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 long term 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: NOEC mortality ( E.albidus ) = 10,000 mg/kg, NOEC mortality ( E.crypticus ) = 6,000 mg/kg, LC50 mortality ( E.albidus, E.crypticus ) could not be estimated, NOEC reproduction ( E.albidus ) = 3,000 mg/kg , NOEC reproduction ( E. crypticus ) = 6,000 mg/kg , EC50 reproduction (E.albidus) = 6,027 mg/kg, EC50 reproduction (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: 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 ) = 3,000 mg/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 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.
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. 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, 5 7.9.2005. Poster 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. 63 64, ISBN 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.
6 Abbreviations ASTM …………..American Society for Testing and Materials ATSDR …………Agency for Toxic Substances and Disease Registry BAF …………….Bioaccumulation factor ECx ……………..Effect concentration for x% effect ERT …………….Enchytraeid reproduction test EU ………………European Union EU RAR ………..European Union Risk Assessment Report HSDB …………..Hazardous Substances Databank IARC …………...International Agency for Research of Cancer ISO .……………..International Organisation for Standardisation Kow …………….Octanol water partition coefficient Kd ………………Partitioning coefficient soil water LCx …………… Effect concentration for x% morthality OECD …………..Organisation for Economic Co operation and Development LOEC …………..The lowest effect concentration NICNAS ………..National Industrial Chemicals Notification And Assessment Scheme NOEC …………..No observed effect concentration NPAH ………… N polycyclic aromatic hydrocarbon PAC …………… Polycyclic aromatic compound PAH …………….Polycyclic aromatic hydrocarbon PCB ……………..Polychlorinated biphenyl POP …………….Persistent organic pollutant RECETOX ……..Research Centre for Environmental Chemistry and Ecotoxicology SCCP ……………Short Chain Chlorinated Parrafin SECOFASE …….Sublethal Effects of Chemicals on Fauna in the Soil Ecosystem UNEP …………...United Nations Environment Programme UN ECE ………...United Nations Economic Commision for Europe WHC ……………Water Holding Capacity WHO ……………World Health Organisation
7 The main goals of the rigorous thesis : • The summary of enchytraeid biology, function and distribution in soils and using of the individual laboratory tests with enchytraeids (history, norms, design, test medium and enchytraeid species, tested pollutants) • The study of toxic effects of short chain chlorinated paraffins to Enchytraeus crypticus and Enchytraeus albidus • The study of toxic effects of toxaphene to Enchytraeus albidus • The study of toxic effects of some N heterocyclic aromatic hydrocarbons (acridine, phenazine, quinoline, 1,10 phenanthroline) to Enchytraeus crypticus
8 1. Theoretical part of the rigorous thesis
9 1.1. Enchytraeids The enchytraeids belong to the phylum Annelida and, along with the lumbricids, to the class Oligochaeta. They may inhabit the terrestric, limnic or sea environment. At present, about 900 species are described world wide. Total of 200–300 species can be estimated for Central Europe only ( Römbke et al., 1997 ). 1.1.1. Biology of enchytraeids Enchytraeids are usually colorless and as adults reach a size of about 2–40 mm. Their body is composed of many segments with a ring or saddleshaped glandular belt (clitellum). The number of segments increases with the age. Enchytraeid´s body is covered by thin cuticule. Setae with specific shape, size and the number of setae per bundles grow up the cover. Enchytraeids do not have eyes. The frontal segment of their body is called prostomium (Buchar et al. 1995; Sedlák, 2000). The food may pass through the pharynx, oesophagus into the gut. It was showed that species Lumbricillus rivalis is able to digestive the proteins, hydrocarbons, sacharides and fats (Learner, 1972). The presence/absence and shape of the oesophageal appendages (peptonephridia) belongs to the deterministic signs. Their possible function as a food moinstening organ or osmoregulatory organ was discussed in some studies (e.g. Schmelz et Westheide, 2000). The residues of food with soil particles may be defecated through the pygidium. Enchytraeids do not have special respiratory system, the gases and ionts permeate the skin. Coelom fluid contains less or more amount of lymphocytes differed by the shape, size, colour or granulation (Nielsen et Christensen, 1959).
b r a i n
p h a r y n x
peptonephridia
ectal glands am pulla spherm ateca ental opening oesophagus
septal glands dorsal blood vessel n e p h r i d i a
oesophageal-intestinal transition origin of dorsal blood vessel sem inal vesicles
m ale funnel
penial bulb o o c y t e s
Schema 1 . Description of enchytraeid body ( O´Connor , 1967).
10 Enchytraeids may also have chloragogen cells (Nielsen et Christensen, 1959), which have several important function in worm’s metabolism (Sedlák, 2000): fat or glykogen deposition, the production of hemoglobin, the fixation and neutralisation of toxins, catabolic degradation of protein and accumulation of ammonium substances. As the next excretion organs could be also nephridie, that should be presented in most segments. Enchytraeids have two or more septal glands in the front part of the body. Their function has not been clear yet (Nielsen et Christensen, 1959). The enchytraeids are mostly hermaphrodits. The significant part of enchytraeid body is clitellum (several glandular segments) with ducts of sexual glands. The clitellum forms at adult worms and should persist the whole life of individuals. The healthy adults have male and female organs. The reproduction process of earthworms, which are the close relatives of enchytraeids (Sedlák, 2000), was taken for a description of reproduction behaviour at enchytraeid worms:
Schema 2. Process of sexual reproduction of oligochaete worms (Sedlák, 2000). During the copulation, two adults are connected by their ventral sides in the opposite position. Worms insert the penial bulb (in area of clitellum) into the spermatheca (III. VI. segment) the others (A). Oocytes shift to the mucous locus (B). Mature eggs in a mucous locus pass over spermatheca and may be fertilized (C). The mucous locus is stripped up the worm as cocoon (D). Cocoons of enchytraeids may sink several eggs and latency time lasts tens days (Christensen, 1956). Juveniles are set free when the cocoon ruptures. Young enchytraeids have not developed sexual organs yet. Therefore, their determination is very difficult and they are recognized only into the genus. However, the reproduction is possible in range from 5 to 25 oC in temperate zone. The temperature over 25 oC may be lethal. Observation of three Enchytraeus species in laboratory conditions from borning to the reaching of maturity may last 65 120 days (Reynolds, 1939). Some species are able to reproduce via parthenogenesis ( C. glandulosa , Buchholzia appendiculata) . Another reproductive strategy is fragmentation ( Enchytraeus fragmentosus , Enchytraeus japonensis , Cognettia sphagnetorum ), where an individual autonomously breaks up into several parts, each of which regenerates into a complete individual (Bell, 1959).
11 1.1.2. The function of enchytraeids in soils The enchytraeids belongs to the significant decomposers in soils. Their function is similar to lumbricids that may be replaced by enchytraeids at strongly acid localities (mostly at forest soils with low pH values). At these areas, enchytraeids may reach very high densities and constitute the main taxon of soil annelids (Westheide and Bethke Beilfuss, 1991). They usually feed on slightly to strongly decomposed remains of plants and microorganisms (bacteria and fungi). 80 % of their diet consisting of microorganisms and 20 % of dead organic matter ( Zachariae, 1965 ; Didden, 1993 ). They are concerned at humus formation, produce excrements and provide the comeback of nutrients to soils, that are available to plants or the following soil decomposers. Worms transport also the mineral particles due to their ingestion (O´Connor, 1967) or overlapping on their body (Ponge, 1984). Mineral particles are so transferd from deeper layers into the cover of soil and organic matter more deeply which may help to processes of decomposition. In additional, enchytraeids consume microorganisms containing immobile nutrients and indirectly help to nutrient involving in the decomposition cycle. 1.1.3. Distribution of enchytraeids in soils Enchytraeids may appear in high abundance in different soils with varying species compositions (ranging from 1 to 30 different species per site). In terrestrial ecosystems, their average annual abundance lies between 20,000 and 60,000 individuals/m 2, but is subject to strong seasonal fluctuations (Römbke et al. 1997). The density (Didden, 1993) is influenced by many abiotic (soil acidity, humidity, temperature, etc.) and biotic (competition, predation, parasitismus, vegetation, etc.) factors. Enchytraeids have thin cuticule permeable for water and therefore they need a suitable humidity of the soil. They living mainly in close contact with soil pore water as well as soil nematodes (Achazi et van Gestel, 2003). The enchytraeids mostly prefer from slightly acid to slightly alcaline soils (Didden, 1993). However, some species as Cognettia sphagnetorum or Marionina clavata favour strongly acid soil environment. They may be a dominant taxon of soil oligochates and are able to replace earthworms at such localities (Healy, 1980). Most of enchytraeids is found in the upper layer of soil (to 20 cm from cover), the number of enchytraeids decreases with a higher depth (Peachey, 1963, O’Connor 1971, Chalupský 1989, Kobetičová et Schlaghamerský, 2002). A. Federschmidt and J. Römbke (1992) has mentioned that 70 % of enchytraeids occurs in the upper 8 cm of soil profile. Some species prefered higher depths than the others. J. A. Springett described that three enchytraeid species ( C. sphagnetorum, Marionina simillina, Achaeta eiseni ) preferably inhabitat the different soil layer (Springett, 1963). The occurence of enchytraeids under the cover is obvious because many organisms, biological activities and decomposer processes are there proceeded. The upper 10 cm of soil offers to worms optimal condition including enough of food and oxygen. The vertical migration of soil organisms depending on season and temperature inversion during the year was well described by Prof. Rusek (Rusek, 2000). Enchytraeids are able to move also horizontally. They have tendency to clustering (the random or regular distribution is unlikely). It was showed that they appear in multispecies aggregation centres (Chalupský et Lepš, 1985). The explanation for such behaviour might be presence of food, the favourable physical condition in a heterogenous soil environment, massive breeding of juveniles from cocoons (Chalupský et Lepš, 1985) or heterogenous contamination of soil (Salminen et Sulkava, 1996).
12 1.2. Tests of toxicity Enchytraeids have been using in the soil ecotoxicology since 70tieth years of the last century (Römbke, 2003). For enchytraeids there have been developed several different laboratory tests, but they can be divided in two groups: short term tests in aqueous or agar medium (Römbke and Knacker, 1989; Westheide et al., 1991) and short to medium term soil test (Römbke, 2003). However, the test with artificial medium is not very suitable for testing of chemicals in soils as well as for soil quality assessment. From this reason, the chronic Enchytraeid Reproduction Test (ERT) was developed and validated nationally and internationally. Recently, the ERT was standardised (ASTM, 2000; ISO, 2002; OECD, 2003). In addition, some of the other soil tests as Avoidance or Bioccumulation test are still under a development. And why the enchytraeids…. ? The objectives of ecotoxicological tests are, on the one hand, to be able to understand and predict the effects of chemical stress on the ecosystem level and, on the other hand, to be able to interpret field data in terms of ecological relevance of xenobiotic substances present (Didden et Römbke, 2001). The following lines gives the main reasons why the enchytraeids should be considered as part of a battery of ecotoxicological tests (Römbke, 2003): • they have a high ecological relevance • they play a key role in the functioning of the soil ecosystem • they cover an exposure route different to those of other test organisms (via the soil solution, the solid phase, and the gaseous phase in soil) • they are sensitive to many chemicals and other stressors • they are easily culturable • the practicability of enchytraeids tests has been proven (small amount of test substrate, relatively short duration) • the laboratory results can be extrapolated to higher test levels like semi field and field studies with relative ease, since the same or similar species are used at all levels. In the following subchapters (1.2.1; 1.2.2.; 1.2.3.; 1.2.4.; 1.2.5.; 1.2.6.), the individual tests are described in more detail (using enchytraeid species, design of test, measured endpoints, advantages/nonadvantages of tests, review of groups of test pollutants).
