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Laboratory Tests of Toxicity with Enchytraeids

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Laboratory Tests of Toxicity with Enchytraeids MASARYK UNIVERSITY FACULTYOFSCIENCE RECETOX ResearchCentreforEnvironmental ChemistryandEcotoxicology Laboratory tests of toxicity with enchytraeids RIGOROUS THESIS Brno,2007MSc.KláraKobetičová 1 Acknowledgements: Mythanks belongstodr.JakubHofmanfor manyconsultationsatfieldofsoilecotoxicology; toMgr.Blanka Holubářová forimplementationofenchytraeidbreedings andtoxicitytest; todr.JitkaBezchlebová,dr.IvanaSochová,Mgr.JanLánafortesting thechemicalsontheearthworms,nematodesandspringtails; todr.JitkaBezchlebováfor practicaladviceinlabofsoilecotoxicology andpreparationoftwosubmittedarticles; tootherstudents andcoleaguesforfriendlyatmosphereat theworkplace ofRECETOX; tomyparents andfriendsfortheirloveandsupport. 2 Tableofcontents Abstract…………………………………………………….….……..4 Abstract (in Czech)…………………………………………………..5 List of original papers……………………………………………….6 International meetings………………………………………..……..6 Abbreviations………………………………………………….……..7 The main goals of the rigorous thesis………………………………8 1. Theoretical part of the rigorous thesis……………………….9 1.1. Enchytraeids……………………………………………………………10 1.1.1.Biologyofenchytraeids……………………………………………………………...10 1.1.2.Functionofenchytraeidsinsoils……………………………………………………12 1.1.3.Distributionof enchytraeidsinsoils………………………………………………..12 1.2. Laboratory tests on enchytraeids……………………………………...13 1.2.1. Watertest…………………………………………………………………………….14 1.2.2. Agartest………………………………………………………………………...……14 1.2.3. Sublethaltestwith Cognettia sphagnetorum (Vejdovský)1877 ……………….…...16 1.2.4. Enchytraeidreproductiontest (ERT) ………………………………………….…. .16 1.2.5. Avoidancetest………………………………………………………………………..18 1.2.6. Bioaccumulationtest…………………………………………………………….…..20 1.3. References………………………………………………………...……. .22 2. Experimental part of the rigorous thesis……………………27 2.1. Toxicity of selected organic pollutants: short-chain chlorinated paraffins; toxaphene; NPAHs ( acridine,phenazine,1,10phenanthroline, quinoline) to Enchytraeus albidus /Enchytraeus crypticus ……………...28 2.1.1.Abstract..……………………………………………………………………………..28 2.1.2. Introduction………………………………………………………………………….28 2.1.3. Materialsandmethods……………………………………………………………...32 2.1.4. Resultsanddiscussion………………………………………………………………33 2.1.5. References…………………………………………………………………………...41 3. Conclusions…………………………………………………...45 4. Appendices……………………………………………….…...47 3 Abstract Enchytraeids (Oligochaeta,Annelida) belongtoanimportant group of soil decomposers that have beenusinginthe soil ecotoxicologyfor more thanfiftyyears.Several short tolong term laboratorytests withvarious test medium (water,agar, artificial or fieldsoil) have been realizedduringthis time.However,the tests withartificial medium are not verysuitable for the assessment of chemicals insoil or for soil qualityassessment.Therefore, the chronic tests have been developed for enchytraeids in soil (SECOFASE, 1994 and ASTM, 2000/ ISO, 2002/OECD,2003). A few of enchytraeidspecies (genus Enchytraeus ,species Cognettia sphagnetorum ) are commonlyusedin the soil tests.The durationof the test ranges from 4to10weeks depending onthe usedspecies. The measuredendpoints are mostlyreproduction(number of juveniles, cocoons, fragments) and adult mortality. The research have been focusing on toxicity of organic compounds, heavy metals, wastes or soil quality assessment since the time. In addition,the studies have more oftenprogressedfrom pure measurement the toxic effects of individual pollutants (expressed as LCx, ECx values) into the more comprehensive investigationof bioavailabilityaffectingthe toxicity. Some of the methods as Avoidance test or Bioaccumulationtest have beenjust under a development. The first of them usually lasts 2 days and is based on the presumption that enchytraeids are able to avoidor escape the unsuitable soils (under some environmental or antropogenic stress factors).The bioacumulationtest has concernedwiththe kinetic patternof the chemical uptake andelimination,the analyse of the sublethal concentrationinsite of toxic actionorinthewholeorganism. The toxiciyof selectedorganic pollutants (toxaphene,short-chain chlorinated paraffins, N-polycyclic aromatic hydrocarbons: quinoline, acridine, phenazine, 1,10phenanthroline) to enchytraeidspecies E. crypticus , E. albidus (or tobothspecies) is presentedinthe following chapture. The chronic soil test was used for testing of all organic pollutants and artificial (OECD) soil as a test substrate.The test withshort-chainchlorinatedparaffins affordedthese results: NOEC mortality ( E.albidus ) = 10,000 mg/kg, NOEC mortality ( E.crypticus ) = 6,000 mg/kg, LC50 mortality ( E.albidus, E.crypticus ) couldnot be estimated,NOEC reproduction ( E.albidus ) = 3,000mg/kg , NOEC reproduction ( E. crypticus ) = 6,000mg/kg , EC50 reproduction (E.albidus) = 