Reka SAVA DOLINKA in BELCA ČN Tabre in MČN Belca (Stalni Letni Monitoring Od Leta 2001)

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Reka SAVA DOLINKA in BELCA ČN Tabre in MČN Belca (Stalni Letni Monitoring Od Leta 2001) LABORATORY OF PLANT CYTOGENETICS Use of Allium M test for purpose of establishing quality of waters; since 1998 LABORATORIJ ZA RASTLINSKO APLIKATIVNO CITOGENETIKO Testna rastlina: navadna čebula (Allium cepa L.) Splošna strupenost (dolžina korenin) in genotoksičnost (indukcija kromosomskih poškodb) Reka SAVA DOLINKA in BELCA ČN Tabre in MČN Belca (stalni letni monitoring od leta 2001) Naročnik: Občina Kranjska Gora Kolodvorska 1b, SI – 4280 Kranjska Gora April 2015 Testno poročilo z rezultati raziskav obsega 9 strani, 2 preglednici in 4 fotografije 1 LABORATORY OF PLANT CYTOGENETICS Use of Allium M test for purpose of establishing quality of waters; since 1998 PROTOKOL METODE ALLIUM METAFAZNI GENOTOKSIČNI TEST ZA TESTIRANJE OKOLJSKIH VZORCEV, KEMIKALIJ IN PITNE VODE Peter Firbas, univ. dipl. biol., Zasebni raziskovalec, Laboratorij za rastlinsko citogenetiko E-pošta: [email protected] 1. Uvod ALLIUM metafazni test je test za ugotavljanje splošne celične strupenosti (citotoksičnosti) in ravni genotoksičnosti v vodnih, kopenskih in zračnih ekosistemih, kjer dokazujemo potencialne genotoksične snovi. Test je kratkotrajen in pokaže usklajen in celokupen učinek onesnaževanja in medsebojno delovanje med testno rastlino (Allium cepa L.) in potencialnimi genotoksiki, nato se še rezultati statistično ovrednotijo. Rezultate raziskav s statistično kalkulacijo prikazuje Fisher’s Exact Test. V 2x2 frekvenčnih tabelah dvosmerna p-vrednost determinira statistično značilnost ali neznačilnost dveh kategoričnih vzorcev. Biološki test ALLIUM ali čebulni test razkriva celosten vpliv na rast in razvoj živih celic ali organizmov ter zaznava prisotnost škodljivih snovi v koncentracijah, ki so bistveno nižje od 1 ppb (part per billion – en del snovi na eno milijardo raztopine ali 1 µg na liter ali kilogram). Od približno 700 prepoznavnih snovi, ki se lahko znajdejo na primer v pitni vodi, jih z običajnimi fizikalno-kemijskimi analizami nadzorujemo le okrog 10 odstotkov (Vir: EU Chemical Bureau, Natural Resources Defence Council: Think before you drink). »Višje« rastline so odlični genski modeli za odkrivanje citotoksičnih in/ali mutagenih v okolju; in se pogosto uporabljajo za biomonitoring (oz. spremljanje kakovosti okolja, predvsem vodnih vzorcev) v različnih ekosistemih (Leme in Marin-Morales, 2009). Med rastlinami ima vodilno mesto navadna čebula (Allium cepa L.), ki je indikatorska testna rastlina za oceno motenih procesov v celični delitvi (mitozi), kar na makroskopski raveni zavira rast korenin; na mikroskopski raveni pa povzroča številčne in strukture poškodbe kromosomov (Firbas 2011; Firbas in Amon, 2013, 2014). Meristemske celice koreninskih vršičkov vsebujejo reduktazne encime (MFO – mixed function oxidase), ki pretvorijo promutagene snovi v mutagene, kar tudi zasledimo v živalskih organizmih in tako omogočajo spremljanje pretvorb genotoksičnih snovi in njihovih metabolitov v drugimi bioloških testnih sistemih. Mednarodni program rastlinskih bioloških testov (IPPB – International Program on Plant Bioassays) je čebulni (Allium) test sprejel za biomonitoring in testiranje okoljskih vzorcev (z morebitnimi onesnaževalci). 