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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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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: LC50 (E.albidus, E.crypticus) could not be estimated, EC50 (E.albidus) = 6,027 mg/kg, EC50 (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 (LC50 = 1,692-2,610 mg/kg ; EC50 = 796mg/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.).

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1. Short-chain chlorinated paraffins (industrial chemicals),

2. Toxaphene (pesticide),

3. Polyaromatic hydrocarbons with one or two nitrogen in place of carbon atom (byproducts 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 C10 to C13 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 C10H10Cl8 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).

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1 PHYSPROP Database: http://www.syrres.com/esc/physdemo.htm 2 log Koc was estimated by formula: logKoc = 0.1 + 0.81 × log Kow (Sabljic et al., 1995) 3 Kd was calculated by formula: Kd = Koc × foc, where foc is the fraction of organic carbon in our artificial soil (0.048).

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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 CaCO3).

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.

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