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«RECETOX Research Centre for Environmental Chemistry and Ecotoxicology Laboratory tests of toxicity with enchytraeids RIGOROUS THESIS Brno, 2007 MSc. ...»

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MASARYK UNIVERSITY

FACULTY OF SCIENCE

RECETOX

Research Centre for Environmental

Chemistry and Ecotoxicology

Laboratory tests of toxicity

with enchytraeids

RIGOROUS THESIS

Brno, 2007 MSc. Klára Kobetičová

1

Acknowledgements:

My thanks belongs to dr. Jakub Hofman for many consultations at field of soil ecotoxicology;

to Mgr. Blanka Holubářová for implementation of enchytraeid breedings and toxicity test;

to dr. Jitka Bezchlebová, dr. Ivana Sochová, Mgr. Jan Lána for testing the chemicals on the earthworms, nematodes and springtails;

to dr. Jitka Bezchlebová for practical advice in lab of soil ecotoxicology and preparation of two submitted articles;

to other students and coleagues for friendly atmosphere at the workplace of RECETOX;

to my parents and friends for their love and support.

2 Table of contents 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. Biology of enchytraeids……………………………………………………………...10 1.1.2. Function of enchytraeids in soils……………………………………………………12 1.1.3. Distribution of enchytraeids in soils………………………………………………..12

1.2. Laboratory tests on enchytraeids……………………………………...13 1.2.1. Water test…………………………………………………………………………….14 1.2.2. Agar test………………………………………………………………………...……14 1.2.3. Sublethal test with Cognettia sphagnetorum (Vejdovský)1877……………….…...16 1.2.4. Enchytraeid reproduction test (ERT)………………………………………….…..16 1.2.5. Avoidance test………………………………………………………………………..18 1.2.6. Bioaccumulation test…………………………………………………………….…..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, 1,10-phenanthroline, quinoline) to Enchytraeus albidus/Enchytraeus crypticus……………...28 2.1.1. Abstract..……………………………………………………………………………..28 2.1.2. Introduction………………………………………………………………………….28 2.1.3. Materials and methods……………………………………………………………...32 2.1.4. Results and discussion………………………………………………………………33 2.1.5. References…………………………………………………………………………...41

3. Conclusions…………………………………………………...45

4. Appendices……………………………………………….…...47 3 Abstract Enchytraeids (Oligochaeta, Annelida) belong to an important group of soil decomposers that have been using in the soil ecotoxicology for more than fifty years. Several short to longterm laboratory tests with various test medium (water, agar, artificial or field soil) have been realized during this time. However, the tests with artificial medium are not very suitable for the assessment of chemicals in soil or for soil quality assessment. Therefore, the chronic tests have been developed for enchytraeids in soil (SECOFASE, 1994 and ASTM, 2000/ ISO, 2002/ OECD, 2003).

A few of enchytraeid species (genus Enchytraeus, species Cognettia sphagnetorum) are commonly used in the soil tests. The duration of the test ranges from 4 to 10 weeks depending on the used species. The measured endpoints are mostly reproduction (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 often progressed from pure measurement the toxic effects of individual pollutants (expressed as LCx, ECx values) into the more comprehensive investigation of bioavailability affecting the toxicity.

Some of the methods as Avoidance test or Bioaccumulation test have been just under a development. The first of them usually lasts 2 days and is based on the presumption that enchytraeids are able to avoid or escape the unsuitable soils (under some environmental or antropogenic stress factors). The bioacumulation test has concerned with the kinetic pattern of the chemical uptake and elimination, the analyse of the sublethal concentration in site of toxic action or in the whole organism.