6,027mg/kg,EC50 reproduction (E.crypticus) = 7,809mg/kg. Toxaphene didnot show anytoxic effect for E. albidus because the highest tested concentration 620 mg/kg corresponded to NOEC value for mortality and reproduction.The N-polycyclic aromatic hydrocarbons showedrelativelythe same order of magnitude for the toxic effects.Nevertheless,the toxicity(nominal concentrationinmg/kg) decreasedin this order for the bothmeasuredendpoints: 1,10phenanthroline > quinoline > phenazine ≥ acridine.After the data recalculationoneffect concentrationinsoil pore water (µmol/l),thereversedrankoftoxicitywasobtained.Thesefindingsare inaccordancewiththe higher toxicity of more lipophilic compounds in aquatic environment described in some literature. Key–words : enchytraeids, Enchytraeus albidus , Enchytraeus crypticus , tests of toxicity,toxic effects,short-chainchlorinatedparaffins,toxaphene,NPAHs,acridine,quinoline,phenazine, 1,10phenanthroline. 4 Abstrakt (Česky) Roupice (máloštětinatci, kroužkovci) jsou důležitouskupinoupůdníchdekompozitorů, která je vekotoxikologii půdy využívána již více než 50 let. Během tohoto období bylo realizováno několikkrátkodobýchaž déletrvajícíchlaboratorníchtestůsrůznými testovacími médii (voda, agar, umělá nebo přírodní půda). Avšak testy sumělým médiem nejsou příliš vhodné proodhadnegativníchvlivů vyplývajícíchz přítomnosti chemikálií vpůdě nebopro posouzení půdní kvality. Ztěchto důvodů byl pro roupice vpůdě vyvinut chronický test (SECOFASE,1994;ASTM,2000/ISO,2002/OECD,2003). V půdních testech se běžně používá několik druhů roupic (rod Enchytraeus , druh Cognettia sphagnetorum ). Testy trvají většinou 410 týdnů vzávislosti na použitém druhu. Sledovanými parametry bývají nejčastěji reprodukce (počet mláďat, kokonů, fragmentů) a mortalita dospělců.Test s roupicemi je takodtédobyvyužívánve studiíchzaměřujícíchse na výzkum toxicityorganickýchlátek,kovů,aplikace odpadůneboktestování biologické kvality přírodních půd. Navíc, studie stále častěji přechází od pouhého zjišťování toxicity jednotlivých látek (vyjadřované hodnotami LCx, ECx) ke komplexnějšímu studiu biodostupnostia tímisamotnétoxicity. Některé z testůjako„avoidance“ test nebobioakumulační test jsoustále ještě vrozvoji. První znichtrvá obvykle 2dnya je založenna předpokladu,že roupice jsouschopné vyhýbat se nevhodné půdě nebo zní unikat (pod vlivem environmentálních anebo antropogenních stresových faktorů). Bioakumulační test je zaměřen na kinetiku příjmu a eliminaci látky, stanovením neletální koncentrace látky vcílovém místě nebo její celkovou koncentrací vtestovanémorganismu. Následující kapitola se věnuje toxicitě vybraných organických polutantů (toxafen, chlorované parafíny skrátkým C-řetězcem, dusíkaté-heterocyklické aromatické uhlovodíky: chinolin,akridin, fenazin,1,10fenantrolin) propůdní roupice druhu E. crypticus , E. albidus (nebo pro oba druhy). Kotestování látek byl použit chronický půdní test a jako testovací substrát artificiální (OECD) půda. Test schlorovanými parafíny poskytl tyto výsledky: NOEC mortalita ( E.albidus ) = 10,000 mg/kg, NOEC mortalita ( E.crypticus ) = 6,000 mg/kg, LC50 mortalita (E.albidus, E.crypticus ) nemohl být vypočítán,NOEC reprodukce ( E.albidus ) = 3,000mg/kg , NOEC reprodukce ( E. crypticus ) = 6,000 mg/kg , EC50 reprodukce ( E.albidus ) = 6,027 mg/kg, EC50 reprodukce ( E.crypticus ) = 7,809 mg/kg. Toxafen neměl negativní vliv na roupici E. albidus ani na jeden ze sledovanýchparametrů,protože hodnotě NOEC odpovídala až nejvyšší testovaná koncentrace 620 mg/kg. Dusíkaté polycyklické aromatické uhlovodíky vpřípadě druhu E .crypticus ukázaly řádově podobný efekt. Jejich toxicita (nominální koncentrace v mg/kg) se přesto snižovala vtomtopořadí: 1,10fenanthrolín> chinolín> fenazin≥ akridin.Popřepočtutěchto dat na efektivní koncentraci vpůdní pórové vodě (µmol/l) se toxicita látekzcela obrátila.To odpovídá závěrům o vyšší toxicitě lipofilnějších látek ve vodním prostředí, jak je uváděno vliteratuře. Klíčová slova: roupice, Enchytraeus albidus , Enchytraeus crypticus , testy toxicity, toxický efekt, chlorované parafíny skrátkým řetězcem, toxafen, NPAHs, akridin, chinolin, fenazin, 1,10fenantrolin. 5 List of original papers • Bezchlebová,Jitka,Černohlávková,Jitka ,Kobetičová,Klára, Lána,Jan,Sochová,Ivana, Hofman, Jakub. Effects of short-chain chlorinated paraffins on soil organisms. EcotoxicologyandEnvironmentalSafety,67(2):206211.June2007(AppendixI.). • Bezchlebová,Jitka,Černohlávková, Jitka, Lána, Jan,Sochová , Ivana, Kobetičová,Klára , Hofman, Jakub . Effects of toxaphene on soil organisms . Ecotoxicology and EnvironmentalSafety,Acceptedashighlightedarticle(AppendixII.). • Kobetičová, Klára ,Bezchlebová,Jitka,Sochová,Ivana,Lána,JanandHofman, Jakub. Toxicityof four polycyclic aromatic nitrogen heterocycles (PAHs) to soil organisms. EcotoxicologyandEnvironmentalSafety,submitted(AppendixIII.).
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