electromagnetic fields besides those at the workplaces (e.g: electronic equipment, mobile phones, residence location from the existing electric networks or radio antennas, etc.) have been also obtained. The information relating to the sleep-wake rhythm of the subjects, residence and workplace night lighting have been put down as they have an extremely important influence on certain homeostasy values (circadian hormonal rhythm of melatonin).


28

The clinical examination has also included an electrocardiographic examination by means of the ocilloscope of the PULSONIC- 2000 type apparatus. The structure of the investigated groups of subjects is given in the following tables. Subject age Substation No. of persons 20 – 30

years 31-40 years

41–50 years > 50 years

Braşov 16 3 10 2 1 Argeş 17 8 5 2 2 Brăila 16 3 5 5 3 Uninterrupted work time Substation 1 – 5 years 6 – 10 years 1 – 15

years 16–20 years > 20

years Braşov 12,5 % 18,7 % 31,2 % 31,2 % 6,2 % Argeş 35,2 % 11, 7% 17,6 % 5,8 % 29,4 % Brăila 6,2 % 6,2 % 31,2 % 12,5 % 43,7 % The biochemical investigations have concentrated on establishing: • The degree the lipide metabolism has been affected, by means of trigliceride, cholesterol, HDL cholesterol determinations; • The degree the liver has been affected, by means of serum transaminase determination (TGO si TGP); • Calcium and magnesium; • Blood sedimentation rate (BSR) The investigated personnel has been mostly ( about 80%) the same since 2001 until 2006. The results of the investigations have pointed out that: • The lipide metabolism has been affected (values of cholesterol and triglyceride concentration higher than the risk values of 200 mg/dl for triglyceride and 260 mg/dl for cholesterol) in the case of one or two persons by each investigated substation (Braila, Brasov, Pitesti) over the entire investigation period. • Off-balance lipide metabolism correlated with liver affection has been encountered in only one case from the Braila substation. • The other biochemical parameters have not been significantly altered. We consider that the minor modifications that have been registered in the biochemical examinations cannot be correlated with the occupational exposure to the electromagnetic fields. As regards the hematological investigations, they do not present significant modifications against the normal values. 6. The hormonal exams A special protocol has been developed for urine harvesting in order to establish the doses of hormonal levels of the 6–hydroximelatonin sulphate. As all the subjects are active persons with a 12 hour work program followed by a 24 hour break and also considering the circadian rhytm (day/night of the physiological secretion of this hormon), in order to point out the influence of the occupational exposure fields, urine harvesting has been carried out on each subject for 3 days uninterruptedly, for each urination. The registered values point out the influences of all the respective overlapping factors. This method is also advantageous because it compares the values registered for the same subject


29

during similar periods of time. It is important to be able to make this comparison as the reliability of the secretion cycle for each person is very low; in other words, the comparison between the hormone levels of different individuals can be avoided, as it may generate confusions, considered as physiological. Part of the affections that have been identified during the medical exeminations, such as osteoarticular affections, certain digestive diseases (ulcer, gastritis) caused by stress or depression, may be related to the work conditions elements, etc., but are not directly influenced by the intensity of the fields. Exception from the previous assertion make the hormone dosages. In this respect, in most of the subjects that have worked for less than 5 years this seems to be a phenomenon of secretion arrythmia within the circadian rhythm, a situation that becomes less obvious in the patients with a longer professional activity. From the biological point of view, the aspect relating to the secretion curves could be interpreted as a phenomenon of organism adaption and entering into operation of the homeostatic type mechanisms. A relatively constant phenomenon, encountered in the case of the subjects who have worked more than 5 years, is secretion dephasing with the work hours. Less constant, but also significant from the statistical point of view, has been the phenomenon of melatonin secretion amplitude diminishing during the work hours in comparison wih the periods of time when the subjects have not been exposed. If we compare the three substations, relatively constant modifications have occurred in the subjects that come from the Lacu Sărat substation. The following report presents the values registered at the beginning, middle and at the end of the period of study. The values from the intermediate years are within the same curves and, for better underlining the registered modifications, the variant of the synthetic presentation of the above mentioned years has been chosen. The synthesis and secretion of melatonin has been evaluated through the dosage of 6-sulfateximelatonin, the urinary metabolite of melatonin. In the case of the human being 90% of melatonin is metabolized in 6-hidroximelatoninsulphate (6-OHMS) and that is why the 6-OHMS concentrations in the urine quantitatively reflect the concentrations of serum melatonin. In the first sets of determinations 64.70% of the subjects have registeres normal values for 6-OHMS, 29.41% presented dephasings of acrophase (maximum secretion) and 5,88% have presented arythmic secretions of melatonin.

Variation 6-OHMS in the years 2000-2001

64,70%

normal5,88%arythmic

0% decreases

of conc.

29,41%

dephasing

The determinations carried out in 2003 pointed out that 21.42% of the subjects presented dephasing of the acrophase, 28.57% of the subjects had lower maximum concentration of 6-OHMS, especially during occupational exposure to electromagnetic radiation and 7.14% of the subjects had an arythmic secretion of melatonin.


30

Variation 6-OHMS in the year 2003

21,42%

dephasing

28,57%

decreases of

conc.

7,14%

arythmics

42,85%

normal

In 2005, 17.39% of the subjects presented acrophase dephasing, 17.39% of the investigated workers have had low values of the maximum 6-OHMS concentration, while 13.04% of the subjects had an arythmic melatonin secretion.


52,17%

normal

13,04%

arythmics

17,39%

decreases of

conc.

17,39,42%

dephasing

In 2006, 14.28% of the subjects presented acrophase dephasing, 24.69% of the investigated workers had low values of the maximum 6-OHMS concentration, while 15.63% of the subjects had an arythmic melatonin secretion.


14,28%

dephasing

24,69%

decreases of

conc.15,63%

arythmics

45,60%

normal

The analysis of the 6-OHMS concentration data has pointed out that the maximum secretion dephasing continued to occur (in 2007) in about 17 % of the subjects, by delaying it, and in about 14 % of the subjects, by secretion arythmias. These deregulations (disorders) have persisted even between the shifts, during the rest-time, especially in the subjects under the age of 40.


31

The phenomenon of metabolite concentration diminishing has continued to occur, especially then, when the comparison is made with the values registered in the previuous years, in the same subject (19%).


50%

normal

19% arythmics

14% decreases

of conc.