13 1.2.1. Water test Water test was firstly described by Weuffen (Weuffen, 1968 in Römbke et Moser, 2002). It has been again used by Römbke and Knacker (1989) after the next 20thy years (see Table 1). The test condition were chosen in analogy to the Daphnia Acute Immobilization Test (OECD, 1984). Water test has been using several times since the time (see Table 2). The measured endpoint is mostly mortality but behavioral or pathological symptoms may be also recorded every day (Römbke a Knacker, 1989; Šuteková, in press). Its advantages are simple design and short duration (4 days). However, it was found that a chemical in water could be 600 times more toxic than in soil, although the same enchytraeid species is used in both experiments (Römbke a Knacker, 1989). Table 1 . Water test on enchytraeids . Guideline or Reference Römbke and Knacker, 1989 Test principle Acute laboratory test Test parameter Mortality, behavioral or pathological changes Test duration 96 hours Test species Enchytraeus albidus or other species of this genus; in all cases originating from mass culture Test substrate Destilled water or reconstituted water (222 mg CaCL2, 123 mg MgSO 4.7 H 2O, 65 mg NaHCO 3, 5.8 mg KCl per litre) Application of test Aquatic solution of contaminant substance Test conditions 10 adult (= clitellate) worms per test vessel (glass with lid; temperature: 20 ± 2 °C; permanently no light; worms are not feed during the test period Control Untreated destilled or reconstituted water Validity criteria (control) Mortality < 20 % (adults) Test assessment NOEC or ECx (treatment versus control) Reference substance K2Cr 2O7: LC50 (mortality) = 1.9 mg/l Limitations and remarks Possible modifications depending on the Enchytraeus species and on the case study Table 2 . Overview of water test on enchytraeids. Reference and year Type of test, enchytraeid species, chemical Weuffen (1968)* Acute, E. albidus , various veterinary drugs Römbke et Knacker (1989) Acute, E. albidus , and four other species, eight chemicals Graefe (1991)* Acute, E. minutus, E. lacteus, soil extracts Willuhn et al. (1994) Acute, E. buchholzi , Cadmium Christensen et Jensen Sub lethal, E . bigeminus , three pesticides (1995)* Šuteková et al. (in press) Acute, E. albidus , E. crypticus , six chemicals (biomarkers) * in Römbke et Moser, 2002. 1.2.2. Agar test The first reference about the using of agar medium originates from Westheide and his colleagues (Westheide et al., 1989). The test design was established during the following years (Westheide et Bethke Beilfuss, 1991) (see Table 3) and some metals, pesticides and other chemicals have been tested (see Table 4).
14 Table 3 . Agar test on enchytraeids. Guideline or Reference Westheide et Bethke Beilfuss,1991 Test principle Sublethal laboratory test Test parameter Cocoon production, hatchability (number of fertiled cocoons) Test duration 30 days: 25 days for cocoon production, the next 5 days for hatchability Test species Genus Enchytraeus ; in all cases originating from synchronized culture (8 days old mature worms) Test substrate 1.5% agar (1 g Ca(NO 3)2, 0.25 g MgSO 4 . 7H 2O, 0.25 g KNO 3, 0.25 g KH 2PO 4, traces of FeSO 4 per 1 L of destilled water) Application of test Mix the compound concentration with the agar before the poured into substance the Petri dishes Test conditions 3 adults per glass Petri dish with lid (diameter 5 cm); temperature: 21 °C; permanently in dark; flaked oats are used for food during the test period) Control Untreated agar medium Validity criteria (control) Mortality < 20 % (adults) Test assessment NOEC or ECx (treatment versus control) Limitations and remarks Possible modifications depending on the Enchytraeus species and on the case study Table 4. Overview of agar test on enchytraeids. Reference and year Type of test, enchytraeid species, chemical Westheide et al. (1989)* Sub lethal, E. crypticus , nine chemicals, liquid manure Ruther et Greven (1990)* Acute, E. buchholzi , four metals Pursche et al. (1991)* Sub lethal, E. crypticus, various pesticides Westheide et Bethke Beilfuss Sub lethal, E. crypticus , three pesticides (1991) Westheide et al. (1991) Prolonged, E. crypticus, E. mintuus , Benomyl Kristufek et al. (1995)* Sub lethal, E. crypticus , seven chemicals Arratea et al. (2004) Sub lethal, E. coronatus , carbendazim, three chemicals * in Römbke et Moser, 2002 The positive side of the agar test is that the parameters as cocoon production and hatching success are observable better in this artificial medium than in soils. In addition, the sublethal effects of some tested compound as fungicide benomyl (Westheide et al., 1991) could be reconciled with soil population tests: lower abundance was due to a very low number of juveniles in the benomyl treated cultures. In the last mentioned study (see Table 4), the effect of carbendazim, 4 nitrophenol and potassium dichromate was measured on survival and on several sublethal variables as number of juveniles and cocoons per adult, number of eggs per cocoon, percent of hatching and adult biomass in chronic agar tests. The number of juveniles per adult was the most sensitive variable measured for the three substances. The reduction in the number of juveniles could be explained mainly by a negative effect on cocoon hatching due to carbendazim. The effect of 4 nitrophenol on the number of juveniles was interpreted as a consequence of a reduction in the number of eggs per cocoon, associated with a reduction in parental biomass. Potassium dichromate decreased reproduction by a reduction in cocoon production due rather to a failure of worm reproductive physiology. The agar test showed various effects of tested compounds on worm reproduction biology and contributed to better understanding of the effects of these substances measured in ERT soil toxicity tests (Arratea et al., 2004).
15 1.2.3. Sublethal test with species Cognettia sphagnetorum (Vejdovsky) 1877 The authors of the test are Rundgren and Augustsson (1998) (see Table 5). The test organism is exclusively enchytraeid Cognettia sphagnetorum (Vejdovsky) 1877. This species occurs mostly in poor coniferous forest soils but is also frequent in grassland or acid meadows (Springett, 1970; Standen, 1984 in Runtgren et Augustsson, 1998). Some authors refered to the sexual reproduction (e. g. Schlaghamerský, 2002) but main reproductive strategy is fragmentation (Nielsen et Christensen, 1959). This implies that an individual breaks up into a number of fragments, which have potencial to develop into new worms (Bell, 1959). In spite of that two experiments with litter as substrate was recorded (see Table 6), the test has been used firstly as a toxicity test in Rundgren and Augustsson study (1998). Table 5. Sublethal test with species Cognettia sphagnetorum (Vejdovsky) 1877. Guideline or Reference Rundgren and Augustsson (1998) Test principle Sublethal laboratory test Test parameter Mortality (adults), asexual reproduction fragmentation (number of survival fragments), growth (number of segments per individual fragment) Test duration Effects are determinated after once a week over 10 weeks Test species Cognettia sphagnetorum (Vejdovsky) 1877 Test substrate Sphagnum peat (75 %) + LUFA 2.2 (25 %); pH about 4 Application of test Mix with the soil substance Test conditions 2 grames of soil and 1 worm per test vessel (glass vials with screw caps; 5 cm 3); temperature: 15 ± 1 °C; 24 hours light; moisture: 80 % of the WHCmax., food: Baker´s yeast or fungae Mortierella isabellina , extraction of the worms and fragments by flushing the content of vials with water; determination using the microscope, the whole procedure is repeated once a week, the studied worms or fragments are then introduced into the soil in the new test vessels with the same test concentration from the previous week Control Untreated test substrate Validity criteria (control) Mortality < 10 % (adults); growth rate is ≥ 1 segments per week Test assessment ECx (treatment versus control) Reference substance Copper chloride: various ECx values are described in Rundgren and Augustsson (1998) Table 6 . Overview of laboratory test with Cognettia sphagnetorum (Vejdovsky) 1877. Reference and year Substrate, type of test, chemical Heugens (1984)* Litter, acute, pH and salt Huhta (1984) * Litter, soil, acute, pH and nutrients Rundgren et Augustsson Soil, sub lethal, three chemicals (1998) * in Römbke et Moser, 2002 1.2.4. Enchytraeid reproduction test (ERT) The Enchytraeid Reproduction Test (ERT) has carried out in well – defined artificial soil such as the commonly used OECD soil from earthworm acute tests (OECD, 1984). Enchytraeus albidus is mostly used species in ERT. E. albidus has been used in ecotoxicology as well as physiology, biochemistry and genetics for more than 50 years (Römbke et Moser,
16 2002). This is due to its world wide distrubution and relatively high size (15 40 mm). However, the other species of genus Enchytraeus (usually smaller species as E. crypticus , E. luxorius or E. bulbosus ) have been also used in the toxicological tests. In ERT, a sublethal parametr fecundity (number of juveniles) is a main endpoint. Lethal effect and behavioural changes should be also recorded. The test can be divided into two steps: (a) a range finding test in which adult mortality is established after 2 weeks and the results allow to find a suitable range concentration for a definitive reproduction test (b). The mortality of adults is studied in a half of test period and reproduction after finishing of the definitive test. The application of test substance differes depending on its solubility. Water soluble substances are applicated as aquatic solution into a test soil. Chemicals little soluble in water are dilluted in organic solvent that is evaporated after application into the soil. The other chemicals are mixed with a little amount of sand and after that with other components (for more details of test design see Table 7). Table 7. The Enchytraeid Reproduction Test (ERT; Römbke, 2003). Guideline or Reference Draft Guideline according to ASTM E 1676 97 (2000), ISO 16387 (2002) or OECD 220 (2003) Test principle Chronic, sublethal laboratory test Test parameter Mortality (adults), reproduction (number of juveniles) Test duration Range Finding Test: 2 weeks; Definitive test: variable, depending on the species; E. albidus : 6 weeks; others species: 4 weeks Test species Enchytraeus albidus ( Enchytraeidae ) or other species of the genus Enchytraeus ; in all cases originating from mass culture Test substrate Artificial soil: quarz sand, kaoline, sphagnum peat, calcium carbonate and destillled water (OECD 1984); also field soils is possible Application of test Mixed into the artificial soil; mixtures of contaminated and control soil substance also possible Test conditions 10 adult (= clitellate) worms per test vessel (glass with lid; 0.2 – 0.25 L volume); temperature: 20 ± 2 °C; permanently no light; moisture: 40– 60% of the WHCmax., pH of soil = 6.0 ± 0.5; rolled oats are mixed with soil once a week, extraction of the juveniles using Bengal red Control Untreated test substrate (e.g. artificial soil or a reference soil such as the German LUFA 2.2) Validity criteria (control) Mortality < 20 % (adults); number of juveniles per test vessel (at the end of the test) > 25 ( E. albidus ) or > 50 (other species), a coefficient of variation < 50 % at the end of the definitive test Test assessment NOEC or ECx (treatment versus control) Reference substance EC50 (reproduction) of Carbendazim: 1.2 ± 0.8 mg/kg Limitations and remarks Possible modifications depending on the Enchytraeus species and, in the case of soil quality assessment, on the test soil The staining with Bengal red has been mostly used for extraction of enchytraeids from soils but wet funnel extraction and floatation method are recommended as the other possible techniques (e.g. ISO, 2002). Wet extraction could be used in laboratory test as well as ecological or deterministic research, however, due to the properties of artificial soil this method is sometimes to difficult to perform (Römbke et Moser, 2002). In addition, some results show that the Ludox Flotation Method is more efficient, faster, and less laborious than the wet extraction method (Phillips et al., 1999) but it has been the least using extraction method for the present. As reference substance was choosen fungicide Carbendazim, which is very toxic for oligochaetes. Its LC50 value overreached 10 mg/kg but EC50 value was established in range
17 from 0.4 to 2.0 mg/kg in an international Ring test. This reference substance should be tested either at regular intervals to verify that response of the test organisms has not changed significantly over time (Römbke et Moser, 2002). ERT has been used for testing of many individual organic compounds, heavy metals or some pharmaceuticals (see Table 8). Later, the test was also validated and modificated for soil quality assessment, usually in cases where the soils were contaminated with mix of polycyclic aromatic compounds, heavy metals or TNT (e.g. Achazi et al., 1996; Schaefer et Achazi., 1999; Filimonova et Pokarshevskii, 2000; Kupermann et al., 2003; Pokarshevski et al., 2003; Frouz et al., 2005). The enchytraeid test was also used in process of soil remediation (Juvonen et al., 2000). In addition, the studies have more often progressed from pure measurement the toxic effects of individual pollutants (LCx, ECx values) into the more comprehensive investigation of bioavailability affecting the toxicity. The aspects as structure of chemical, test species and physico chemical properties of soil, test design or influence of ageing has been investigated (see Table 8). Table 8 . Overview of Enchytraeid Reproduction Test (ERT). Chemical Measured effects Reference and year (number of studies**) Pharmaceuticals (4) toxicity Achazi et al. (1997); Baguer et al. (2000); Diao et al.(2007), Kolar et al. (in press) Explosive material (5) ageing, toxicity, juveniles Schaefer et Achazi (1999), Dodard et al., adult differencies (2003; 2004), Kuperman et al., (2003, 2006) Surfactants (2) toxicity Geiljsberg et al. (2001), Holmstrup et Krogh (2001) Halogenated flame sensitivity of soil organisms Sverdrup et al., (2006) retardants (1) PACs (4) toxicity, mode of action Bleeker et al. (2003) Sverdrup et al. (2002a,b, 2007) Heavy metals (10) ageing, soil properties, soil Kratz et Brose (1997); Lokhorst, 1997; types, adaptation processes, Posthuma et al. (1997); Posthuma et bioavailability Notenboom, (2000)*; Beylich (2001)*; Lock (2002)*, Lock et Janssen, (2003 a,b,c); Amorim et al. (2005) Pesticides (10) influence of soil moisture, Puurtinen et Martikainen (1997); influence of pH on effects, Amorim et al. (1999, 2005); Collado et ring test, enchytraeid species al. (1999)*; Kuperman et al. (1999); sensitivity, different soil types, Hund Rinke et al. (2002c)*; Lock et al. soil/species differences (2002); Römbke et Federschmidt (1995); Römbke et Moser (2002) * in Römbke et Moser, 2002, ** the numbers of studies are only orientation, it is possible that more works exist (works of students, articles in bulletins and national papers, etc). 1.2.5. Avoidance test The first study about using of enchytraeids in an Avoidance test came up in the mid 1990s (Achazi et al., 1996). Generally, the avoidance or escape behaviour of soil invertebrates is based on the fact that the organisms dispose of chemoreceptors sensitive to unsuitable antropogenic or environmental condition (e.g. Römbke et Schmidt, 1999 in Amorim et al., 2005b).
