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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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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.

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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 (LCx) and x% effect concentration for the reproductive output (ECx) 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, LC50 values could not be estimated. Reproduction was more sensitive endpoint than survival of adults. The EC50 = 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.

( ) 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).

( ) 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.


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 LC50/EC50 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.

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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 LC50 and EC50 values are described in the Table 15.

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4 400 2 200 0 0 0 100 500 1000 1500 2000 2500 ( ) 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 10 500

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) 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 10 500

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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).

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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-octanolwater 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:

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L(E)Cwater (mmol/L) = effective concentration in soil pore-water, L(E)Csoil (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 porewater 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)Cwater ( 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 1,10phenanthroline (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 EC10 = 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 (LC50 value = 0.4 mol/L) (compare with Table 15) and about one order of magnitude higher for quinoline (LC50 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 EC50 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).

402.1.5. 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.

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