17% dephasing

In 2008, 19% of the subjects presented acrophase dephasing, 16% of the investigated workers had low values of the maximum 6-OHMS concentration in while 21% of the subjects had an arythmic melatonin secretion. Variation 6-OHMS in the year 2008

By analyzing the data obtained during these 8 years there results that about 50% of the subjects have had a normal rate of production and secretion of 6-hidroxymelatonisulphate, therefore melatonin, with normal values of the maximum registered concentrations. The number of persons who have registered normal curves of 6-OHMS has decreased with time, thus between 2001 and 2002, the normality percentage was of 64.70%, while in 2008 it decreased to 44%. Part of the investigated subjects have had maximum secretion shifts (acrophase) before, or later than the usual hour, thus influencing the circadian rhythm. Between 2001 and 2002, 29.41 % of the investigated subjects registered dephasings in the melatonin mqaximum secretion, the percentage slightly decreased in the years 2003 and 2005 to 21.42%, and 17.39%, respectively, the decrease being more accentuated in 2006, when 14.28% of the subjects registered dephasing in the acrophase registration hour. In 2007 a slight increase (17 % ) in its value was registered and in 2008 the acrophase was 19%. A tendency towards the decrease in the maximum concentration of 6-OHMS has been noticed. The decreases in the 6-OHMS concentration were registered in 2003 (28.57%), 2005 (17.39%), 2006 (24.69%), 2007 (19%) and 2008 (21%). This has not been a constant phenomenon; it has appeared slowly, after several years of exposure and we are bot cettain whether it has been caused by the exposure to the electromagnetic fields or it is the result of ageing. The maximum melatonin secretion dephasings registered in a great number in the first years of the study seem to have changed into arythmic secretions in the last years, after a prolonged exposure to the electromagnetic fields. This is also supported by the results we have obtained pointing out that the percentage of arrythmic secretions increases in the course of time. Thus, 5.88% of the subjects have presented arrhythmias in 2001-2002, the


32

percentage increased to 7.13% in 2003 and to 13.045% in 2005, while in 2006 this increase was greater, attaining 15.63% of the investigated subjects, in 2007 the percentage was of 14% and in 2008 the value was 16%. The modifications that have occurred in the production and secretion of melatonin seem to be more accentuated in the cold season than in the warm one. The results of the melatonin determinations have been monitored for each of the analyzed subject during the seven year period of time. The results have pointed out that in 28.88% of the workers whose melatonin has been constantly determined, the secretion of 6-OHMS, that was normal in the first year or presented a slight shift in the maximum concentration, turned into an arythmic secretion to the end of the investigation period. The aspects presented above could be of the result of the exposures to the electromagnetic fields (knowing their effect on the immune system), but, in order to support this assertion, supplementary determinations and analyses, that, on the other hand, are scheduled for the following phases of the project, have to be carried out. 7. Conclusions Electromagnetic field intensity can be reduced only by positioning the current paths in a relatively efficient way and/or locating the sources of the field as far from the protected zones as possible. This is true in the cases where the available space is not restricted, the case of the overhead electric lines more specifically. Nevertheless, in the case of the existing and future electric installations, where the available space is limited, the screening (passive or active) seems to be the only possible way to diminish the electromagnetic field. For seven consecutive years three groups of subjects, selected from the personnel of the Lacul Sarat, Bradu, Darste substations, that have been exposed to the electromagnetic fields, have been placed under medical supervision. The medical supervision has been based on protocol of examination including clinical, functional (EKG, PFV) examinations, psychological evaluation, laboratory (hematological, biochemical) analyses and dosage of the urinary metabolite (6-OHMS) of the melatonin hormone. The selection has considered the following criteria: the age (knowing that after the age of 45 the secretion level of this hormone decreases), occupational exposure to the electromagnetic fields (the personnel worked in three shifts). At the same time, the possible associated medical affections, random exposures to fields of this type (even the low intensity ones), such as the mobile phones, household appliances, antennas or other relays, located in the nearby of their resiedences, have also been considered. From among the monitored parameters the melatonin hormone has represented the main point of interest, as its antioxidant role is well known, as well as the possibility to be influenced by the electromagnetic fields. Part of the research studies in the literature in the field underline the modifications of the circulating secretion levels of this hormone and, consequently, the possible negative influence on the human body protection against the free radicals and their effects (in oncogenesis, as well). The synthesis and secretion of melatonin has been assessed by 6-OHMS dosing, which is the urinary metabolite and represents about 90% of the secreted melatonin, its concentrations following with fidelity the hormone curves (with a 2-3 hour shift). A major argument in favor in this respect has been the fact that both harvesting and dosing (from urine) are easy to perform, as the monitoring process has had to cover a long period of time and repeated biannually. The 6-OHMS dosage has required the development of a special protocol for urine harvesting. As all the selected subjects are active people, with a 12 hour (24 hour break) working program, in general, and considering the day/night circadian rhythm of the physiological secretion of the hormone, urine harvesting has been carried out for each subject over a 3 day time interval without any interruption, at each urination, in order to be able to point out any possible influence of the occupational exposure to the fields. Two periods of harvesting (in spring and in autumn) have been organized each year during the period of the study.


33

This method is also advantageous as it enables the comparison between the registered values for the same subject in similar periods. In the case of some subjects the results of the medical and laboratory examinations (biochemical and hematological) have pointed out a series of modifications suggesting different affections (osteoarticular, cardiovascular, digestive, etc.), that, statistically, do not present significant modifications neither against the personnel of the three substations, nor against the general population. These modifications do not seem to be directly related to the work conditions, and are not generated by them, but they could be influenced by certain factors, characteristic of the workplace (work in shifts, stress, etc.). Extremely interesting results have been obtained in connection with the melatonin concentrations. Until now, these results are to be found only partly in the literature in the field. As known, the circadian secretion rhythm of this hormone registers a maximum concentration at night and a minimum during daytime, with rather great individual variations within the physiological range of values. From the analysis of the obtained data the first effect that can be noticed is the presence of certain dephasings in the acrophase (maximum secretion) usually by delay. In the years of the study the percentage amounted to 29% (of the values), but in 2006 the percentage did not surpass 15%, in 2007 it amounted to 17% and in 2008 the percentage was 19%. This decrease in the number of dephasings has an inverse evolution with the frequency of arrythmias (up to rhythm inversions). During the first year of the study, arrythmias represented about 5%, then 7.4%, in 2003 while in 2006 they surpassed 15%, in 2007 they amounted to 14% and in 2008 was 16%. This may be one and the same phenomenon, namely the dephasing of acrophasia up to the occurrence of an actual arythmia, sometimes even with the inversion of the circadian rhythm. This is a progressive deregulation of the secretion and the phenomenon appears especially in the younger subjects with shorter uninterrupted work duration (about 10 years). This functional deregulation does not seem to be the result of the inversion of the circadian sleep /wake cycle due to the night shifts, as it keeps occurring during the periods of rest after the night shifts. Another important result (this time mentioned in other studies in the field, as well) is the one relating to the melatonin metabolite concentration diminishing. Over the seven years period of time, diinishings in the melatonin concentration have been noticed in 22.91% of the subjects. It is worth mentioning that the concentration values that have been determined have been also compared with the values from the same patient (and not only to the values considered normal, in general); thus, the comparison is much more representative and accurate. At the same time, the fact that the age of these subjects varied between 35 and 47 at the time of the study, is also important as the diminishing caused by physiological factors (ageing) is not to be taken into account. The fact that, in general, the samples with lower concentrations have been harvested during the period of work implies the influence of the occupational exposure factor. As regards the fact that the modifications in the melatonin concentrations have been more evident in the samples harvested in the autumn months than in those harvested in spring, no plausibile expalantion has been found yet, as the level of light during daytime, the ratio day/night, or the temperature variations have not differed so much to account for this. The results obtained and presented in this study are partially confirmed by other similar studies. In the course of time, the initial functional disorders of the melatonin circadian secretion rhythm have been followed by circulating concentration diminishing, and, after this, by the imbalance in the human body mechanisms protecting it against the free radicals. This effect could be compensated by increasing the intake of natural antioxidants such as food (vegetal) rich in carotenoids.