18 Enchytraeids have been very rearly used in Avoidance test (Achazi et al., 1996; Achazi et al., 1999; Amorim et al., 2005) and most of the studies are not commonly available since some of them are diploma thesis (e.g. Panneck, 2000; Wagner Vaske, 2000). The existing works are briefly described in the Table 9. Table 9. Overview of Avoidance test with enchytraeids. Reference and year species endpoint test chemical or soil Achazi et al. (1996)* E. crypticus Avoidance and PCB and metals escape reaction Achazi et al. (1999)* E. crypticus Avoidance reaction Former irrigation fields, sewage sludges Panneck (2000)* E. crypticus Escape reaction Cu acetate, Cd chloride Wagner Vaske (2000) * E. crypticus Escape reaction Phenmediphan,Ethiofencarb, Carbofuran, cypermethrin, Schafer et Achazi (2004) E. crypticus Escape reaction Hexyl Amorim et al. (2005) E. albidus Avoidance reaction OECD and several natural soils, Carbendazim, Phenmediphan, Kobetičová et al., (2007) E. albidus , Avoidance reaction OECD soil + 5 natural soils E. crypticus * in Römbke et Moser, 2002 The results from the mentioned studies indicate that avoidance tests are useful as method for the assessment of chemicals in soils (see Table 9) or for soil quality assessment (Amorim et al., 2005b), however, this test is suitable only for chemicals that cause irritant effects to test organisms (Yeardly, 1996). The avoidance test with enchytraeids is still under a development and some differencies in test design are under a disccusion (Wagner Vaske 2000 in Römbke, 2003, Amorim et al., 2005b, Kobetičová et al., 2007). Nevertheless, the proposal from one of the first publicized study (Achazi et al., 1999) is described in the Table 10. and short description of test design is visible in the Schema 3. Table 10. The proposal of design of Avoidance test with enchytraeids (Achazi et al., 1999 in Römbke, 2003). Guideline or Reference Achazi et al. (1999) Test principle Determination of the site choice of the enchytraeids Test parameter Behaviour (movement activity, stay at the end of the test) Test duration 48 hours Test species E. crypticus or another Enchytraeus species; adult individuals from synchronised laboratory mass cultures Test substrate Spiked control soil or contaminated field soils (10 replicates) Application of test Mixed into the artificial soil; mixture of contaminated and control soil substance also possible Test conditions 20 adult (= clitellate) worms per test vessel (10 g control and 10 g test soil, separated by gaze (1 mm); temperature 20 ± 1 °C; permanently no light; moisture: 50 % WHCmax Control Untreated field soil (LUFA standard soil 2.2) Validity criteria (control) Mortality ≤ 20 % Test assessment Different distribution compared to the control (LUFA soil on both sides) or number of worms in the test soil ≤ 20 % Reference substance Not specified Remarks Avoidance test: 20 worms are set on the control soil; Escape test: 20 worms are set on the test soil; Combined test: Either 20 worms on the line between the two sides or 10 worms on both sides of the test vessel
19
Schema 3 . Schematic representation of the experimental procedures of enchytraeid avoidance test: (1) introduction of the movable wall in the centre of the test vessel; (2) introduction of the soils to be tested; (3) movable wall is removed; (4) placement of the enchytraeid worms in the centre of the soils; (5) covering the test vessel with a lid (perforated); (6) reintroduction of the wall to separate the soils and counting of the organisms present in each side (Amorim et al., 2005b). 1.2.6. Bioaccumulation test The bioaccumulation is defined as the increasing a concentration of the test substance in or on an organism relative to the concentration of the test substance in the surrounding medium, including uptake of a chemical from both medium and food via all possible pathways (e.g. OECD, 1996). Its degree could be expressed as the bioaccumulation factor (BAF), which is calculated as the quocient of the concentration of the test chemical in the animal divided by the concentration in the soil. Bioaccumulation is a direct measure of chemical concentrations in an organism resulting from the net flux of chemicals from the soil due to the balance between uptake and depuration processes (Lanno et al., 2004). Bioaccumulation is associated with chemical residues accumulated at the site of toxic action but below a toxic threshold, as well as residues in other tissues containing no site of toxic action (Lanno et al., 2004).
Schema 4 : Schematic model of bioavailability (Lanno, 2003).
The significance of research of bioaccumulation at soil invertebrates consists in the detection and understanding the potencial enriching of pollutant concentration through a food chain (see Schema 5):
Cl
Cl Cl
Cl Cl Cl Schema 5 . Schema of biomagnication the food chain by chemical.
20 The insecticide Lindane and the fungicide Hexachlorobenzene (HCB) were used in a bioaccumulation study from Germany (Bruns et al., 2001). The test was performed with two enchytraeid species ( E. albidus, E. luxuriosus ) as model organisms with an artificial soil and a natural standard soil (LUFA 2.2) as test substrates. Uptake as well as the elimination of the test substances were examined in a closed test system for 42 days. The smaller species E. luxuriosus accumulated the two chemicals to a greater degree than E. albidus . The authors suggested two possible explanation for this: 1. E. luxorius may selectively feed on small particles, which may carry higher amounts of chemical than coarser material. 2. The smaller size of E. luxorius leads to a large body surface area relatively to the weight which supports an enhanced diffusion of test substance via the body wall. In any case, the bioaccumulation pattern was considerable for both the enchytraeid species (Bruns et al., 2001). In the other study (Amorim et al., 2002), the uptake and elimination of lindane in Enchytraeus albidus was analysed in OECD soil and a natural agricultural soil. The uptake and elimination phases lasted 10 days. The results indicated the correlation among the total soil concentration, the biovailable concentration in soil (water extracts) and concentration in worms (the concentration decreased in all substrates equally) in OECD soil. On the contrary, the bioavailable fraction of lindane in the natural soil decreased faster than the total soil concentration. The higher bioaccumulation occured in the natural soil than in the artificial soil due to a higher organic matter content of OECD soil, which caused a lower bioavailability and accumulation in this soil (see Table 11). However, the higher elimination rate was observed in the OECD soil. The authors supposed that the large amount of sand particles in OECD soil was in charge of raising elimination the soil particles with lindane from worm‘s gut . Table 11 . BAF values of lindane and Hexachlorobenzene (HCB). E. albidus E. luxorius Reference Lindane OECD soil 6.4*; 12 ± 1.1 Bruns et al., 2001*; 12 ± 0.7 Amorim et al. 2002 LUFA 2.2 22 ± 0.4 36 ± 1.1 Bruns et al., 2001 Agricultural soil 9.6 Amorim et al. 2002 HCB OECD soil 14 ± 4.4 27 ± 0.9 Bruns et al., 2001 LUFA 2.2 28 ± 0.8 35 ± 0.7 Bruns et al., 2001 Independently from the two works described above, bioaccumulation tests with heavy metals using enchytraeids have been done by Postuma et al. (1998b) and Lock et Janssen (2001), confirming the suitability of enchytraeids as model organisms in bioaccumulation tests (see Table 12). Table 12 . Overview of the Bioaccumulation test. Reference and year Test species, Chemical Postuma et al. (1998)* 2 species, Zn Bruns et al. (2001) E. albidus , E. luxorius , Lindane and HCB Lock et Janssen (2001)* E. albidus , Zn, Cd Amorim et al.(2002) E. albidus , Lindane * in Römbke et Moser, 2002 .
21 1.3. References Achazi, R. K., Chroszcz, G., Pilz, B., Rothe, B., Steudel, I., Throl, C. (1996): Der Einfluss des pH Werts und von PCB52 auf Reproduktion und Besiedlungsaktivität von terrestrischen Enchytraeen in PAK , PCB und schwermetallbelasteten Rieselfeldböden. Verhandlungen der Gesellschaft für Ökologie, 26: 37–42. Achazi R. K., Van Gestel C. A. M. (2003): Uptake and Accumulation of PAHs by Terrestrial Invertebrates. PAHs: Edited by Peter E.T. Douben. An Ecotoxicological Perspective: 173 190. Amorim, M. J. D., Römbke, J., Schallnass, H. J., Mortagua, A., Soares, V. M. (2005c): Effect of soil properties and aging on the toxicity of copper for Enchytraeus albidus, Enchytraeus luxuriosus, and Folsomia candida. Environmental Toxicology and Chemistry, 24: 1875 1885. Amorim, M. J., Römbke, J., Scheffczyk, A., Soares, A. M.V. M. (2005a): Effect of different soil types on the enchytraeids Enchytraeus albidus and Enchytraeus luxuriosus using the herbicide Phenmedipham. Chemosphere, 24: 1875 1885. Amorim, M. J., Sousa, J. P., Nogueira, A. J. A., Soares, A. M. V. M. (2002): Bioaccumulation and elimination of 14 C lindane by Encyhtraeus albidus in artificial (OECD) and a natural soil. Chemosphere, 49: 323 329. Amorim, M., Soares, A., Römbke, J. (2005b): Avoidance behaviour of Enchytraeus albidus: Effects of Benomyl, Carbendazim, phenmedipham and different soil types. Chemosphere, 59: 501 510. Arratea, J. A., Rodrigueza, P., Martinez Madrida, M. (2002): Effects of three chemicals on the survival and reproduction of the oligochaete worm Enchytraeus coronatus in chronic toxicity tests. Pedobiologia, 46: 136 149. ASTM (American Society for Testing and Materials) (2000): Standard guide for conducting laboratory soil toxicity or bioaccumulation tests with the lumbricid earthworm Eisenia fetida and the enchytraeid potworm Enchytraeus albidus . ASTM Guideline No. E 1676– 97 (Draft). Baguer, A. J., Jensen, J., Krogh, P. H. (2000): Effects of the antibiotics oxytetracycline and tylosin on soil fauna. Chemosphere, 40: 751 757. Bell, A.W. (1959): Enchytraeus fragmentosus, a new species of naturally fragmenting oligochoeta worm. Science (Wash. D. C.),129: 1278. Bleeker, E. A. J., Wiegman, S., Droge, S. T. J., Kraak, M. H. S., van Gestel, C. A. M. (2003): Chronic toxicity of PACs to Folsomia candida and Enchytraeus crypticus. In: Towards an improvement of the risk assessment of polycyclic (hetero)aromatic hydrocarbons. Report 2003 01 of the department of Aquatic Ecology and Ecotoxicology, University of Amsterdam, Amsterdam, The Netherlands. Report 2003 04 of the Institute of Ecological Science, Vrije Universiteit, Amsterdam, The Netherlands: 27 32.. Bruns, E., Egeler, P., Römbke, J., Scheffczyk, A., Spoerlein, P. (2001): Bioaccumulation of lindane and hexachlorobenzene by the oligochaetes Enchytraeus luxuriosus and Enchytraeus albidus (Enchytraeidae, Oligochaeta, Annelida). Hydrobiologia, 463: 185 196. Buchar, J., Ducháč, V., Hůrka, K., Lellák, J. (1995): Klíč k určování bezobratlých. Scientia: 285 pp. Diao, X., Jensen, J., Duus Hansen, A. (2007): Toxicity of the anthelmintic abamectin to four species of soil invertebrates. Environmental Pollution, 148: 514 519. Didden, W. A. M. (1993): Ecology of terrestrial Enchytraeidae. Pedobiologia, 37: 2 29. Didden, W., Römbke, J. (2001): Enchytraeids as Indicator Organisms for Chemical Stress in Terrestrial Ecosystems. Ecotoxicology and Environmental Safety, 50: 25 43.