2. Material in metode Test se izvaja po: Al-Sabti 1989, Firbas 2004, 2011; Kumar in Panneerselvam (2007); Ragunathan in Panneerselvam (2007); Panneerselvam in sod. (2012). Citogenetske raziskave potekajo na raziskovalnem mikroskopu znamke OLYMPUS – BX 41 Japonska) s samodejnim foto sistemom PM 10 SP, pri povečavi 400X in 1000X na Leica formatu 24 mm X 36 mm). Parametri ALLIUM metafaznega testa podajajo: splošno strupenost in raven genotoksičnosti SPLOŠNA STRUPENOST (Slika 1, 2) podaja dolžino korenin testnih rastlin mlade čebule (Allium cepa L.); je obratno sorazmerni z dolžino korenin testnih rastlin. Daljše kot so korenine manjša je splošna toksičnost in krajše kot so korenine testnih rastlin večja je splošna strupenost (toksičnost). 2 ZASEBNI LABORATORIJ ZA RASTLINSKO CITOGENETIKO Uporaba Allium metode za določanje kakovosti okolja in ocene tveganja Slika 1 – Inhibicijsko stimulacijski test mlade Slika 2 – Inhibicijsko stimulacijski test mlade čebule A. cepa L. gojen 72 ur v 1 ppm 1 (mg/l) oz. čebule A. cepa L. gojen 72 ur v 1 ppb (1µg/l) oz. 1 ppm (part per milion) MMS 1ppb (part per bilion) MMS. RAVEN GENOTOKSIČNOSTI (Slika 3 in 4) podaja poškodbe kromosomov v celicah koreninskih vršičkov testnih rastlin mlade čebule (Allium cepa L.); je odstotkovno razmerje med vsemi metafaznimi celicami in s celicami s poškodbami kromosomov (Al-Sabti 1985, 1989; Firbas 2004; 2006). Identificira se 200 ali več metafaznih celic. Pri visoki ravni genotoksičnosti pa manj. Rezultati se podajajo v odstotnih točkah (Odst. t.). Celica se šteje za poškodovano, če je v kromosomski garnituri poškodovan vsaj en kromosom. STATISTIČNA KALKULACIJA: Fisher’s Exact Test. V 2x2 frekvenčnih tabelah dvosmerna p-vrednost determinira statistično značilnost ali neznačilnost dveh kategoričnih vzorcev. 2. 1. Kontrola ALLIUM metafaznega testa in inhibicija rasti korenin Ugotovitev, da test ALLIUM sploh deluje, potekata vzporedno ob raziskanem vzorcu še dva testa, ki nam zagotavljata: kakšna je sama odzivnost testa ALLIUM in to, ali test ALLIUM sploh deluje (Slika 1 in 2). Negativna kontrola pokaže, kolikšna je stopnja strupenosti pri ne izpostavljenih čebulah in hkrati kontrola, da test sploh deluje. Pozitivna kontrola pa se uporablja z znano kemično snovjo, ki v večji meri povzroča stopnjo strupenosti in je potrebna za kontrolo odzivnosti testa. Za pozitivno kontrolo se je uporabljala koncentracija 10 mg/L ali 10 ppm metil metansulfonat 66- 27-3 200-625-5 – MMS 4016 SIGMA. Za negativno kontrolo pa se je uporabila 10.000 krat nižja koncentracija (1 µg/l) iste snovi. 3 ZASEBNI LABORATORIJ ZA RASTLINSKO CITOGENETIKO Uporaba Allium metode za določanje kakovosti okolja in ocene tveganja Slika 3. Nepoškodovani metafazni kromosomi navadne čebule (Allium cepa L.) v koreninskih celicah. Celica vsebuje 16 kromosomov (2n = 16). Kromosom tvorita dve vzdolžni kromatidi (kt) ali Slika 4. Poškodovani kromosomi v metafaznih vzdolžni polovici. Centromera (cm) razmeji celicah koreninskih vršičkov mlade čebule kromosom v krajši (p) krak in daljši (q) krak. (Allium cepa L.). Kraka kromosoma sta lahko tudi enaka. 