34

Considering the experimental results obtained and corroborating them with the results at the international level, the research could be continued over a new period of time on the same subjects, in the same substations. References [1] S. Hurdubeţiu, St. Ioan Instrucţiuni privind natura şi rezolvarea problemelor de impact al reţelelor de înaltă şi medie tensiune (linii şi staţii) asupra mediului ambiant, nov. 1996.

[2]Ungureanu M., Cristescu D., Magureanu G., Ioan S, Contract 7 - 97 - 15/poz.S43001- " Procedură de evaluare a impactului asupra mediului in vederea obţinerii acordului şi/sau autorizaţia de mediu pentru LEA şi staţii de înaltă tensiune (220-750 kV conform legii nr 137/95 de protecţie a mediului şi ordinului MAPPM nr.125/96 (reactualizarea metodologiei elaborate in sept.1995)”.

[3] S. Hurdubeţiu, St. Ioan, Metodologie şi program de calcul al câmpului magnetic tridimensional al liniilor electrice aeriene. (ISPE, PEC 143-97).

[4] CENELEC - Electromagnetic fields in the human environement. Low frequencies 0 - 10 kHz .ENV 50166 - 1 /1995.

[5] CENELEC - Electromagnetic fields in the human environement. High frequencies 0 - 10 kHz. .ENV 50166 - 2 /1995.

[6] CISPR- Publicatia 18-1 - Caracteristiques des lignes et des equipements a haute tension relatives aux perturbations radioelectriques. Partie I-a: Description des phenomenes. CEI 1982.

[7] CISPR- Publicatia 18-2 - Caracteristiques des lignes et des equipements a haute tension relatives aux perturbations radioelectriques. Partie II-a: Methodes de mesure et procedures d’etablissement des limites. CEI 1986.

[8] CISPR - Publicatia 18-2 Amendament -1 - Caracteristiques des lignes et des equipements a haute tension relatives aux perturbations radioelectriques. Methodes d’etablissement des limites pour les perturbationsr adioelectrique

[9] CISPR - Publicatia 18-3 Amendament -3 - Caracteristiques des lignes et des equipements a haute tension relatives aux perturbations radioelectriques. Code practique de reduction du bruit radioelectrique. CEI 1996.

[10] CISPR - C.9.1 - Establishment of limits for the radio noise produced by overhead power linesin the range of frequency 0,15 - 30 MHz. CEI - 1996

[11] CIGRE - Interferences produced by corona effect of electric system. . Addendum to CIGRE Document nr.20 (1974)- WG 36.01.

[12] PE-104/93 - Normativ pentru constructia liniilor aeriene de energie electrica cu tensiuni peste 1000 V. Regia Autonoma de Electricitate - RENEL..

[13] CIGRE - Electric and magnetic fields produced by transmission systems. Practical Guide for calculation. WG 36.01, Paris 1980.

[14] Shperling I.B. , Menemenlis-Hopkins , L. , Fardanesh B. Claairmont B. , Childs, D. : Reduction des champs magneriques de lignes de transport utilisant des boucles passives; CIGRE, Sesion-1996.


35

STRUCTURAL CHARACTERIZATION AND IN VITRO CYTOTOXIC POTENTIAL OF COAL

DUST IN A ROMANIAN POWER PLANT Alice Raducanu11, Aurica Suvergel1, George Darie2, Ileana Rau2, Constantin

Grigoriu3, Cristian Viespe3, Traian Vasiu5, Ionela Daniela Popescu4, Eleonora Codorean4

1 Institute for Energy Research and Development, No.8 Bld., Bucharest 3, Postal Code 032092, Romania 2 Politehnica University from Bucharest, 1 Polizu Street, 011061, Bucharest, Romania 3National Institute for Laser, Plasma and Radiation Physics, 409 Atomistilor Street, 077125, Bucharest-Magurele, Ilfov County, Romania 4 “Victor Babes” National Institute of Pathology, 99-101Splaiul Independentei, 050096, Bucharest, Romania 5 SC Electrocentrale DEVA SA, 1 Santierului Street, Mintia, Hunedoara County, Romania Abstract A number of specific measures have been undertaken in Romania in the power generation sector in order to limit or eliminate the pollutants and to prevent their adverse health effects of occupational exposure. However these measures did not result to the expected efficiency, and a number of workplaces still exist within the thermal power plants where the employees work under improper conditions. The infliction of the working environment, dust and ash fine particles from coal-fired power plants has a detrimental effect upon the exposed subjects depending on the pollutants toxicity, the concentration, exposure duration, and health state of the subjects exposed. The paper presents a quantitative and qualitative assessment of dust, with estimation of chemical-structural composition and particle size distribution within a case study on Deva-Romania power plant. The potential to modulate cell proliferation and to induce cytotoxic effects in vitro performed on BALB/c 3T3 fibroblast cell line is also presented, as a measure of the biological hazardous compounds existent in coal dust and ash sampled from the investigated site. The preliminary in vitro data indicate that even at the lowest dose some cytotoxic effects were detectable, dust and ash samples significantly inhibiting cell proliferation and increasing LDH release.

Key words: coal dust and ash, physicochemical parameters, in vitro cytotoxicity.

1. Introduction

Dust defined as particulate matter is "any airborne finely divided solid or liquid material with a diameter smaller than 100 micrometers". Dust and smoke are the two major components of particulate matter. Fly ash consists in particles released into the environment in effluents from the combustion of coal and provides a matrix for the interaction of a great variety of substances during the combustion and emission processes. Specific pollutants from

1 Author to whom all corespondence should be addressed: e-mail: [email protected]; tel: +40212700469; fax :+40213035903


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thermal power plant industry, coal dusts and ashes are complex particles of a variable composition, often dependent on the source of coal and the precipitation technique (Borm, 1997).

The toxicity of the environmental particles has received significant attention along the time. The industrial ambient air monitoring collected key data of existing aerosol composition, and structure in the workplace, their particulate matter (PM) count, size distribution and morphology that may link to toxicological studies. Particle research is closely connected to industrial activities of materials, such as coal power plant, asbestos, man-made mineral fibers, and recently ambient particulate matter (PM) (Borm, 2002). The size distributions of indoor aerosols showed less variability than the outdoor aerosols and the distributions of indoor ultrafine particles may have implications concerning personal exposure and can be associated with health consequences (Zhu et al., 2005).

There is a strong probability that biological activity of particles will depend on physico-chemical parameters routinely considered in toxicity screening studies (Oberdoester et al., 2005). Several studies indicate that is necessary to evaluate and control any possible releases or peaks of nanoscale particles (NP), ultrafine particulate matter having a diameter less then 100 nm. There is a growing consensus that the complexity of these issues requires a multidisciplinary approach to particle toxicology risk assessment screening for these specific pollutants from the thermal power plant industry (Borm, 1997; 2002; Oberdoester et al., 2005; 2007).