22 Dodard, S. G., Powlowski, J., Sunahara, G. I. (2004): Biotransformation of 2,4,6 trinitrotoluene (TNT) by enchytraeids (Enchytraeus albidus) in vivo and in vitro. Environmental Pollution, 131: 263 273. Dodard, S. G., Renoux, A. Y., Sunahara, G. I. (2003): Lethal and Sub Chronic Effects of 2,4,6 Trinitrotoluene (TNT) on Enchytraeus albidus in Spiked Artificial Soil. Ecotoxicology and Environmental Safety, 54: 131–138. Federschmidt, A., Römbke, J. (1992): Bodenbiologische Untersuchungen an einer Streuobstwiese und einem Eichen Hainbuchenwald: Die Enchyträen und Tubificidenzönose. Verhandlungen der Gesellschaft für Ökologie, 21: 59–64. Filimonova, Z. V., Pokarzhevskii, A. D., Zaitsev, A. S. (2000): Ecological Mechanisms of Soil Biota Tolerance to Metal Contamination. Biological Sciences, 370: 77 80. Frouz, J., Krištůfek, V., Bastl, J., Kalčík, J., Vaňková, H. (2005): Determination of toxicity of spoil substrates after brown coal mining using a laboratory reproduction test with Enchytraeus crypticus (Oligochaeta). Water Air and Soil Pollution, 162: 37 47. Gejlsbierg, B., Klinge, C., Samsoe Petersen, L., Madsen, T. (2001): Toxicity of linear alkylbenzene sulfonates and nonylphenol in sludge amended soil. Environmental Toxicology and Chemistry, 20 (12): 2709 2716. Healy, B. (1980): Distribution of terrestrial Enchytraeidae in Ireland. Pedobiologia, 20: 159 175. Holmstrup, M., Krogh, P. H. (2001): Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 3. Sublethal effects on soil invertebrates. Environmental Toxicology and Chemistry, 20: 1673 1679. Chalupský, J. (1989): Metody stanovení živočišných populací v půdě. In: D. Dykyjová (ed.): Metody studia ekosystémů. Academia, Praha: 503–512. Chalupský J., Lepš, J. (1985): The spatial pattern of Enchytraeidae (Oligochaeta). Oecologia (Berl.), 68: 153 157. Christensen, B. (1956): Studies on Enchytraeidae. 6. Technique for culturing Enchytraeidae, with notes on cocoon types. Oikos, 7: 302 307. ISO (International Organisation for Standardisation) (2002): Soil Quality – Effects of Pollutants on Enchytraeidae ( Enchytraeus sp.). Determination of effects on reproduction. ISO No. 16387. Juvonen, R., Martikainen, E. A. T., Schultz, E., Joutti, A., Ahtiainen, J., Lehtokari, M. (2000): A Battery of Toxicity Tests as Indicators of Decontamination in Composting Oily Waste. Ecotoxicology and Environmental Safety, 47: 156 166. Kobetičová, K., Hofman, J., Holoubek, I. (2007): Vliv půdních vlastností na chování roupic (Enchytraeus albidus , Enchytraeus crypticus ) v Avoidance testech. In: Sborník recenzovaných příspěvků, 1. svazek – biologická a environmentální sekce. Univerzita Komenského v Bratislave; Prírodovedecká Fakulta, Slovakia: 261 263. Kobetičová, K., Schlaghamerský, J. (2003): On the efficiency of three schemes of enchytraeid wet funnel extraktion. Newsletter on Enchytraeidae, 8: 25–31. Kolar, L., Kozuh Erzen, N., Hogerwerf, L., van Gestel, C. A. M. (in press): Toxicity of abamectin and doramectin to soil invertebrates. Environmental Pollution, xx: 1 8. Kratz, W., Brose, A. (1997): Bodenokologische Untersuchungen zur Wirkung und Verteilung von organischen Stoffgruppen (PAK, PCB) in ballungsraumtypischen Okosystemen. Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie, Berlin. Kuperman, R. G., Checkai, R. T., Simini, M., Phillips, C.T., Kolakowski, J. E., Kurnas, C. W., Sunahara, G. I. (2003): Survival and reproduction of Enchytraeus crypticus (Oligochaeta, Enchytraeidae) in a natural sandy loam soil amended with the nitro heterocyclic explosives RDX and HMX: The 7th international symposium on earthworm ecology, Cardiff, Wales, 2002, Pedobiologia, 47: 651 656.
23 Kuperman, R. G., Checkai, R. T., Simini, M., Phillips, C. T., Kolakowski, J. E., Kurnas, Davis, E. A. (2006): Toxicity of emerging energetic soil contaminant CL 20 to potworm Enchytraeus crypticus in freshly amended or weathered and aged treatments. Chemosphere, 62: 1282 1293. Kuperman, R.G., Simini, M., Philips, C. T., Cheackai, R. T. (1999): Comparison of Malathion toxicity using enchytraeid reproduction test and earthworm toxicity test in different soil types. Pedobiologia, 43: 630 634. Lanno, R. P., LeBlanc, S. C., Knight, B., Tymowski, R., Fitzgerald, D. G. (2003): Application of body residues as a tool in the assessment of soil toxicity. In: Advances in Earthworm Ecotoxicology. SETAC. Lanno, R., Wells, J., Conder, J., Bradham, K., Basta, N. (2004): The bioavailability of chemicals in soil for earthworms. Ecotoxicology and Environmental Safety, 57: 39 47. Learner, M. A. (1972): Laboratory studies on the life histories of four enchytraeid worms (Oligochaeta) which inhabit sewage percolating filters. Annals of Applied Ecology, 70: 251 266. Lock, K., Janssen, C. R. (2002a): Ecotoxicity of Chromium (III) to Eisenia fetida , Enchytraeus albidus and Folsomia candida . Ecotoxicology and Environmental Safety, 51: 203 205. Lock, K., Janssen, C. R. (2002b): Ecotoxicity of nickel to Eisenia fetida , Enchytraeus albidus and Folsomia candida . Chemosphere, 46: 197 200. Lock, K., Janssen, C. R. (2002c): Multi generation toxicity of zinc, cadmium, copper and lead to the potworm Enchytraeus albidus . Environmental Pollution, 117: 89 92. Lokhorst, A. (1997): Heavy metal and fungicide toxicity in E. albidus Evaluation of a proposed enchytraeid toxicity test with artificial OECD soil. Studentenverslag Landbouwuniversiteit,Vakgroep Terrestrische Oecologie en Natuurbeheer, Wageningen. Nielsen, C. O., Christemsen, B. (1959): The Enchytraeidae, critical revision and taxonomy of European species. Naturalia Jutlandica, 8–9: 1–160. O´Connor, F. B. (1967): The Enchytraeidae. In: Burges, A., Raw, F. (eds) Soil biology. Academic Press, London, New York: 213 257. O´Connor, F. B. (1971): Methods of Study in Quantitative Soil Ecology: population, production and energy flow 7. The Enchytraeids. IBP Handbook 18: 83 106. OECD (Organisation for Economic Co operation and Development) (1984): Earthworm, acute toxicity tests. OECD guideline for testing of chemicals, No. 207. OECD. Earthworm toxicity tests, Paris, France. OECD (Organisation for Economic Co operation and Development) (1984): Daphnia sp. acute immobilisation test and reproduction test. OECD Guideline for testing of chemicals, No. 202. OECD, Paris, France:16 pp. OECD (Organisation for Economic Co operation and Development) (1996): Guidelines for Testing of Chemicals No. 305. Bioconcentration: Flow through Fish Test. OECD, Paris: 9 pp. OECD (Organisation for Economic Co operation and Development)(2003): OECD Guideline for Testing of Chemicals No. 220. Enchytraeidae Reproduction Test. Paris. Peachey, J. E. (1963): Studies on the Enchytraeidae (Oligochaeta) of moorland soil. Pedobiologia, 2: 81 95. Phillips, C. T., Kuperman, R. G., Checkai, R. T. (1999): A rapid and highly efficient method for extracting enchytraeids from soil. Pedobiologia, 43: 523 527. Pokarzhevskii, A. D., Goryachev, O. A., Filimonova, Z. V., Pushnikova, Z. N (2003): Toxic effects of crude oil saturated soils on the survival and reproduction of the enchytraeid Enchytraeus cryptius in a short term testing experiment. Fresenius Environmental Bulletin, 12: 844 847.