The toxiciy of selected organic pollutants (toxaphene, short-chain chlorinated paraffins, N-polycyclic aromatic hydrocarbons: quinoline, acridine, phenazine, 1,10-phenanthroline) to enchytraeid species E. crypticus, E. albidus (or to both species) is presented in the following chapture. The chronic soil test was used for testing of all organic pollutants and artificial (OECD) soil as a test substrate. The test with short-chain chlorinated paraffins afforded these results: NOECmortality (E.albidus) = 10,000 mg/kg, NOECmortality (E.crypticus) = 6,000 mg/kg, LC50mortality (E.albidus, E.crypticus) could not be estimated, NOECreproduction (E.albidus) = 3,000 mg/kg, NOECreproduction (E. crypticus) = 6,000 mg/kg, EC50reproduction (E.albidus) = 6,027 mg/kg, EC50reproduction (E.crypticus) = 7,809 mg/kg. Toxaphene did not show any toxic 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 showed relatively the same order of magnitude for the toxic effects. Nevertheless, the toxicity (nominal concentration in mg/kg) decreased in this order for the both measured endpoints: 1,10-phenanthroline quinoline phenazine ≥ acridine. After the data recalculation on effect concentration in soil pore water ( mol/l), the reversed rank of toxicity was obtained. These findings are in accordance with the 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-chain chlorinated paraffins, toxaphene, NPAHs, acridine, quinoline, phenazine, 1,10-phenanthroline.

4 Abstrakt (Česky) Roupice (máloštětinatci, kroužkovci) jsou důležitou skupinou půdních dekompozitorů, která je v ekotoxikologii půdy využívána již více než 50 let. Během tohoto období bylo realizováno několik krátkodobých až déletrvajících laboratorních testů s různými testovacími médii (voda, agar, umělá nebo přírodní půda). Avšak testy s umělým médiem nejsou příliš vhodné pro odhad negativních vlivů vyplývajících z přítomnosti chemikálií v půdě nebo pro posouzení půdní kvality. Z těchto důvodů byl pro roupice v půdě 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 4-10 týdnů v závislosti na použitém druhu.

Sledovanými parametry bývají nejčastěji reprodukce (počet mláďat, kokonů, fragmentů) a mortalita dospělců. Test s roupicemi je tak od té doby využíván ve studiích zaměřujících se na výzkum toxicity organických látek, kovů, aplikace odpadů nebo k testování biologické kvality přírodních půd. Navíc, studie stále častěji přechází od pouhého zjišťování toxicity jednotlivých látek (vyjadřované hodnotami LCx, ECx) ke komplexnějšímu studiu biodostupnosti a tím i samotné toxicity.

Některé z testů jako „avoidance“ test nebo bioakumulační test jsou stále ještě v rozvoji.

První z nich trvá obvykle 2 dny a je založen na předpokladu, že roupice jsou schopné vyhýbat se nevhodné půdě nebo z ní unikat (pod vlivem environmentálních anebo antropogenních stresových faktorů). Bioakumulační test je zaměřen na kinetiku příjmu a eliminaci látky, stanovením neletální koncentrace látky v cílovém místě nebo její celkovou koncentrací v testovaném organismu.

Následující kapitola se věnuje toxicitě vybraných organických polutantů (toxafen,

chlorované parafíny s krátkým C-řetězcem, dusíkaté-heterocyklické aromatické uhlovodíky:

chinolin, akridin, fenazin, 1,10-fenantrolin) pro půdní roupice druhu E. crypticus, E. albidus (nebo pro oba druhy). K otestování látek byl použit chronický půdní test a jako testovací

substrát artificiální (OECD) půda. Test s chlorovanými parafíny poskytl tyto výsledky:

NOECmortalita (E.albidus) = 10,000 mg/kg, NOECmortalita (E.crypticus) = 6,000 mg/kg, LC50mortalita (E.albidus, E.crypticus) nemohl být vypočítán, NOECreprodukce (E.albidus) = 3,000 mg/kg, NOECreprodukce (E.

crypticus) = 6,000 mg/kg, EC50reprodukce (E.albidus) = 6,027 mg/kg, EC50reprodukce (E.crypticus) = 7,809 mg/kg. Toxafen neměl negativní vliv na roupici E. albidus ani na jeden ze sledovaných parametrů, protože hodnotě NOEC odpovídala až nejvyšší testovaná koncentrace 620 mg/kg. Dusíkaté polycyklické aromatické uhlovodíky v případě druhu E.crypticus ukázaly řádově podobný efekt. Jejich toxicita (nominální koncentrace v mg/kg) se přesto snižovala v tomto pořadí: 1,10-fenanthrolín chinolín fenazin ≥ akridin. Po přepočtu těchto dat na efektivní koncentraci v půdní pórové vodě ( mol/l) se toxicita látek zcela obrátila. To odpovídá závěrům o vyšší toxicitě lipofilnějších látek ve vodním prostředí, jak je uváděno v literatuře.