Nanoparticles have higher mobilities, they may diffuse easier than solid particles and behave more like gas molecules in the air and large molecules in solutions, being less subject to sedimentation than bigger particles. This may have implications also for the movement of nanoparticles in tissue. Whether nanoparticles enter and transfer within the body to different organs might have a significant importance for the impacts of nanoparticles on human health and in the environment. Dissolution, translocation, and disposition have been shown to play a key role in the fate and effects of inhaled nanoparticles and may be a critical step for some nanoscale materials in determining fate in the environment and within the body (Borm et al., 2006). Similar to all other contaminants, it is essential that levels of nanoscale materials to be determined and controlled in order to reduce their exposure in the workplaces.

The quantitative measurement of particles in the nano range is still in the development phase. The World Health Organization (WHO) protocols refer to the PM health risk including the exposure concentrations, particle size, target populations, endpoints, and length of the exposure (European Commissionm, 2005). Many studies focused on environmental and occupational exposure of humans to nanoparticles frequently examined respiratory tract and pulmonary toxicity as target organ. The animal studies, routinely show an increase of the pulmonary parameters of inflammation, immunotoxicity and genotoxicity that are mainly considered as indicators of the health effects of exposure (Castranova, 2000; Oberdörster., 1994, 2001, 2005).

Laboratory-based studies have investigated the effects of a large range of PM including nanoparticules through in vivo exposures using various animal models as well as cell-culture-based in vitro experiments. Toxic constituents in these particles are considered to be metals, polycyclic aromatic hydrocarbons and silica. Several in vitro and in vivo data on coal fly ash and dusts were related to studied endpoints to the role of (crystalline) silica, considering its recent classification as a human carcinogen. For most of the effects coal mine dust was chosen as a reference, since it contains up to 15% of crystalline silica (ot-quartz) and is well studied both in vivo and in vitro (Borm, 1997; Stone et al., 2007).

There are indications that silica dioxide can penetrate the cells and disrupt functions of the nucleus, most of the effects leading to its recent classification as a human carcinogen. The hazards relevant to humans, provides some of the newly emerging literature on fate and behavior of nanoparticles in the environment, as well as their potential risks to humans and the environment. For many studies, crystalline silica was chosen as a reference, and is well


37

studied both in vivo and in vitro (Albulescu, 2009; Oberdörster et al., 2005; 2007; Unfried et al., 2007).

In vitro techniques allow specific biological and mechanistic pathways to be isolated and tested under controlled conditions, in ways that are not feasible in in vivo tests. Non-cellular assessment of nanoparticle durability, protein interactions, complement activation, and pro-oxidant activity is also considered, nanoparticles having a higher potency than particles from the same substance in the micrometer range (fine dust) (Stone et al., 2007).

To assess the intrinsic cytotoxic effects of chemical substances, several in vitro tests have been developed by measuring different biological endpoints such as cell viability, cell metabolism and membrane leakage (Bakand et al, 2006; Fotakis and Timbrell, 2006; Stone et al., 2007; 2009). Recent studies were focused to share knowledge relating to particle toxicity testing in order to provide advice and support for the interferences of the particles in the cell metabolism events and may predict in vivo toxicity. The predictive value of in vitro cytotoxicity tests is based on the idea that toxic chemicals affect basic functions of cells which are common to all cells, and that toxicity can be measured by assessing cellular damage (Lambroussis et al., 2009; Stone et al., 2009; Unfried et al., 2007).

The infliction of the working environment has more or less detrimental effect upon the exposed persons depending on the toxicity of the existing pollutants, the concentration, exposure duration, and constitution of the subjects exposed. At present in Romania, a number of specific measures have been undertaken in the power generation sector in order to limit or eliminate the pollutants, with the aim to prevent the employee professional diseases, however these measures did not result in the expected efficiency (Raducanu et al., 2008).

This paper presents a quantitative and qualitative assessment, estimating composition, physico-chemical characteristics, size distribution of dusts and ashes collected from the Deva (Romania) power plant. A special attention was paid to realize variable time and dose in vitro exposure to the coal dust and ash samples and to evaluate their effects at the cellular level. In this way we performed the assays of cytotoxic and proliferative capacities in vitro, as most frequently used tests for evaluating early preliminary biological reactivity of particles in vitro.

2. Experimental 2.1. Structural characterization

The work places under investigation from physical and physical - chemical points of

view (coal dust - powder) are located both indoors in closed spaces and outdoors, in a configuration specific to each operation section and to the specific technological processes carried out on site.

The powder characterization was performed by physical-chemical analyses. The investigations established the major constituents and their mass concentration.

For size distribution assessment, a laser sizer was used, model Mastersizer 2000. The evaluation of crystallinity degree was made by X ray diffraction analysis, using a

Bruker-AXS, D8 Advance diffractometer (Cu Kα1).

2.2. In vitro cytotoxicity 2.2.1. Cell cultures and treatments

BALB/c 3T3 fibroblast cell line was used as in vitro test system to evaluate the cytotoxic effects of dust and ash samples collected from coal-fired power plant from Deva-Romania. BALB/c 3T3 fibroblast cell line, from ECACC (European Collection of Cell Cultures), preserved in “Victor Babes” Institute cell cultures collection) was used. Cells were grown in MEM (modified Eagle’s medium) supplemented with 10% fetal bovine serum, 1% MEM non essential amino acid solution, and 1% penicillin / streptomycin solution in 0.9% NaCl) in a humidified atmosphere of 5% CO2, at 35

0 C in a incubator from SHEL LAB, Sheldon


38

Manufacturing, Inc., USA). Cells were cultured in 96-well plates (200µL of cell solution/well). The optimum cell concentration as determined by the growth profile of the cell line was 105 or 2×105cells/ml. Cells were allowed to attach for 24 h before treatment with dust or ash samples. All plates containing cells were incubated 3h at 37°C in an environment with 5% CO2 in a SHEL LAB incubator. Sterile conditions were maintained throughout the duration of the experiment (UV laminar flow hood – HOLTEN A/S MAXI SAFE 2010, Denmark, was used). Dust and ash samples were sterilized by heating at 1600C for 90min and suspended in sterile phosphate buffer saline (PBS). BALB/c 3T3 fibroblast cell line was treated with dust and ash suspensions, respectively, within a range of concentration from 0, 25, 50, 100, 150, 200 µg/mL and incubated at 37°C with 5% CO2 for 24 h in order to evaluate cell proliferation capacity and LDH leakage.

2.2.2. Cell proliferation assay (MTS test)

MTS:3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, novel tetrazolium compound is bioreduced by living cells (NADPH or NADH, produced by dehydrogenase enzymes) into a colored formazan that is soluble in tissue culture medium product. The quantity of formazan product as measured by the absorbance at 490nm is directly proportional with metabolically active cells. The test was conducted by using Promega CellTiter96 Non-Radioactive Cell Proliferation Assay following Promega technical bulletin. The detection reagent is composed of solutions of MTS (Tetrazolium salt) and PMS (Phenazine methosulfate, an electron coupling reagent). Both substances were initially dissolved in PBS (Phosphate Buffered Saline) at ratios of 2 1 (w/v, MTS: PBS) and 0.92 1 (w/v; PMS: PBS), filter sterilized and stored separately in light protected containers at –20 °C. The plate exposed to ash and dust samples were incubate 1-4 hours with MTS solution (20 µL to 100 µL of cell in culture) in dark and then read at 490nm on a Microplate Reader (PR 3100 TSC, Biorad, USA).