24 Puartinen, H. M., Martikainen, E. A. T.(1997): Effects of soil moisture on pesticide toxicity to an enchytraeid worm, Enchytraeus sp.. Archives of Environmental Contamination and Toxicology, 33: 34 41. Ponge, J. F. (1984): Étude écologique d´un humus forestier par l´observation d´un petit volume, premier résultats. I. – La couche Ll d´un moder sous pin sylvestre. Revue d´Écologie et de Biologie du Sol, 21: 161 187. Posthuma, L., Baerselman, R., Van Veen, R. P. M., Dirven Van Breemen, E. M. (1997): Sinngle and Joint Toxic Effects of Copper and Zinc on Reproduction of Enchytraeus crypticus in Relation to Sorption of Metals in Soils. Ecotoxicology and Environmental Safety, 38: 108 121. Reynolds, T. B. (1939): On the life history and ecology of Lumbricillus lineatus Mull. (Oligochaeta). Annual of Applied Biology, 26: 782 798 Römbke, J., Beck, L., Forster, B., Frund, H.C., Horak, F., Ruf, A., Rosciczewski, K., Scheurig, M., Woas, S. (1997): Boden als Lebensraum fur Bodenorganismen und die bodenbiologische Standortklassifikation: Eine Literaturstudie. Texte und Berichte zum Bodenschutz 4/97. Landesanstalt fur Umweltschutz Baden Wu rttemberg (Karlsruhe). Römbke, J., Federschmidt, A. (1995): E!ects of the fungicide Carbendazim on Enchytraeidae in laboratory and field tests. Newsletter on Enchytraeidae, 4: 79 96. Römbke, J., Knacker, T. (1989): Aquatic toxicity test for enchytraeids. Hydrobiologia, 180: 235 242. Römbke, J., Moser, T. (2002): Validating the enchytraeid reproduction test: organisation and results of an international ringtest. Chemosphere, 46: 1117 1140. Rungren, S., Augustsson, A. K. (1998): Test on the Enchytraeid Cognettia sphagnetorum (Vejdovsky) 1877. In Lokke, H., van Gestel, C. A. M. (Eds.), Handbook of Soil Invertebrate Toxicity Tests. John Wiley & Sons Ltd., Chichester. Rusek, J. (2000): Živá pùda (5). Sukcesní vývoj pùdy a ekosystémù. Živa, 5: 217 221. Salminen, J., Sulkava, P. (1996): Distribution of soil animals in patchily contaminated soil. Soil Biology and Biochemistry, 28: 1349 1355. SECOFASE (1994): Progress Report 1994 of SECOFASE. Third Technical Report. ISBN: 87 7772 204 3. Sedlák, E. (2000): Zoologie bezobratlých. Skriptum. PřF, MU v Brně: 336 pp. Schäfer, R., Achazi, R. K. (1999): The toxicity of soil samples containing TNT and other ammunition derived compounds in the enchytraeid and collembola biotest. Environmental Science and Pollution Research, 6: 213 219. Schlaghamersky, J. (2002): The Enchytraeidae of spruce forest plots of different exposure and acid deposition in a German mountain range. European Journal of Soil Biology, 38: 305 309. Schmelz, R. M., Westheide, W. (2000): Ultrastructure of oesophageal appendages („peptonephridia“) in enchytraeids (Annelida, Clitellata). Invertebrate Biology, 119: 94 103. Springett, J. A. (1963): The distribution of three species of Enchytraeidae in different soils. In: Doecken J. et Drift J. (eds), Soil organisms. Amsterdam: 414–419 pp. Springett, J. A. (1970): The distribution and life histories of some moorland Enchytraeidae (Oligochaeta). Journal of Animal Ecology, 39: 725–737. Sverdrup, L. E., Hartnik, T., Mariussen, E., Jensen, J. (2006): Toxicity of three halogenated flame retardants to nitrifying bacteria, red clover (Trifolium pratense), and a soil invertebrate (Enchytraeus crypticus). Chemosphere, 64: 96 103. Sverdrup, L. E., Jensen, J., Kelley, A., Krogh, P. H., Stenersen, J. (2002b): Effects of eight polycyclic aromatic compounds on the survival and reproduction of Enchytraeus
25 crypticus (Oligochaeta, Clitellata). Environmental Toxicology and Chemistry, 21: 109 114. Sverdrup, L. E., Krogh, P. H., Nielsen, T., Stenersen, J. (2002c): Relative sensitivity of three terrestrial invertebrate tests to polycyclic aromatic compounds. Environmental Toxicology and Chemistry, 21:1927 1933. Šuteková, E., Hilscherová, K., Hofman, J. (in press): Oxidative stress in enchytraeidae ( E. albidus, E. crypticus ) Establishment and optimalization of methods. Folia Facultatis scientiarum naturalium Universitatis Masarykianae Brunensis. Westheide, W., Bethke Beilfuss, D. (1991): The sublethal enchytraeid test system: guidelines and some results. Modern Ecology: 497 508. Westheide, W., Bethke Beilfuss, D., Hagens, M., Brockmeyer, V. (1989): Enchytraeiden als Testorganismen – Voraussetzungen fur ein terrestrisches Testverfahren und Testergebnisse. Verhandlungen der Gesellschaft für Ökologie, 17: 793 798. Yeardley, R. B., Lazorchak, J. M., Gast, L. C., 1996. The potential of an earthworm avoidance test for evaluation of hazardous waste sites. Environmental Toxicology and Chemistry, 15: 1532–1537. Zachariae, G. (1967): Die Streuzersetzung im Köhlgartengebiet. In: Graff, O., Satchell, J.E. (eds) Progress in Soil Biology. Braunschweig, Amsterdam: 490 506.
26 2. Experimental part of the rigorous thesis
27 2.1. Toxicity of selected organic pollutants: toxaphene; short chain chlorinated paraffins and NPAHs (1,10 phenanthroline, acridine, phenazine, quinoline) to Enchytraeus albidus /Enchytraeus crypticus 2.1.1. Abstract Effects of short chain chlorinated paraffins (industrial chemicals), Toxaphene (insecticide) and four N heterocyclic aromatic hydrocarbons acridine, phenazine, quinoline, 1,10 phenanthroline (by products of combustion processes) were studied in soil chronic test (ERT). The species Enchytraeus crypticus and/or Enchytraeus albidus were used as the test organism and artificial soil as test substrate. The measured endpoints were reproduction (number of juveniles) and adult mortality. The results were expressed as ECx and LCx values or NOEC values, respectively. The test with short chain chlorinated paraffins demonstrated the very similar results for both enchytraeid species: LC 50 ( E.albidus, E.crypticus ) could not be estimated, EC 50 (E.albidus) = 6,027 mg/kg, EC 50 (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 experimental NOEC value for mortality and reproduction. The tests with N heterocyclic PAHs showed relatively the same order of toxic effects for all compound (LC 50 = 1,692 2,610 mg/kg ; EC 50 = 796 1,412 mg/kg). The toxicity (nominal concentration in mg/kg) decreased in the rank for mortality and reproduction: 1,10 phenanthroline > quinoline > phenazine ≥ acridine. After data recalculation on equilibrium pore water concentrations (ųmol/l), the reversed order of toxicity was obtained. These findings are in accordance with the data from literature describing a higher toxicity of more lipophilic compounds in aquatic tests with invertebrates 2.1.2. Introduction The persistent organic pollutants (POPs) are distributed in all matrices (soils, sediments, air, limnic, sea and underground waters, ice) world wide due to their long range atmospheric transport. They are stable in an environment and have tendency to acummulation and toxicity for aquatic and terrestrial organisms including human (UN ECE, 1994). From these reasons, they have been under a strong attention of many research teams. Some POPs have occured naturally in an environment but many of them have spread thanks to anthropogenic activities (production and using of industrial chemicals, drugs, pesticides, coal and waste burning, mining of coal, metals or oils, by products of industry and combustion products, accidents, etc.). POPs have usually tendency to sorption into the soil matter. Their high sorption is related to their structure, higher lipofilicity (log Kow), lower solubility in water and other physico chemical properties. On the other hand, to the mechanisms of their lossing from soils belong the anaerobic and aerobic microbial degradation, abiotic degradation, volatilisation, leaching into the groundwaters and uptake, bioaccumulation and metabolisation by terrestrial organisms (UNEP, 1999). Generally, heavy metals, polycyclic aromatic hydrocarbons (PAHs) and some selected pesticides (Lindane, Carbendazim) have been relatively frequently studied in enchytraeid ecotoxicology (see chapture 1.2.). However, some of organic pollutants (e.g. halogenated flame retardants, N,S,O heterocyclic aromatic hydrocarbons, pharmaceuticals) have not been studied so far and only a few of works about their toxic effects have been published (see Table 8 in the chapture 1.2.4.).
28 +++The experiments with some of the unexplored chemicals are introduced in this rigorous thesis: 1. Short chain chlorinated paraffins (industrial chemicals), 2. Toxaphene (pesticide), 3. Polyaromatic hydrocarbons with one or two nitrogen in place of carbon atom (by products of anthropogenic activities). The more information about the tested chemicals are described in the following lists: Short chain chlorinated paraffins (SCCPs) Short chain chlorinated paraffins (SCCPs) are synthetic chlorinated alkanes of chain lenght C 10 to C 13 inclusive . They have never been occured naturally in the environment. They have been produced since thirties last century and belongs to industrial chemicals using as flame retardants, additives in paints, coatings, adhesives, sealant materials or in a boot industry (NICNAS, 2001). They replaced the polychlorinated biphenyls (PCBs) in which the toxicity had confirmed (e.g. Johansson et al., 2001). Recently, short chain chlorinated paraffins were wrote up the list of prior persistent pollutants and the list of carcinogen chemicals (Europian Commision, 2002; UNEP, 2006). The physical chemical properties of SCCPs are affected by different scale of chlorination, its log Kow may ranged from 5 to 12.6 (Tommy et al., 1998). SCCPs are able to long distance atmospheric transport, strong sorption into solid matrices as soils, sediments or sewage sludges and have also bioaccumulation and toxicological potencial (Europian Commision, 2002). SCCPs levels were analysed in some European waters and sediments (EU RAR, 1999). The concentration reached up the tens mg/kg in sediments at some industrial localities or tens mg/kg of dry mass of sewage sludge from waste waters. Levels of SCCPs were evaluated by using the regional model in natural (10.8 g/kg) or in field and industrial soils (11.5 g/kg) (EU RAR, 2000). The toxicity of SCCPs to aquatic organisms is relatively well known (EU RAR, 2000). However, analogous to toxaphene, there is a limited data on the toxicity of SCCPs to soil enchytraeids because only one article about toxicity of short chain chlorinated paraffins and the other two chlorinated flame retardants to Enchytraeus crypticus has been publicated (Sverdrup et al., 2006). Toxaphene Toxaphene (CAS: 800 35 2) is pesticide (insecticide) consisting of more than 670 congeners, mainly polychlorinated bornanes. Its formula is C 10 H10 Cl 8 and is also known like camphechlor, chlorocamphene, polychlorcamphene or chlorinated camphene. Toxaphene belongs to prior persistent organic pollutants. Its industrial production started in the 1945 and was very frequently used in protection of cotton, fruit, vegetable, corn or as acaricid (ATSDR, 1998). It was applicated in concentration 1 4 kg/ha into some agricultural soils (HSDB, 2003). Toxaphene was also very often used in the last Czechoslovakia. It was the third most used pesticide in our Republic before it was banned. Pilot screening showed concentrations
29 below 1.6 ppb of toxaphene in arable or meadow soils with lower organic matter but the levels of toxaphene were detected up to 10 ppb in forest soils from Czech mountains contaminated due to the long distance atmospheric transport (Kosubová et al., 2003). Toxaphene distribution is world wide as well as SCCPs due to the mentioned air transport. It was detected in ice and fat of arctic animals far from the original sources (Bidlemann et al., 1989). Toxaphene is also very persistent in soil, its half life could up to 14 years (ATSDR, 1998). That's just it the using of toxaphene was banned in many countries due to its high persistence in an environment, accumulation and toxic effects (nephrotoxicity, neurotoxicity, hepatotoxicity, effects on reproduction, mutagenity, endocrine toxicity) for mammals, birds, fishes or the other aquatic organisms (UN ECE 1994; ATSDR, 1998). In addition, toxaphene is also possible carcinogen for human (Group 2B, IARC, 2001). However, toxaphene has never been studied under laboratory test design with soil invertebrates and the data about its possible toxicity to soil organisms are not available. N polycyclic aromatic hydrocarbons (NPAHs) Although most research on polycyclic aromatic compounds (PACs) focused on homocyclic compounds, two thirds of the known organic compounds are heterocycles (Adrian et Suflita, 1994). Nitrogen containing polycyclic aromatic hydrocarbons (NPAHs), in which one or more carbon atom has been replaced by a nitrogen atom, are such a family of heterocyclic compounds (Bleeker et al., 2003). Both, heterocyclic and homocyclic hydrocarbons tend to occur in a strong association in the environment due to same sources, in which belong incomplete combustion of fossil fuels, spills, refining, storage, wood preservation, fires (Adams et Giam, 1984), pesticide use (Kuhn and Suflita, 1989). In addition, NPAHs have been also occured naturally e.g. as alkaloids (Kaiser et al., 1996). NPAHs are ubiqitous and were detected in all matrices (Jones et al., 1989; Sanders et al. 1993; Kawamura et al., 1994; Van Genderen et al., 1994). However, concentrations of NPAHs have been predominantly measured in air (Wild and Jones, 1995, Chuang et al., 1991) or in waters and sediments (Pereira et al., 1987; van Gender et al., 1994; Osborne et al., 1997). Strong accumulation of these compounds was also described for soils (WHO, 2004), in which the concentrations of PAHs and NPAHs mixtures may reach several tens or hundreds mg/kg at industrial sites. The substitution of a nitrogen atom in the fused ring structure has a large effect on the physical/chemical properties of N heterocycles. NPAHs are, for instance, more polar and better soluble in water in comparison with their parental compounds (Dijkman et al., 1997; Pearlman et al., 2002). In spite of that NPAHs are present in the environment in levels up to 1 10 % of their homocyclic analogues (Nielsen et al., 1999), the toxic effects of both, heterocyclic and homocyclic compounds are comparable (Bleeker et al., 2003). The effects of heterocyclic aromatic hydrocarbons in water environment have been relatively well described (e.g. Bleeker, 1999; de Voogt et al., 1999; Feldmanová et al., 2006). In addition to narcosis, PACs can negatively influence the reproduction (Van Brummelen et al., 1996) and development (Bleeker et al., 2003) or evoke teratogenity (e.g. Millemann et Ehrenberg, 1982; Warshavsky, 1992, Burýšková et al., 2006). In addition, the PACs may be transformed some abiotic and biotic processes. Biotransformation usually lead to excretion of products from body but it was also found that some products of metabolisation may be more reactive and toxic than parental hydrocarbons (Warshavsky, 1992). It was also found that the UV radiation photoenhanced toxicity of some N heterocyclic compounds in the water environment (e.g. Bleeker et al., 2003). Further, the
30 genotoxic potencial of N heterocycles and their metabolits has been also recorded in the bacterial Mutatox test (e.g. Warshavsky, 1992; Bleeker, 1999) and some studies refered to carcinogenic activity of some N heterocyclic compounds (e.g. Hirao et al., 1976; Warshawsky, 1992). In contrast to aquatic toxicology, rare studies describing effects of several selected NPAHs were published for soil microorganisms (Sverdrup et al., 2002a), terrestrial plants (Sverdrup et al., 2003; Pašková et al., 2006), springtails (Sverdrup et al., 2001; Bleeker et al., 2003), earthworms (Sverdrup et al., 2002c), enchytraeids (Sverdrup et al., 2002b; Bleeker et al., 2003), and snails (Sverdrup et al, 2006). However, except the study of Pašková (Pašková et al., 2006), compounds containing only one nitrogen atom in place of in ring carbon atom (mainly acridine, quinoline and phenanthridine once time) have been tested in the mentioned studies.