3. REZULTATI RAZISKAV, ZAKLJUČEK IN RAZPRAVA Rezultati splošne strupenosti in ravni genotoksičnosti so podani v Preglednici 1 in 2. Vzorčenje: 26. marec 2015 Z metodo Allium so bili raziskani naslednji vzorci: I. ZELENCI – izvir Save dolinke II. Iztok komunalno-meteornih vod v reko Pišnico III. SAVA DOLINKA 50 m po sotočju Pišnice IV. Čiščena komunalno-urbana voda (iztočna voda ČN Tabre) V. Čiščena komunalno-urbana voda (iztočna voda MČN* Belca) VI. BELCA (50 m za iztokom MČN Belca) VII. SAVA DOLINKA: 100 m po sotočju Belce in Bistrice v kraju Dovje (Mlinci) *MČN: mala čistilna naprava 3.1. Splošna strupenost (dolžina korenin testnih rastlin in meritve) Rezultati splošne strupenosti so podani v preglednici 1. Raziskani rečni vzorci (Vzorci I, III in VI) za splošno strupenost (dolžina korenin testne rastline) ne kažejo statistično značilne razlike (p < 0,05) po iztoku čiščenih voda iz ČN (Vzorec IV in V). Povečana raven splošne strupenosti je značilna za komunalno-meteorne vode (Vzorec II) z iztokom v reko Pišnico. Vsi vzorci imajo bistveno nižjo stopnjo splošne strupenosti od vzorca pozitivne kontrole (p < 0,01). 4 ZASEBNI LABORATORIJ ZA RASTLINSKO CITOGENETIKO Uporaba Allium metode za določanje kakovosti okolja in ocene tveganja Preglednica 1. Povprečna dolžina korenin testne rastline Allium cepa L. – raziskava splošne strupenosti in citološki učinki raziskanih vzorcev - raziskava ravni genotoksičnosti Razmerje pregledanih Raven Povprečna celic in celic s Genotoksičnos dolžina korenin VZOREC / MERNO MESTO poškodovanimi ti (odst. t.) (mm) kromosomi 39 ٭I- ZELENCI (izvir SD*) 300/10 3,00 II – IZTOK komunal.-meteor. vod v Pišnico 300/28 9,33 33 III – SD 100 m po sotočju s Pišnico 300/14 4,66 38 IV – Iztok čiščene vode iz ČN Tabre 300/23 7,66 37 V – Iztok čiščene vode iz MČN Belca 300/14 4,66 37 38 ٭VI – BELCA; 50 m za iztokom MČN Belca 300/13 4,33 VII – SD; v kraju Dovje (Mlinci) 300/11 3,66 39 VII-neg.kontrola 200/5 2,5 40 ٭VIII-pozit.kontrola 200/42 21,0 25 *SD: reka Sava Dolinka 3.2. Raven genotoksičnosti (poškodbe kromosomov in opazovanja) Rezultati citogenetskih raziskav so podani v preglednici 2. Reka Sava Dolinki po sotočju z reko Pišnico v primerjavi z Zelenci statistično ni značilna, oz kakovost se ne poslabša (p = 0,5328 > 0,05 ), kar tudi velja za Savo Dolinko v kraju Dovje (p = 1). 4. ZAKLJUČEK Reka Sava Bolinka s pritokom reke Pišnice ne kaže poslabšanje kakovosti po iztoku odpadne komunalno-meteorne vode (Čičare; na lokaciji ob golfišču). Delovanje MČN Belca in ČN Tabre je zelo visoko optimizirano, saj se raven genotoksičnosti čiščeni iztočni vodi sprejemnemu vodotoku ne poslabša oz. spremeni. Iz rezultatov študije lahko zaključimo, da ČN Tabre in MČN Belca optimizirano deluje, in zagotavljajo veliko čistilno moč odpadnim komunalno-urbani vodi iz okoliških krajev. To tudi potrjuje kakovost reke Save Dolinke v kraju Dovje, obdrži visoko kakovost površinskih tekočih voda v primerjavi s kakovostjo izvirske vode v Zelencih. 5. PRILOGA Monitoring reke Save Dolinke s testom ALLIUM 5 ZASEBNI LABORATORIJ ZA RASTLINSKO CITOGENETIKO Uporaba Allium metode za določanje kakovosti okolja in ocene tveganja Monitoring za obdobje 2001 do 2013 je bil tudi objavljen v znanst. reviji v sklopu drugih okoljskih vzorcev v Sloveniji: FIRBAS P (2015). A survey of Allium cepa
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