2.2.3. LDH release assay

The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage.

By using solution reagents contained in a Promega Kit CytoTox96 Non-Radioactive Cytotoxicity Assay, released LDH in culture supernatants is measured with a 30 minutes coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product; the amount of color is proportional to number of lysed cells.

After exposure, the microplate was centrifuged 250 x g for 4 minutes by using a microplate centrifuge Centra CL3, Thermo IEC, Thermo Labsystems, Finland. 50 µL alliquots were transferred from all wells in an enzymatic assay 96-well plate with 50 µL substrate, incubated in dark 30 minutes. 50 µL stop solution were added and within one hour after the addition the absorbance at 490nm was recorded at PR 3100 TSC Microplate Reader, Biorad, USA.

2.2.4. Statistical analysis

Data were expressed as mean +/-standard deviation (SD); Student’s t test were applied for individual comparison.

3. Results and discussion

The measurements of the powders have been carried out for each work place in the points where the operating personnel has to carry out different operations or different other activities specific to the technological process.

Concerning the composition of the dry ashes collected from power plant of Deva-Romania electrofilter, Table 1 shows the mass concentration of each constituent.


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Table 1. The mass concentration of the dry ashes collected from power plant of Deva-Romania electrofilter

Compound Mass Concentration [%]

Silica Sulfates Fe2O3 Al2O3 Lime - CaO Magnesia Total vanadium expressed asV2O3 Nickel oxide Copper oxide Zinc oxide Manganese oxide Cadmium oxide Massicot Total chromium expressed as Cr2O3 Mercuric oxide Arsenic oxide Sodium oxide Potassium oxide

52.63 0.382 7.411 5.467 3.105 2.453 0.024 0.014 0.022 0.014 0.028 6.10-5

0.007 0.032 6.10-5

6.6.10-4

0.384 2.435

Highlighted compounds had a concentration higher than 1%. It is noteworthy that

SiO2 is predominant, being over 50%. The other significant constituents are Fe2O3 > 7%, Al2O3 >5%, etc.

The composition of the dust coal and crushing coal powder that supply the combustion furnace SC Elecrocentrale DEVA SA is shown in Table 2.

Table 2. The composition of the dust coal and crushing coal powder that supply the combustion furnace SC Elecrocentrale DEVA SA

Compound Mass concentration [%]

Silica Sulfates Fe2O3 Al2O3 Lime - CaO Magnesia Total vanadium expressed asV2O3 Nickel oxide Copper oxide Zinc oxide Manganese oxide Cadmium oxide Massicot Total chromium expressed as Cr2O3 Mercuric oxide Arsenic oxide Sodium oxide Potassium oxide

18.550 4.679 3.485 1.112 1.907 1.221 0.009 0.007 0.008 <0.001 0.012 4.10-5

0.007 0.015 <4.9.10-5

2.1.10-4

0.145 0.685


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The concentration of the highlighted compounds was > 1%.

In Fig. 1 they are shown the X ray diffraction spectra for the dry ashes (a) collected on electrofilter and coal dust (b) collected at the work places.

(a) (b) Fig. 1. XRD spectra of the flue ashes (a) and coal dust (b)

One can note that the silica (SiO2) was a major constituent (confirmed by chemical analysis) and had a crystalline structure (quartz). Aluminum silicate (3Al2O32SiO2 or 2Al2O3 SiO2) is crystalline as well. The other compounds are in amorphous state, or quantitatively insignificant.

In case of coal dust (Fig. 1b), crystalline silica is also present. Besides quartz, other crystalline components could be found, as kaolinite Al2Si2O5(OH)4 and magnesium silicate Mg3Si4O10(OH)2, but at a very low concentration.

The results related to the size distribution of the two powders are presented in Fig 2.

Fig. 2. Size distribution of the powders: (a) flue ashes, (b) coal dust.

The size of the flu ash particles (Fig. 2a) is ranging between 0.24 and 0.63 µm, while for coal dust (Fig. 2b) between 0.27 and 2187 µm.

The samples of dusts and ashes analyzed contained, as confirmed by chemical analysis, numerous heavy metals as well as a high proportion of silica (SiO2) as the major constituent with a crystalline structure (quartz). To evaluate the cytotoxic effects of ash and dust samples from Deva power plant, BALB/c 3T3 cell line were used to quantify measuring two cytotoxicity endpoints in vitro.

BALB/c 3T3 cells are a murine cell line with a morphology resembling fibroblasts with a wide range of receptors and second messenger system, that have been used as a standard cell line for many biochemical and cell biological studies. The performance level of BALB/c 3T3 fibroblasts in cell transformation assays have been reported on about 180 chemicals exposure


41

with good concordance, sensitivity and specificity assay (Muramatsu et al., 2009; Sakai, 2008).

The concentration range 0, 25, 50, 100, 150, 200 µg/mL and the exposure times have been selected based on previous studies performed in our laboratory on heavy metal in vitro toxicity, or in other test systems as well as literature information (Allermann and Poulsen , 2002; Bakand et al., 2006; Codorean et al., 2004; Lambroussis et al., 2009).

The MTS proliferation assay was used to measure the toxicity of test chemicals by determining the number of viable cells, their metabolic state in culture. This assay has been used in our laboratory for toxicity testing of chemicals, alternative new drugs, food contaminants, environmental exposure (Codorean et al., 2009; Meghea et al., 2009; Robu et al., 2009).

Previous laboratory studies have been performed by cell-culture-based in vitro experiments for assessing the cytotoxic and proinflammatory potential of several implantable materials including - silica and titanium nanoparticles on BALB/c 3T3 cells. The results demonstrated the sensitivity of the assay to provide preliminary information about cytotoxicity of the nanoparticulate compounds (Albulescu et al., 2008). MTS proliferation test and LDH leakage are the most frequently used assays for expressing preliminary biological reactivity of nanoparticles in vitro. The MTS test which quantified cell viability through NADH activity measured in presence of tetrazolium salt were correlated with number of live cells and with the metabolic activity as documented in the product information sheet.

Factors that affect the metabolic activity of cells may also affect the relationship between cell number and absorbance. Bioassay data comparing [3H]thymidine incorporation to the MTS-based and the original MTT-based assay demonstrate that tetrazolium reagents can substitute [3H]thymidine incorporation (Promega CellTiter96 Non-Radioactive Cell Proliferation Assay, technical bulletin). The results on MTS and LDH in vitro cytotoxicity assays are presented in Figs. 3 and 4, as concentration – effect behaviour induced by test samples on BALB/c 3T3 cell fibroblasts.

MTS assay in dust exposure

0,7

0,75

0,8

0,85

0,9

0,95

1

1,05

control 25μg/mL 50μg/mL 100μg/mL 150μg/mL 200μg/mL

Ab

s 4

90

nm

MTS assay in ash exposure

0,7

0,75

0,8

0,85

0,9

0,95

1

1,05

1,1

1,15


ab

s 4

90

nm

(a) (b)

Fig. 3. The concentration – effect curves of dust and ash samples on BALB/c 3T3 fibroblast proliferation using MTS assay.(a) dust inhibited fibroblast proliferation


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LDH in dust exposure

1,5

1,7

1,9

2,1

2,3

2,5

2,7

2,9

3,1

3,3


OD

49

0n

m

`

LDH in ash exposure

1,5

1,7

1,9

2,1

2,3

2,5

2,7


OD

49

0n

m

(a) (b) Fig.4. LDH release in BALB/c 3T3 cell (a) after dust and (b) ash exposure; different compared to control (p<0,01)

The MTS assay revealed significant cytotoxicity, at lower concentrations compared to the LDH leakage, expressed by a higher difference between control and the lowest (25µg/mL) concentration for MTS compared to LDH.