Tested NPAHs
Some physico chemical properties of tested compounds (Quinoline, Acridine, Phenazine, 1,10 phenanthroline) are described in Table 13.
Table 13. The tested chemicals, their structure and their main physico-chemical properties: molecular weight (Mw), water solubility (Sw), octanol-water partitioning coefficient (log Kow), organic carbon-water partitioning coefficient (log Koc), soil-water partitioning coefficient for our artificial soil (Kd).
1 Sw 1 2 3 Chemical Structure Mw Log K ow Log K oc Kd (mg/l)
Quinoline N CAS: 99 22 5 129.16 6,110 2.03 1.74 2.41
N Acridine 179.22 38.40 3.40 2.85 33.98 CAS: 260 94 6
Phenazine N CAS: 92 82 0 180.21 16.00 2.84 2.40 12.06 N
1,10 Phenanthroline N 180.21 2,690 1.78 1.54 1.66 CAS: 66 71 7 N
1 PHYSPROP Database: http://www.syrres.com/esc/physdemo.htm 2 log K oc was estimated by formula: logK oc = 0.1 + 0.81 × log Kow (Sabljic et al., 1995) 3 Kd was calculated by formula: K d = K oc × f oc , where f oc is the fraction of organic carbon in our artificial soil (0.048 ).
31 2.1.3. Materials and methods Experimental soil Artificial soil (OECD, 1984) was used as experimetal soil in tests with all organic pollutants. This soil consist of 70 % sand, 20 % caoline clay, 10 % sphagnum peat (pH value was set to 6.0 ± 0.5 by CaCO 3). Species Enchytraeus crypticus Westheide & Graefe, 1992 and/or Enchytraeus albidus Henle, 1837 were used in the experiments. The enchytraeids are permanently maintained at RECETOX (Brno, Czech Republic) and during the time, the breeding and test condition of the both species were optimalized (Holubářová, 2004; Kozlová, 2006; Bezchlebová et Kobetičová, unpublicated). Finally, E. crypticus has been breeding in an artificial soil and E. albidus (original breedings from Germany) in a mix of garden substrate and arable soil. Dried oat flakes were added weekly on the cover of the breeding soil with both species. Chemicals and spiking procedure The following chemicals were used in the individual tests with enchytraeids: 1. Chlorinated paraffins (labeled as C12, 64 % chlorine content by weight; a viscous honey like liquid) providing by Novácké závody Inc. (Slovakia) as an industrial product, 2. Toxaphene standard (Sigma Aldrich, Ltd., Czech Republic), 3. Acridine standard (Sigma Aldrich, Ltd., Czech Republic), 4. Quinoline standard (Sigma Aldrich, Ltd., Czech Republic), 5. Phenazine standard (Sigma Aldrich, Ltd., Czech Republic), 6. 1,10 phenanthroline standard (Sigma Aldrich, Ltd., Czech Republic). All chemicals were dissolved in acetone (HPLC purity; Chromservis Inc., Czech Republic) to obtain the highest test concentrations and then the appropriate dilutions were prepared. The solutions were mixed with artificial soil (1 ml per a test vessel). The test vessels were kept in the fume hood for 24 hours to evaporate the solvent. Then, spiked and control soil was moistened by destilled water on 50 % of its WHC. Test design The range finding tests with all tested chemicals were done by Holubářová, the appropriate procedures and results are written in her diploma thesis (Holubářová, 2004). In terms of the results, the test concentration in final tests were proposed. The final test was performed according to norm (OECD, 2004). The differencies among the individual tests are described in Table 14. Five replicates for control (without chemical) + 5 replicates for acetone + 5 replicates for each concentration was prepared. At the start of test, dry oat flakes (food) was mixed with soil and 10 adults with clitellum were introduced into the test vessel. The test vessels were closed by lid and were stored by 20 ± 2 0C and under 16/8 light–dark cycle. The food was added every week into the soil. At the end of test, the worms were killed by ethanol (5 ml) and coloured by Bengal red. The next day, the survival of adults and number of juveniles were manually counted.
32 Table 14. The summary of toxicological tests with E. albidus or E. crypticus :test chemicals, enchytraeid species, test duration and test concentration. Chemical Species Test duration Test concentration (mg/kg) (days) SCCPs E. albidus 42 500; 1,000; 3,000; 6,000; 10,000 SCCPs E. crypticus 28 500; 1,000; 3,000; 6,000; 10,000 Toxaphene E. albidus 42 10; 25; 50; 100; 250; 500;1,000 4 individual NPAHs E. crypticus 28 100; 500;1,000; 1,500; 2,000; 2,500 Statistical analyses Program STATISTICA 6.0 software ( StatSoft, Inc., 2004 ) was used for data evaluation. No observed effect concentration (NOEC) was determined by analysis of variance (ANOVA) and Dunnett’s procedure at a 5% significant level. The concentration at which x% of adult survival was observed (LC x) and x% effect concentration for the reproductive output (EC x) were calculated according to Haanstra et al. (Haanstra et al., 1985) using logistic regression analysis. 2.1.4. Results and discussion Short chain chlorinated paraffins The results indicate that SCCPs did not affect enchytraeid survival for species Enchytraeus albidus . The 30% mortality was occured in test with E. crypticus. From this reason, LC 50 values could not be estimated. Reproduction was more sensitive endpoint than survival of adults. The EC 50 = 6,027 (3,576 8,478) mg/kg and 7,809 (4,381 11,237) mg/kg were estimated for E. albidus and E. crypticus , respectively.
SCCPs - E. albidus
10 50 8 40 n n n n o o o o i i i i l l l l t t 6 30 t t a a a a c c c c v v v v i i u u i i u u v v v v d d d d r r r r 4 20 o o o o u u u u r r r r p p p p S S S S e e 2 10 e e R R R R 0 0 0 500 1000 3000 6000 10000
Concentration mg/kg ( ( ) ) ( ( ) ) Survival Number of juveniles
Figure 1 . Toxicity test with Short chain chlorinated paraffins (SCCPs) and enchytraeid species E. albidus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates .
33 In conclusion, the used species were similarly low sensitive to SCCPs exposure in the artificial soil. In addition, the results are in a good accordance with the data from literature (Sverdrup et al., 2006a). They tested SCCPs in an agricultural soil (1.6 % of organic carbon) with species E. crypticus . Similarly, the highest test concentration (1,000 mg/kg) has not any effect on enchytraeid survival and reproduction in their test (Sverdrup et al., 2006a). SCCPs - E.crypticus
10 500 8 400 n n n n o o o o i i i i l l l l t t t 6 300 t a a a a c c c c v v v v u u i i u u i i v v v v d d d d r r r r 4 200 o o o o u u u u r r r r p p p p S S S S e e e 2 100 e R R R R 0 0 0 500 1000 3000 6000 10000
Concentration mg/kg ( ( ) ) ( ( ) ) Survival Number of juveniles
Figure 2 . Toxicity test with Short chain chlorinated paraffins (SCCPs) and enchytraeid species E. crypticus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates . Its possible that low toxicity of SCCPs to enchytraeids is influenced by their relatively high molecule (Mw = 320 500), their low water solubility (0.15 0.47 mg/l) and high sorption into soil matter. In any case, SCCPs most probably does not cause negative effects for enchytraeids in soils. Toxaphene The results of test with toxaphene are showed in the figure 3. Adult survival and reproduction (number of juveniles) were not markedly affected in the highest test concentration and LC 50 /EC 50 values could not be estimated. Nevertheless, the NOEC values (620 mg/kg) for both the endpoints were estimated by ANOVA analysis at least. The species Enchytraeus albidus was similarly unsensitive to tested pesticide as well as the second enchytraeid species E. crypticus (see Table 2 in the Appendix II.)(the toxaphene test with E. crypticus was made by Dr. Bezchlebová and from this reason, the results from this experiment are not presented in the chapture „Results and disccusion“ of my rigorous thesis but they are only included in the appropriate article – see Appendix II.). Unfortunatelly, there are no literature data on toxaphene toxicity to soil enchytraeids under laboratory test condition and hence, the data on enchytraeids are compared with the results from the other invertebrate tests from the mentioned article.
34 Toxaphene - E. albidus
10 50
8 40
6 30 4 20 urvival S
survival number of juveniles eproduction
2 10 R
0 0 0 10 25 50 100 250 500 1000
Concentration mg/kg ( ( ) ) ( ( ) ) Figure 3 . Toxicity test with Toxaphene and enchytraeid species E. albidus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates . The both enchytraeids species were unsensitive to toxaphene exposure in comparison to the other test invertebrates as springtails, earthworms or nematodes (see Appendix II.). The sensitivity of invertebrates decresing in the order: springtails > earthworms > nematodes > enchytraeids. The possible explanation for the results could be: 1. Toxaphene is an insecticide with more specific mode of toxic action (neurotoxicity) and that is why springtails (Arthropoda: Hexapoda) could be more sensitive than worms. 2. It has been shown that arthropods are able to metabolise pollutants better than soil worms (van Brummelen and van Straalen, 1996) which have higher tendency to bioaccumulation (Jager et al., 2000) and biotransforming animals could be more sensitive to than bioaccumulating ones (Van Straalen, 2004). 3. Lower bioavailability of low soluble toxaphene to soil enchytraeids via soil pore water however, these possibilities did not explain the higher toxicity of toxaphene to earthworms or nematodes. The sensitivity of nematodes would have been decreased if we had used the OECD soil in nematode test as well as in enchytraeid test. The used natural soil with the lower content of organic matter and different design of nematode test could finally increased bioavailability and toxicity of toxaphene to nematode worms in comparison to enchytraeids in OECD soil with a higher content of organic matter (see Table 2 in Appendix II.). On the other hand, the various toxicity betwen oligochaetes (earthworms versus enchytraeids) could not be explained satisfactory. In some studies (Römbke et Moser, 2002), the sensitivity of earthworms and enchytraeids to various chemicals was comparable but in the others, earthworms were more sensitive than their smaller relatives (Sverdrup et al., 2002b). There, one explanation is maybe disposable: smaller enchytraeids are in very close contact with soil pore water similarly as nematodes (Achazi et van Gestel, 2003) but bigger earthworms may uptake more toxaphene via ingestion of contaminated soil particles and food (horse manure) due to a higher sorption of toxaphene into solid phase (organic matter) in comparison to soil pore water. These hypothesis are partly supported by a field study, where the decreasing of abundance was
35 attributed to indirect effect via lack of food (this aspect was not relevant in our study because all organisms had enough food during the individual test periods) or toxaphene exposure via ingestion or a sensitivity of studied organisms (Bäumler et al., 1978 in Didden et Römbke, 2001). N polycyclic aromatic hydrocarbons (NPAHs) The dose response curves were different for the individual compounds (see Figure 4 7). The effect of compounds on adult survival was in the same order of magnitude, nevertheless their toxicity decreased in the rank: 1,10 phenanthroline > quinoline ≥ phenazine ≥ acridine. The reproduction was more sensitive endpoint than survival of adults. The toxicity of compounds decreased nearly in the same rank as at survival: 1,10 phenanthroline ≥ quinoline > phenazine > acridine. The LC 50 and EC 50 values are described in the Table 15. Table 15 . Summary results of the toxicity of NPAHs: survival (LC 50 ) and reproduction (EC 50 ) of soil invertebrates and their 95% confidence intervals (in parentheses). Compound LC 50 (mg/kg)* LC 50 ( mol/l)** EC 50 (mg/kg)* EC 50 ( mol/l)** Quinoline 2093 6.72 990 3.18 (2012 - 2173) (744 1237) Acridine 2610 0.43 1412 0.24 (2189 - 3031) (1187 1638) Phenazine 2488 1.15 1073 0.50 (2377 - 2599) (916 1229) Phenanthroline 1692 5.66 796 2.66 (1433 - 1951) (653 939) * values based on nominal concentrations (mg/kg soil dry weight). ** LC 50 and EC 50 values recalculated on concentrations in soil pore-water according to equilibrium partitioning method: L(E)Cwater (mmol/L) = L(E)Csoil (mg/kg) / Mw / Kd (Di Toro et al., 1991). Quinoline - E.crypticus 10 1000 n n n n o o o o l l l l i i i 8 800 i t t t t a a a a c c c c v v v v i i i i u u u 6 600 u v v v v d d d d r r r r o o o o u u u u r r r 4 400 r S p p S S p S p e e e e R R R 2 200 R 0 0 0 100 500 1000 1500 2000 2500 Concentration mg/kg ( ( ) ) ( ( ) ) Adult survival Reproduction
Figure 4. Toxicity test with Quinoline and enchytraeid species E. crypticus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates .
36 Acridine - E.crypticus 10 500 n n n n o o o o i i i 8 400 i t t t t l l l l c c c c a a a a u u u u v v v v d i d i d i d i 6 300 o v o v o v o v r r r r r r r r p u p u p u p u 4 200 e e e e S S S S R R R R 2 100 0 0 0 100 500 1000 1500 2000 2500 Concentration mg/kg ( ( ) ) ( ( ) ) Adult survival Reproduction Figure 5. Toxicity test with Acridine and enchytraeid species E. crypticus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates . Phenazine - E.crypticus 10 500 n n n n o o o o i i i 8 400 i l l l l t t t t a a a a c c c c v v v v u u u u i i i i 6 300 d d d d v v v v r r r r o o o o r r r r u u u u 4 200 p p p p S S S S e e e e R R R 2 100 R 0 0 0 100 500 1000 1500 2000 2500 Concentration mg/kg ( ( ) ) ( ( ) ) Adult survival Reproduction Figure 6. Toxicity test with Phenazine and enchytraeid species E. crypticus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates .
37 Phenanthroline - E.crypticus 10 500 n n n n o o o o i i i 8 400 i t t t t l l l l c c c c a a a a u u u 6 300 u v v v v d d d d i i i i o o o o v v v v r r r r r r r r p p p 4 200 p u u u u e e e e S S S S R R R 2 100 R 0 0 0 100 500 1000 1500 2000 2500 Concentration mg/kg ( ( ) ) ( ( ) ) Adult survival Reproduction Figure 7. Toxicity test with 1,10 phenanthroline and enchytraeid species E. crypticus . The results (adult mortality and reproduction = number of juveniles) are expressed as mean values ± standard error (SE), n = 5 replicates . The effects of phenazine and 1,10 phenanthroline on soil enchytraeids have never been studied. Thus, comparison with the literature data is possible only for acridine and quinoline, which have been tested in two studies among some other PAHs (Sverdrup et al. 2002b; Bleeker et al., 2003). It was found that acridine has lower toxic effects than quinoline (Bleeker et al., 2003) and this is in agreement with our data (compare Table 15 and 16). Toxicity described in this holand work (Bleeker et al., 2003) was also higher than toxicity in our study. This might be related to the lower organic matter content in natural soil using in Bleker´s study. Surprisingly, the results from the second study (Sverdrup, 2002b) were relatively similar to ours but reason for that remains unclear (toxicity from this study should have been the highest in comparison with Bleker´s and our results due to the lowest organic carbon content of agricultural soil see Table 16). Table 16. Initial soil concentrations (mg/kg) of acridine and quinoline at which 50% mortality (LC 50 ) and 50% inhibition of reproduction in comparison to control (EC 50 ) with 95% confident intervals for E.crypticus ; origin and content of organic carbon (%) of tested soil and literature sources. Species Compound LC 50 EC 50 Soil Source (95%CI) (95%CI) (origin, %OC) (mg/kg) (mg/kg) E. crypticus Acridine 3,600 1,500 Agricultural, Sverdrup (2,700 4,000) (1,200 1,600) 1.6 % OC (2002b) E. crypticus Acridine 904* 172 904* LUFA 2.2, Bleeker et al. 2.3 % OC (2003) E. crypticus Quinoline 526* 285* LUFA 2.2, Bleeker et al. (243 810) (257 313) 2.3 % OC (2003) * the original data are expressed in mol/kg (Bleeker et al. 2003). It is very well known that only bioavailable part of nominal concentration (mg/kg) applied to soil could cause toxicity to soil organisms. The bioavailability of compounds is influenced by many factors as structure of chemicals, physico chemical properties of soil and
38 surrounding environment, ageing and sensitivity of the test species (e.g. Lanno, 2003). No less importance have also the exposure pathways for the organism (uptake of chemical in or on an organism). Enchytraeids could be exposed via contaminated food or soil pore water whereas contamination through a gase phase is mostly neglectable (Achazi et van Gestel, 2003). The pore water exposure is treated as the most important route because oligochaete worms are in close contact with soil solution. The exposure via contaminated food raises significance rather for compounds with higher lipophilicity (Log Kow over 5.5) (Achazi et van Gestel, 2003). All organic compounds are able to cause narcosis and many studies have shown that narcosis is strongly related to the lipophilicity of the compound, expressed as the n octanol water partitioning coefficient = Log Kow (e.g. Chen et al., 1997). These conclusions were confirmed in aquatic as well as terrestrial environment (e.g. Sverdrup et al., 2002c; Bleeker et al., 2003). The effective concentration from soil tests of toxicity has been more often overestimated on effective concentration in soil pore water according to the formula: L(E)C water (mmol/L) = L(E)C soil (mg/kg) / Mw / Kd, where: L(E)C water (mmol/L) = effective concentration in soil pore water, L(E)C soil (mg/kg) = nominal effective concentration in soil, Mw = molecular weight, Kd = partitioning coefficient soil water (overtaken from Sverdrup et al., 2002c). Theoretically, this concentration express the concentration of compound in soil pore water that may cause the x% effect in soil. However, the using of this method assumes that organisms are exposed exclusively via pore water in soils. The other disadvantage is that the final value ( LC or EC (mmol/L) ) is strongly influenced by Kd value of the test soil. This aspect makes comparison the experimental data with the data from literature more difficult because the coefficients often differs in orders of magnitude due to the various physico chemical properties of used soils and different values of experimental or estimated Log Koc (e.g. Doucette, 2003). In my study, the recalculated L(E)C water ( mol/L) values showed the opposite toxicity in comparison to primary results from the tests. Acridine was the most toxic compounds followed by phenazine, quinoline and the least toxic compound seemed to be 1,10 phenanthroline (see Table 15). It is obvious that the more lipophilic compounds may potencially cause the higher toxicity as well as in the aquatic tests (e.g. Bleeker et al., 2003). The similar conclusions were found also for soil nematodes in aquatic test of toxicity (Sochová et al., in press). Nematode species Caenorhabditis elegans was sensitive to tested compounds in this order (from the most toxic to the least toxic compound): acridine > phenazine > 1,10 phenanthroline > quinoline. On the other hand, the nematode LC50 values (Sochová et al., in press) were one or two orders of magnitude higher than the overestimated LC50 values for enchytreids in my tests. This could be caused the different design of the both tests (duration of test, species sensitivity, water or soil medium, etc.). The EC 10 = 1.77 ( mol/L) from the other test with E. crypticus (Sverdrup et al., 2002b) showing lower toxicity for acridine in their Danish natural soil in comparison with toxicity in artificial soil from my study. However, the comparison of my data with the ones from acute aquatic tests with Chironomus riparius (Bleeker et al., 2003) showed almost the same toxicity for acridine (LC 50 value = 0.4 mol/L) (compare with Table 15) and about one order of magnitude higher for quinoline (LC 50 value = 37.9 mol/L) for larvae of chironomids (Bleeker et al., 2003). Their results of toxicity for Chironomus riparius were also very well in accordance with the
39 EC 50 values for E. crypticus in pore water in LUFA 2.2 soil from the same study (Bleeker et al., 2003). On the other hand, the toxicity from acute or chronic aquatic tests with daphnids showed higher differencies in toxicity (one order of magnitude lower, higher or in the same order of magnitude) as for enchytraeids for different NPAHs (Feldmannová et al., 2006). In addition, these findings are relevant for narcosis but NPAHs are thanks to incorporation of nitrogen atom candidates also for the more specific mode of toxic action. Nevertherless, NPAHs indicated rather narcosis to soil invertebrates in the present available studies (Sverdrup et al., 2002b,c; Bleeker et al., 2003).
40 2.1.5. References Adams, J. and Giam, C. S. (1984): Polynuclear azaarenes in wood preservative wastewater. Environmental Science and Technology, 18: 391 394. Adrian, N. R., Suflita, J. M. (1994): Anaerobic biodegradation of halogenated and nonhalogenated N , S , and O heterocyclic compounds in aquifer slurries. Environmental Toxicology and Chemistry, 13: 1551 1557. ATSDR (1998): Toxicological profile for toxaphene. Atlanta, GA, Agency for Toxic Substances and Disease Registry. Centre for Desease Control and Prevention. Bidleman, T. F., Patton, G. W., Walla, M. D., Hargrave, B. T., Vass, W. P., Erickson, P., Fowler, B., Scott, V., Gregor, D. J. (1989): Toxaphene and other organochlorines in Arctic Ocean fauna: evidence for atmospheric delivery. Arctic, 42: 307–313. Bleeker, E. A. J. (1999): Toxicity of Azaarenes: Mechanisms and Metabolism. Disertation thesis. Universiteit van Amsterdam, Amsterdam, The Netherlands. 145 pp. Bleeker, E. A. J., Wiegman, S., Droge, S. T. J., Kraak, M. H. S., van Gestel, C. A. M. (2003): Chronic toxicity of PACs to Folsomia candida and Enchytraeus crypticus. In: Towards an improvement of the risk assessment of polycyclic (hetero)aromatic hydrocarbons. Report 2003 01 of the department of Aquatic Ecology and Ecotoxicology, University of Amsterdam, Amsterdam, The Netherlands. Report 2003 04 of the Institute of Ecological Science, Vrije Universiteit, Amsterdam, The Netherlands: 27 32. Burýšková, B., Hilscherová, K., Bláha, L., Maršálek, B., Holoubek, I. (2006): Toxicity and modulations of biomarkers in Xenopus laevis embryos exposed to polycyclic aromatic hydrocarbons and their N heterocyclic derivatives. Environmental Toxicolology, 21: 590 1598. Chen, J. , Wang, L., Lu, G., Zhao, T. (1997): Quantitative structure activity relationship studies of selected heterocyclic nitrogen compounds. Bulletin of Environmentall Contamination, and Toxicollogy, 58: 372 379. Chuang, J. C., Mack, G. A., Kuhlman, M. R., Wilson, N. K. (1991): Polycyclic aromatic hydrocarbons and their derivatives in indoor and outdoor air in an 8 home study. Atmospheric Environment Part B Urban Atmosphere, 25: 369 380. de Voogt, P., Bleeker, E. A. J., van Vlaardingen, P. A. L., Fernandéz, A., Slobodník, J., Wever, H., Kraak, M. H. S. (1999): Formation and identification of azaarene transformation products from aquatic invertebrate and algae metabolism. Journal of Chromatography B: 265 274. Dijkman, N. A., Van Vlaardingen, P. L. A., Admiraal, W. A. (1997): Biological variation in sensitivity to n heterocyclic PAHs, effects for acridine on seven species of micro algae. Environmentall Pollution, 95 (10): 121 126. EU RAR (1999): Alkanes, C10 13, Chloro, CAS No.:85535 84 8, EINECS No.: 287 476 5. European Union Risk Assessment Report Publication: EUR 19010 EN. European Chemicals Bureau. Web site: http: //ecb.jrc.it/DOCUMENTS/ExistingChemicals/RISK ASSESMENT/REPORT/sccpreport010.pdf. European Commission (2000): European Commission Joint Research Centre. European Union Risk Assessment Report. Vol. 4: alkanes, C10–13, chloro . European Chemicals Bureau, Brussels, Belgium: 166 pp. Feldmannová, M., Hilscherová, K., Maršálek, B., Bláha, L. (2006): Effects of N heterocyclic polyaromatic hydrocarbons on survival, reproduction, and biochemical parameters in Daphnia magna. Environmental Toxicolology, 21: 425 431. Gejlsbierg, B., Klinge, C., Samsoe Petersen, L., Madsen, T. (2001): Toxicity of linear alkylbenzene sulfonates and nonylphenol in sludge amended soil. Environmental Toxicology and Chemistry, 20 (12): 2709 2716.
