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

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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).

151.2.3. 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).

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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).

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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).

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1.2.5. Avoidance test The first study about using of enchytraeids in an Avoidance test came up in the mids (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).

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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.

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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 foodchain (see Schema 5):

Cl

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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.

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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.

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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 14C-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.



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