This fact indicates that toxicity detection is earlier with the MTS assay compared to the LDH leakage, due to different sensitivity for each assay, suggesting that the MTS assay is more sensitive in detecting cytotoxic events compared to the LDH leakage assay. The two cytotoxic assays in vitro on BALB/c 3T3 fibroblasts may express high cytotoxicity of dust and ash samples by their potential to modulate proliferation and LDH release inducing values significantly different compared to control (p<0.01).

The differences among the two cytotoxicity assays indicate early intracellular effects due to exposure to noxious compounds detected by MTS assay before arising any permanent cell membrane damage occurred and the loss of intracellular LDH into the culture medium as indicator of irreversible cell death (Fotakis and Timbrell, 2006). The studies investigating toxicity of particles focused mainly on atmospheric exposure of humans to heterogeneous mixtures of environmentally produced nanoparticles, and examined the respiratory tract and pulmonary toxicity associated in the target organisms. Because of their small size nanoparticles inhaled particle effects are no longer confined to the lung, since particles are suggested to translocate to the blood while lung inflammation invokes systemic responses. Tests are suggested for portal-of-entry toxicity for lungs, skin, and the mucosal membranes, and target organ toxicity for endothelium, blood, spleen, liver, nervous system, heart, and kidney (Borm, 2002; 2006).

These data obtained on fibroblast and monocyte cell line cultures will be able to compare with the ex vivo whole blood investigation developed in our laboratory on the target subjects occupationally exposed in Deva power plant. The study will be focused on the pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α secretion, performed by xMAP Luminex® 200™ technology in whole blood cultures, related to subject health status and epidemiological data. While there is a growing body of information about hazards of nanomaterials, little is known about the risks to workers exposed to them. It is difficult to obtain reliable measurements of the exposure to each component in dust and ash samples from different workplaces, since they may vary profoundly in composition.


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Although toxicologic research is still the highest priority, it is time to actively anticipate the health needs of workers. Moreover there are increasing need for development of criteria for extrapolating toxicological data in biological systems to predict the risk of adverse outcomes in humans (Krewski, 2004; Park, 2005; Orthen, 2006; Thidell and Arnfalk, 2004; Trout and Schulte, 2010).

Future study should attempt to replicate the air monitoring protocol and question its assumptions and value for industrial hygiene assessment; at the same time it is important to document the respiratory exposure and to correlate it with toxicological and epidemiological data in order to find out some modern technical solutions, which diminish as much as reasonably possible the chemical and physical noxious species and establish some measures aimed at reducing the occupational exposure in the power plant sector (Raducanu et al., 2008).

4. Conclusions

• The ash collected in Deva power plant site contains various components, with more than 50% SiO2, and also with 7% Fe2O3, 5%, Al2O3 etc. Of these compounds SiO2 and 2Al2O3 2SiO2 are crystalline.

• The ash particles have diameters between ~ 0.3 µm to several hundred microns. • The carbon powders contain various compounds, such as silica, aluminum oxide,

calcium oxide, magnesium oxide, iron oxide or sulfates. Of these compounds SiO2, kaolin Al2Si2O5(OH)4 and magnesium silicate Mg3Si4O10(OH)2 are crystalline.

• Particle diameters of dusts are mostly between ~ 0.3 µm and ~ 100 µm and a smaller part and between ~ 300 µm ~ 1900 µm.

• The two cytotoxic assays in vitro on BALB/c 3T3 fibroblasts may express high cytotoxicity of dust and ash samples by their potential to modulate cell proliferation and LDH release inducing values significantly different compared to control (p<0,01).

• The MTS assay revealed significant cytotoxicity at lower concentrations compared to the LDH leakage, suggesting that toxicity detection with the MTS assay is earlier compared to the LDH leakage assay.

• The high sensitivity of the in vitro test tool – MTS, LDH - makes it useful for early hazard evaluation of the power plants working environment.

• The implementation of a risk management program in workplaces where exposure to coal dust or flue ashes, including nanosized materials, may help to minimize the potential health risk for exposure to such materials.

Acknowledgement The paper was supported by the Romanian National Authority for Scientific Research, Project PNII 22-090/2008.

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CHARACTERIZATION OF ULTRAFINE PARTICLES GENERATED AT “DEVA-MINTIA” POWER PLANT

Alice Raducanu2, Angela Stanca1, George Darie2, Ileana Rau2, Constantin Grigoriu3, Cristian Viespe3, Traian Vasiu4

1 Institute for Energy Research and Development, No.8 Bld., Bucharest 3, Postal Code 032092, Romania

2 Politehnica University from Bucharest, 1 Polizu Street, 011061, Bucharest, Romania 3National Institute for Laser, Plasma and Radiation Physics, 409 Atomistilor Street, 077125,

Bucharest-Magurele, Ilfov County, Romania 4SC Electrocentrale DEVA SA, 1 Santierului Street, Mintia, Hunedoara County, Romania

Abstract Ensure population health, especially the workers exposed to occupational hazards, is an important objective in quality of life. Achieving this goal involves mainly the monitoring of exposure to occupational hazards. A number of workplaces where the employees work under noxes environment exist within the thermal power plants. The infliction of the working environment has more or less detrimental effect upon the personnel exposed depending on the existing noxes toxicity, the concentration, exposure duration, and constitution of the subjects exposed. Evaluation of occupational exposure to dust in a representative is required to obtain information on concentration and properties of existing dust in the air of a given job, in a period of time, in order to take appropriate measures to reduce the risk of illness workers. Exposure to airborne ultrafine and nanoparticles has raised increased interest over the recent years as they may cause adverse health effects. A common way to quantify exposure to airborne particles is to measure particle number size distributions through electrical mobility analysis. The size distribution of nanoparticles with diameter less than 100 nm was made with a system consisting of a Long Differential Mobility Analyzer (LDMA) and a Faraday Cup Electrometer (FCE). The different between the numbers of nanoparticles per cm3 measurement in different place from the Deva power plant differ by one order of magnitude. De completat D. Raducan Introduction Ensure population health, especially the workers exposed to occupational hazards, is an important objective in quality of life. Achieving this goal involves mainly the monitoring of exposure to occupational hazards A number of workplaces where the employees work under noxes environment exist within the thermal power plants. The infliction of the working environment has more or less detrimental effect upon the personnel exposed depending on the existing noxes toxicity, the concentration, exposure duration, and constitution of the subjects exposed. At the identification of the noxes there will be taken into account the physical-chemical properties, such as; the state of aggregation (vapours, gas or suspended matters, aerosols or dust), boiling point, vapour pressure, olubility because the cognition of these parameters determine the working methods to be used.