41 Haanstra, L., Doelman, P., Voshaar, J. H. O. (1985): The use of sigmoidal dose–response curves in soil ecotoxicological research. Plant Soil, 84: 293–297. Hirao, K., Shinohara, Y., Tsuda, H., Fukushima, S., Takahashi, M., Ito, N. (1976): Carcinogenic activity of quinoline on rat liver. CANCER RES, 36: 329 335. Holubářová, B. (2004): Testování ekotoxického vlivu POPs na půdní roupice (Enchytraeidae, Oligochaeta). Diplomová práce, PřF, MU, Brno: 70 pp. HSDB (2001): Toxaphene toxicology data file number 1616 in Hazardous Substances Databank (HSDB). Toxicology data network TOXNET. National Medicine Library (NML). IARC (2001): Toxaphene. World Health Organisation, International Agency for Research of Cancer, Geneva, Switzerland. Jager, T.,.Sanchez, F. A. A., Muijs, B., van der Welde, E. G., Posthuma, L. (2000): Toxicokinetics of polycyclic aromatic hydrocarbons in Eisenia andrei (Oligochaeta) using spiked soil. Environmental Toxicology and Chemistry, 19: 953 961. Johansson, N., Hanberg, A., Bergek, S., Tysklind, M. (2001): PCB in sealant is influencing the levels in indoor air. Organohalogen Compounds, 52: 436 440. Jones, K. C., Stratford, J. A., Tidridge, P., Waterhause, K. S., Johnston, A. E. (1989): Polynuclear aromatic hydrocarbons in an agricultural soil; long term changes in profile distribution. Environmental Pollution, 56: 337 351. Kaiser, J. P., Feng, Y., Bollag, J. M. (1996): Microbial metabolism of pyridine, quinoline, acridine, and their derivates under aerobic and anaerobi conditions. Mikrobiology review, 60: 483 498. Kawamura, K., Suzuki, I., Fujii, Y., Watanabe, O. (1994): Ice core record of polycyclic aromatic hydrocarbons over the 400 years. Naturwissenschaften, 81: 502 505. Kosubová, P., Grabic, R., Holoubek, I. (2003): (HR)GC MS/MS analysis of toxaphene congeners in various matrices from the Czech environment. Fresenius´ Environmental Bulletin, 12: 1303 1308. Kozlová, K. (2006): Ekotoxicita vybraných perzistentních organických polutantů v půdách ve vztahu k jejich biodostupnosti. Diplomová práce, PřF, MU, Brno: 83 pp. Kraak, M. H. S., Ainscough, C., Fernández, A., van Vlaardingen, P. A. L., de Voogt, P., Admiraal, W. (1997): Short term and chronic exposure of the zebra mussel ( Dreissena polymorpha ) to acridine : effect and metabolism. Aquatic toxicology, 37: 9 20. Kuhn, E. P. and Suflita, J. M. (1989): Microbial degradation of nitrogen, oxygen and sulphur heterocyclic compounds under anaerobic conditions. Environmental Toxikology and Chemistry, 8: 1149 1158. Millenmann, R. E., Ehrenberg, D. S. (1982): Chronic toxicity of the azaarene quinoline, a synthetic fuel component, to the pond snail Physa gyrina. Environmental Technology Letters, 3 (5):193 198. NICNAS (National Industrial Chemicals Notification And Assessment Scheme) (2001): Short chain chlorinated paraffins (SCCPs). Priority Existing Chemical Assessment Report No. 16. ISBN 0 642 70983 1. Nielsen, T., Feilberg, A., Binderup, M. L. and Tonnesen, J. (1999): Impact of Regulations of traffic Emissions on PAH Level in the Air. Environmental project report no. 447, Danish Environmental Protection Agency. OECD (Organisation for Economic Co operation and Development) (1984): Earthworm, acute toxicity tests. OECD guideline for testing of chemicals, No. 207. OECD. Earthworm toxicity tests, Paris, France. OECD (Organisation for Economic Co operation and Development) (2004): Test no. 220. Enchytraeid Reproduction Test. OECD Guidelines for the Testing of Chemicals, Organisation for Economic Co operation and Development.
42 Pašková, V., Hilscherová, K., Feldmannová, M., Bláha, L. (2006): Toxic effects and oxidative stress in higher plants exposed to polycyclic aromatic hydrocarbons and their N heterocyclic derivatives. Environmental Toxicology and Chemistry, 25: 3238 3245. Pearlman, R. S., Yalkowsky, S. H., Banerjee, S. (1984): Water solubility of polynuclear aromatic and heteroatomic compounds. J. Phys. Chem. Ref. Data, 13: 555 562. Pereira, W.E., Rostad, C.E., Garbarino, J.R., Hult, M.F., 1983. Groundwater contamination by organic bases derived from coal tar wastes. Environmental Toxicology and Chemistry, 2: 283 294. Römbke, J., Moser, T. (2002): Validating the enchytraeid reproduction test: organisation and results of an international ringtest. Chemosphere, 46: 1117 1140. Sanders, G., Jones, K. C., Hamilton Taylor, J., Dorr, H. (1993): Concentrations and deposition fluxes of polynuclear aromatic hydrocarbons and heavy metals in the dated sediments of a rural English lake, Environmental Toxicology and Chemistry, 12: 1567 1581. Sochová, I.; Hofman, J.; Holoubek, I. (in press): Effects of seven organic pollutants on soil nematode Caenorhabditis elegans . Environment International. StatSoft I. (2004): STATISTICA (data analysis software system), version 6 /http://www.statsoft.com . Sverdrup, L. E., Ekelund, F., Krogh, P. H., Nielsen, T., Johnsen, K. (2002a): Soil microbial toxicity of eight polycyclic aromatic compounds: Effects on nitrification, the genetic diversity of bacteria, and the total number of protozoans. Environmental Toxicology and Chemistry, 21: 1644 1650. Sverdrup, L. E., Hartnik, T., Mariussen, E., Jensen, J. (2006a): Toxicity of three halogenated flame retardants to nitrifying bacteria, red clover (Trifolium pratense), and a soil invertebrate (Enchytraeus crypticus). Chemosphere, 64: 96 103. Sverdrup, L.E., Jensen, J., Kelley, A.E., Krogh, P.H., Stenersen, J. (2002b): Effects of eight polycyclic aromatic compounds on the survival and reproduction of Enchytraeus crypticus (oligochaeta, clitellata). Environmental Toxicology and Chemistry, 21: 109 114. Sverdrup, L. E., Krogh, P. H., Nielsen, T., Kjaer, C., Stenersen, J. (2003): Toxicity of eight polycyclic aromatic compounds to red clover (trifolium pratense), ryegrass (lolium perenne), and mustard (sinapsis alba). Chemosphere, 53: 993 1003. Sverdrup, L. E., Krogh, P. H., Nielsen, T., Stenersen, J. (2002c): Relative sensitivity of three terrestrial invertebrate tests to polycyclic aromatic compounds. Environmental Toxicology and Chemistry, 21: 1927 1933. Sverdrup, L.E., De Vaufleury, A., Hartnik, T., Hagen, S. B., Loibner, A. P., Jensen, J. (2006b): Effects and uptake of polycyclic aromatic compounds in snails ( Helix aspersa ). Environmental Toxicology and Chemistry, 25: 1941 1945. Tomy, G. T., Fisk, A. T., Westmore, J. B., Muir, D. C. G. (1998): Environmental chemistry and toxicology of polychlorinated n/alkanes. Review Environmental Contamination and Toxicolology, 158: 53–128. UN ECE (1994): United Nations Economic Commision for Europe. Descriptions of the Substances Identified in the EN ECE LRTAP Conventoin 1994: Task Force on Persistent Organic Polutants. UNEP (2006): Summary of short chained chlorinated paraffins proposal. United Nations Environment Programme/POPS/POPRC.2/14. Stockholm Convention on Persistent Organic Pollutants, Persistent Organic Pollutants Review Committee, Second meeting, Geneva, Switzerland: 6 10..
43 van Genderen, J., Noij, T. H. M., van Leerdam, J. A. (1994): Inventory and toxicological evaluation of organic micropollutants. RIWA report, Association of Rhine and Meuse Water Supply Companies (RIWA), Amsterdam, The Netherlands. van Straalen, N. M. (2004): The use of soil invertebrates in ecological survey of contaminated soils. In: Vital Soil Function, Value and Properties. Developments in Soil Science, 29: 159 197. Warshawsky, D. (1992): Environmental sources, carcinogenity, mutagenicity, metabolism and DNA binding of nitrogen and sulphur heterocyclic aromatics. Journal of Environmental Science and Health C, 10: 1 71. Wild, S. R., Jones, K. C. (1995): Polynuclear aromatic hydrocarbons in the united Kingdom environment – a preliminary source inventory and budget. Environmetal Pollution, 88: 91 108. WHO (2004): Concise International Chemical Assessment Document 62: COAL TAR CREOSOTE. World Health Organisation, Geneve, Switzerland.
44 3. Conclusions
45 3. Conclusions The existing laboratory tests of toxicity with enchytraeids in water, agar and soil medium were introduced in this rigorous thesis. All these tests have some advantages and disadvantages and differ in their using (study of reproduction, lethality, behavioral changes, biomass, physiological and morphological symptoms, etc.) for an assessment of individual chemicals or mixtures and or for a soil quality assessment (in case of soil tests). The toxicity of six organic pollutants (SCCPs, Toxaphene, four NPAHs) with not very known toxic potencial to soil organisms were studied in the experimental part of rigorous thesis. All of them were tested in chronic test (ERT) with artificial soil under standard condition. The results suggested the different toxicity of pollutants depending on their structure and properties. The toxicity is described by NOEC, LCx or ECx values and discussed with available literature. The results were also presented on several scientist meetings and mainly, they are a part of three accepted/submitted scientist papers about the toxicity of these organic pollutants on soil organisms (Ecotoxicology and Environmental Safety, ELSEVIER).
46 4. Appendices
47 Appendices Appendix I. 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 II. 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 III. Kobetičová, Klára , Bezchlebová, Jitka, Sochová, Ivana, Lána, Jan, Hofman, Jakub. Toxicity of four polycyclic aromatic nitrogen heterocycles (PAHs) to soil organisms. Ecotoxicology and Environmental Safety, submitted.
48