«RECETOX Research Centre for Environmental Chemistry and Ecotoxicology Laboratory tests of toxicity with enchytraeids RIGOROUS THESIS Brno, 2007 MSc. ...»
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
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).
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
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).
111.1.2. 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/m2, 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).
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).
131.2.1. 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 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).
Table 4. Overview of agar test on enchytraeids.