2 Author to whom all corespondence should be addressed: e-mail: [email protected]; tel: +40212700469; fax :+40213035903


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In case several noxes are simultaneously present in the same workplace, after the identification of such noxes, a classification and hierarchy of them will be made while taking into account their cumulated and synergetic action upon the human organism, establishing thus the level of risk it has on the personnel. The choice of pollutants to be investigated will take into account the existence of one (single exhaust) or more hazards in the workplace investigated. Evaluation of occupational exposure to dust in a representative is required to obtain information on concentration and properties of existing dust in the air of a given job, in a period of time, in order to take appropriate measures to reduce the risk of illness workers. Airborne particulate matter can be found in the atmosphere in the form of dust, smoke, fumes, pollen,and other aerosols. Major sources of particulate in the urban and working environments include combustion, materials processing,manufacturing,energy generation, vehicle engine emissions, and construction. Particles play in general a dominant role in many industrial processes and natural phenomena. In 2005, nanoparticles and ultrafine particles" were already appraised to be the mostimportant "emerging risk" for occupational safety in an EU expert survey. Particulate matter is responsible for reduced visibility, the spread of contamination, inhalation of toxic substances and low worker productivity. It is also recognised as a contributory factor to many medical conditions including asthma, bronchitis and lung cancer. Dust and fumes was by far the most common serious health hazard in mining and quarrying, where 86% of the respondents considered it as a problem of some degree - minor or serious - and 56% as a serious problem. Evaluation of occupational exposure to dust in a representative is required to obtain information on existing strength in air and dust properties of a given job, in a period of time, in order to take appropriate measures to reduce the risk of illness workers. Occupational exposure assessment is difficult given the variability of dust-generating processes and types of dust resulting from these processes. Consequently, the variability of exposure is very high. In this complex context, the strategy of air particulate sampling job is responsible for the representativeness of results. Therefore, the location, time and duration of sample taken and sampling equipment are critical elements to ensure quality assessment. The respiratory tract system is the main route for dust entering the human body, followed by ingestion. Dust deposition in the pulmonary system varies considerably according to the granulometry of ultrafine dusts and their airborne behaviour. Studies show that ultra fine particles are more toxic than larger particles with the same chemical composition and the same mass concentration [1-4]. This is beacuse smaller particles have a much larger total surface area than larger particles of the same mass. This nanoparticles can penetrate deep into the respiratory tract and can translocate from the lung into the circulation [5-7], raising the posibility that nanoparticles may facilitate not only lung inflammation but also hemostatic disturbance in the circulation. In prezenta lucare ne-am propus investigarea pulberilor in suspensie, in special al nanoparticulelor in diverse locuri la compania S. C. Electrocentrale Deva. The work in this investigation we proposed matter, especially of nanoparticles in various places in SC Electrocentrale Deva Experimental The methodology for carrying out the powder measurements is based on the norms and standards in the field. The measured values have been compared with the maximum values allowed by GD 355/11.04.2007. Measurements were made with the Casella aparatus - PRO Microdust Aerosol Monitoring System, S / N 0381545.


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The MicroDust pro is an ideal survey instrument for the assessment of real-time particulate concentration in mg/m. It is totally portable and suitable for both fixed site and general survey applications. A user selectable analogue output and alarm are provided that can be connected to external logging devices. Accurate and repeatable dust concentration measurement is achieved using proven forward light scattering techniques. The MicroDust pro offers graphical presentation of concentration trends, internal data logging as standard, simple and clear user interface and digital calibration methods to suit any dust sampling scenario.The WinDust pro supplied with this instrument has been developed to make the downloading and presentation of data as simple as possible. In addition, it offers a PC real time display of particle concentration with a scrolling graph.The MicroDust pro is able to exhibit the highest degree of sensitivity for particle sizes within the respirable domain, while the detachable probe allows readings to be taken in relatively inaccessible areas. All instruments are calibrated at the factory using reference test dust (ISO Fine 12103-1 A2, equivalent to Arizona Road Dust). These facilities allow the calibration concentration to be determined using traceable gravimetric analytical techniques. An optical calibration element is supplied as a reference to confirm the factory calibration point. For optimum calibration against local dust conditions, a sampling pump and optional gravimetric or respirable adapter unit may be used to provide comparative analysis between gravimetric and real time data. The high definition liquid crystal display is capable of showing both text and graphical information. It shows instrument configuration details, contaminant levels, logger information and battery condition. The differentrial mobility analysis (DMA) is commonly used especially for measuring of ultrafine particles [8-10]. The sample air passes a radioactive charger to charge the particles to a defined charge distribution. Then the particles are separated according to their mobility in an electrical field. Only particles with an appropriate charge and size, travel to the sample air outlet, entering a Faraday Cup Electrometer (FCE) as a mono-disperse aerosol. According to the used DMA type, particles are classified in the selected channels by scanning/stepping. This classifier cut out mono-disperse particle size fractions of a poly-disperse aerosol sample. One of the simplest methods to determinate particle concentration at different dimension is by the FCE. It consists of an electrometer and a filter inside a Faraday cage. Charged particles collected by the filter generate an electric current which is measured by the electrometer. The sample air flow is drawn through a particle filter, which is placed in a metal cage which is isolated to the housing of the instrument. The attached particles are transporting also electrical charges to the filter. The amount of the electrical charges in a certain time unit is measured as electric current. If the sample airflow and the charging probability are known, the particle concentration can be calculated. Our interest was to evaluate the size distribution and concentration of the nanoparticles (with diameter less than 100 nm) existing in the aerosols in different work places of power plant. To ease the presentation of the size distribution, it is common to normalize the detected number of particles particularly not with respect to the width of the interval, but rather with respect to the width of the logarithm of the size intervals. That is why the size distribution and concentration is presented as dN/ln(dp)/cm3 (where N is number of particles, dp particle diameter). We used a DMA system from GRIMM AEROSOL consisting of a Long Differential Mobility Analyzer (LDMA) model 5.705 and a Faraday Cup Electrometer (FCE) model 5.706. It is able to measure in the range 4 nm to 1000nm. Results and Discussion The work places that have been investigated from the physical and physical - chemical points of view (coal dust - powder) are located both indoors in closed spaces and outdoors, in a configuration specific to each operation section and to the specific technological process carried out there.


49

The investigation of the work places for physical and chemical noxes (dust) are located both inside and outside enclosed in a specific configuration of each operating sections and developed specific technological process. Place during the technological process produces a large amount of dust in work areas of personnel serving equipment. Dust concentration is variable, depending on the technological process. The coal dust and fly ashes were colected from the foloowing locations: � machinist machine preparation � breakdown room – rock breaker dresser � 1313A/1616A straps – supervision operator � Operating room fuel � 1616A straps – strap operator � 1717A straps – bunker operator � fuel oil station operator � slush station operator � compressor operator � bunker no.13 of 1717A straps The measurements of the powders have been carried out for each work place in the points where the operating personnel has to carry out different operations or different other activities specific to the technological process. Dust present below values determined for each job mentioned above: Item.

Sampling place and time Noxes Concentration (mg/m³ air)

Acceptable concentration (mg/m³

air) H.G.