Klíčová slova: roupice, Enchytraeus albidus, Enchytraeus crypticus, testy toxicity, toxický efekt, chlorované parafíny s krátkým řetězcem, toxafen, NPAHs, akridin, chinolin, fenazin, 1,10-fenantrolin.

–  –  –

• Bezchlebová, Jitka, Černohlávková, Jitka, Kobetičová, Klára, Lána, Jan, Sochová, Ivana, Hofman, Jakub. Effects of short-chain chlorinated paraffins on soil organisms.

Ecotoxicology and Environmental Safety, 67 (2): 206-211. June 2007 (Appendix I.).

• Bezchlebová, Jitka, Černohlávková, Jitka, Lána, Jan, Sochová, Ivana, Kobetičová, Klára, Hofman, Jakub. Effects of toxaphene on soil organisms. Ecotoxicology and Environmental Safety, Accepted as highlighted article (Appendix II.).

• Kobetičová, Klára, Bezchlebová, Jitka, Sochová, Ivana, Lána, Jan and Hofman, Jakub.

Toxicity of four polycyclic aromatic nitrogen heterocycles (PAHs) to soil organisms.

Ecotoxicology and Environmental Safety, submitted (Appendix III.).

International meetings

• Kobetičová, Klára, Lána, Jan, Bezchlebová, Jitka, and Hofman, Jakub. Toxicity effects of PANHs to soil invertebrates Folsomia candida and Enchytraeus crypticus. In Book of Abstracts of ECOTOX 2005. ISBN 80-210-3799-7. p. 158. Brno, Czech Republic, 5Poster presentation.

• Hofman Jakub, Bezchlebová Jitka, Sochová Ivana, Černohlávková Jitka, Kobetičová Klára, Lána Jan, and Holoubek Ivan. SOILETOX - Effects of priority persistent organic pollutants (POPs) on soil organisms. In Book of Abstracts of Soil Protection strategy – needs and approaches for policy support. Pulawy, Poland, 8-12.3.2006. p. 63ISBN 83-89576-03-1. Poster presentation.

• Bezchlebová, Jitka, Černohlávková, Jitka, Sochová, Ivana, Lána, Jan, Kobetičová, Klára, Hofman, Jakub. Effects and ecological risk assessment of short chain chlorinated

paraffins in soil. In Abstract Book of SETAC Europe the 15th Annual Meeting. Brussels :

SETAC, 2005. p. 388-388. Poster presentation.

• Bezchlebová, Jitka, Černohlávková, Jitka, Kobetičová, Klára, Lána, Jan, Sochová, Ivana, Hofman, Jakub. Efekt chlorovaného parafínu s krátkým řetězcem na půdní organismy. In: Sborník Študentské vedecké konferencie, svazok 1 – biologická a environmentální sekce. Bratislava : Univerzita Komenského v Bratislave; Bratislava, Slovensko, 26.4.2006. 203-206 ps. ISBN 80-88870-58-5. Oral presentation.

• Kobetičová, Klára, Bezchlebová, Jitka, Sochová, Ivana, Lána, Jan, Hofman, Jakub and Holoubek, Ivan. Toxicity of four polycyclic aromatic nitrogen heterocycles (PANHs) to soil organisms. In Abstracts of International Conference on Ecotoxicology 2006 Trends and Perspectives. Wisla, Poland, 17.-20.9.2006, p. 75-75. Poster presentation.

• Hofman, Jakub, Bezchlebová Jitka, Sochová Ivana, Černohlávková Jitka, Kobetičová Klára, Lána Jan, and Holoubek, Ivan. Effects of selected priority persistent organic pollutants (POPs) on soil organisms. In The Book of Abstracts of The Seventh European Meeting on Environmental Chemistry EMEC. Brno, Czech Republic: VUT Brno, 2007. p.121.. ISBN 80-214-3320-5. Poster presentation.



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