355/11.04.2007

Work method

1. machinist machine preparation powders 0,634 10 According PO-SME-12

2. breakdown room – rock breaker dresser

powders 26,6 10 According PO-SME-12

3. 1313A/1616A straps – supervision operator

Powders 19,42 10 According PO-SME-12

4. Operating room fuel Powders 0,660 10 According PO-SME-12

5. 1616A straps – strap operator Powders 18,45 10 According PO-SME-12

6. 1717A straps – bunker operator Powders 14,86 10 According PO-SME-12

7. fuel oil station operator Powders 0,609 10 According PO-SME-12

8. slush station operator Powders 0,572 10 According PO-SME-12

9. compressor operator Powders 0,634 10 According PO-SME-12

10.

bunker no.13 of 1717A straps Powders 10,82 10 According PO-SME-12


50

During the performed technological process a great amount of dust is generated in the work places of the personnel that operate the equipment. The concentration of the dust varies with the technological process. In all the situations great amounts of dust are generated whose concentration surpasses the allowable concentration established by the standards in force. The installations and equipment that do not generate dust in amounts surpassing the permissible limits function normally. The value in red signify the sampling points exceeding the acceptable concentration. There are many concerns regarding the possible impacts on human health and the environment that can arise when the smaller constituencies of materials are brought down to the nanoscale. Although these impacts may not be any different from those that can be caused by chemicals, it is not possible today to anticipate the impacts based solely on the chemical composition as such. The measurement was made in 7 different places, where located in distinct places where the job of the employers is for supervise or manipulate the equipment. The concentration of particle at different size distribution is presented in figure 1.

cabin of machinist from unloading/loading platform

coal breaker

bunker operator

belt conveyer 1717A


51

Fig. 1 The nanoparticles size distribution measured and concentration at diferent

places at Deva power plant. From DMA diagrames one cand notice that in general concentration of the coal nanoparticles is higher in the range 8-30 nm, exhibiting with different peaks depending on the investigated workplace. Also, is notable the fact that the highest values of the concentration is for size between 8 and18 nm. From concentration point of view values of order x 1000 nanoparticles/cm3 was observed: cabin of machinist from unloading/loading platform, belt conveyer, or around the electrostatic precipitators. The electrostatic precipitator environment does not exhibit a uncommon concentration or size distribution, different from other places where are coal based aerosols. The highet concentration was measured in two places: in the place where the coal is fragmented into small pieces: mill hall and coal breaker, where 120,000 and 95,000 nanoparticles per cm3. For reducing the harmful environmental effects on the operator, the protection against nanoparticles inhalation is compulsory. The highest concentration of nanoparticles with dimension of 100 nm is in mill hall and in breakdown room. Also the nanoparticles with dimension less than 15 nm are the highest concentration in all the places where was made measurement. Bibliografie 1. G. Oberdorster, Pulmonary effects of inhaled ultrafine partilcles, Int. Arch. Occ. Env. Hea. 2001, 74 (1), 1-8

mill hall operator of the belt conveyer 1717A

electrofilter area


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2. D. M. Brown, V. Stone, P. Findlay, W. Macnee, K. Donaldson, Increased inflamation and intracelular calcium caused by ultrafine carbon black is independent of transition metals or other soluble components, Occup. Enviroment, Medicine 57, 685-691,2000. 3. K. Donaldson, X.Y. Li, W. Macnee, Ultrafine (nanometre) particle mediated lung injury, J. Aerosol Sci. 29, 553-560, 1998 4. K. Donaldson, V. Stone, A. Clouter, L. Renwick, W. Macnee, Ultrafine particles. Occup environ. medicine 587, 553-560, 2001 5. A. Nemmar, P.H. Hoet, B. Vanqiuckenborne, D. Dinsdala, M. Thomeer, M.F. Hoylaerts, Passage of inhaled particles into the blood circulation in humans, Circulation, 105 (4): 411-414, 2002 6. A. Nemmar, M.F. Hoylaerts, P.H. Hoet, D. Dinsdale, T. Smith, H. Xu, Ultrafine particles affect experimental thrombosis in an in vivo hamster model, Am. J. respir. Crit. care. Med. 166 (7):998-1004, 2002 7. A. Nemmar, M.F. Hoylaerts, H.P. Hoet, J. Vermylen, B. Nemery, Size effect of intratracheally instilled particles on pulmonary inflammation and vasular thrombosis, Toxical. Apll. Pharmacol, 186(1):38-45, 2003 8. S. Hasegawa,M. Hirabayashi, S. Koboyashi, Y. Moriguchi, Y. Kondo, K. Tanabe, S. Wakamatsu, Size Distribution and Characterization of Ultrafine Particles in Roadside Atmosphere, J. of Environmental Sceince and Health, part. A, Vol. A39, no 10, pp 2671-2690, 2004. 9. C. Asbach, H. Kaminski, H. Fissan, C. Monz, D. Dahmann, S. Mulhopt, H. R. Paur, H. J. Kiesling, F. Herrman, M. Voetz, T. A. J. Kuhlbusch, Comparision of four mobility particle sizer with different time resolution for stationary exposure measurements, J. Napoparts Res (2009) 10. B. Davison, D. Whyatt, C. Boardman, Aerosol eolution from a busy road in North-west Engleand, Metoorologische Zeitschrift, vol. 18, no. 1, 055-060, 2009 11. Raducanu A., Suvergel A., Stanca A., Meghea A., (2008), The evaluation of performances of installations by power plants from Romania concerning professional risks, WSEAS Transactions on Environment and Development, 4, 210-223. 12. Robu M., Tănase C., Boscornea C., Tomas S., Albulescu R., (2009), Curcumin derivatives with potential biological activity, Revista de Chimie, 60, 76-80. 13. Sakai A., (2008), BALB/c 3T3 cell transformation assays for the assessment of chemical carcinogenicity, Alternatives to Animal Testing and Experimentation Academic Journal, 14 , 367-373. 14. Günter Oberdörster, Eva Oberdörster, and Jan Oberdörster Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles Environ Health Perspect. 2005 July; 113(7): 823–83915. G. Oberdörster, J. Ferin, S. Soderholm, P. E. Morrow Increased Pulmonary Toxicity of Inhaled Ultrafine Particles: Due to Lung Overload Alone? The Annals of Occupational Hygiene Volume38, Issueinhaled particles VII Pp. 295-302. 16. K. Donaldson, D. Brown, Anna Clouter, R. Duffin, W. MacNee, V. Stone. The Pulmonary Toxicology of Ultrafine Particles Journal of Aerosol Medicine. June 2002, 15(2): 213-220. 17. Thomas et al. (2006) Research strategies for safety evaluation of nanomaterials. part VII. Evaluating consumer exposure to nanoscale materials, Toxicological Sciences, 91, 14-19 18. Oberdorster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Kreyling, W., et al. (2004b). Translocation of inhaled ultrafine particles to the brain. Inhalation Toxicology, 16(6-7), 437-445. 19. Kreuter, J. (2001). Nanoparticulate systems for brain delivery of drugs. Advanced Drug Delivery Reviews, 47(1), 65-81. 20. Schins et al. (2002b) Oxidant-induced DNA damage by quartz in alveolar epithelial cells Mut Res, 517: 77-86 21. Powers et al. (2006) Research strategies for safety evaluation of nanomaterials. part IV. characterisation of nanoscale particles for toxicological

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