Heterocyclic Compounds: Toxic Effects Using Algae, Daphnids and the Salmonella/Microsome Test Taking Methodical Quantitative Aspects into Account

Abstract

Heterocyclic aromatic hydrocarbons containing nitrogen, sulfur, or oxygen (NSO-HET), have been detected in air, soil, sewage sludge, marine environments, and freshwater sediments. Since toxicity data on this class of substances are scarce, the present study focuses on possible implications NSO-HET have for ecotoxicity (algae and daphnids) and mutagenicity (Salmonella/microsome test). A combination of bioassays and chemical-analytical quantification of the test compounds during toxicity assays should aid in determination of the hazard potential. Samples of the test concentrations of 14 NSO-HET were taken at the beginning and end of the bioassays; these samples were then quantified by high-performance liquid chromatography. The toxicity potential of the substances was evaluated and compared with the toxicity calculated with the nominal concentrations. Significantly different results were obtained primarily for volatile or highly hydrophobic NSO-HET. The concentration of heterocyclic hydrocarbons can change significantly during the algae and Daphnia test. The EC50 values (effective concentration value: the concentration of a chemical that is required to produce a 50% effect) calculated with the nominal concentrations underestimate the toxicity by a factor of up to 50. Prioritizing the tested compounds according to toxicity, the mutagenic and toxic compounds quinoline, 6-methylquinoline, and xanthene have to be listed first. The greatest ecotoxic potential on algae and daphnids was determined for dibenzothiophene followed by acridine. In the Daphnia magna immobilization test, benzofuran, dibenzofuran, 2-methylbenzofuran, and 2,3-dimethylbenzofuran and also carbazole are ecotoxicologically relevant with EC50 values below 10 mg/L. These substances are followed by indole with a high ecotoxic effect to daphnids and less effect to algae. Only minor toxic effects were observed for 2-methylpyridine and 2,4,6-trimethylpyridine.

Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

“…Eisenträger et al [12] found that out of the tested substances (similar to the set tested here) only quinoline, 6-methylquinoline and xanthene caused mutagenic effects in the Ames assay with Salmonella typhimurium, while these effects were only apparent in treatments with metabolic activation, i.e. when supplemented with rat liver S9.…”

“…Carbazole was demonstrated to be clastogenic by Jha et al [48] but was not mutagenic in the Ames assay [49] or carcinogenic in mice [43]. The O-heterocyclic compound benzofuran showed very interesting effects, although being negative in the Ames assay [12], [50]; DNA damage, as measured with the Comet assay, as well as the formation of micronuclei was higher in female specimens, both in vivo in rat kidney and in vitro using primary rat and human kidney cultures [51]. Dibenzofuran, a substance that was previously reported not to be genotoxic in a mammalian system was identified to be potent genotixicant in the fish system [52].…”

“…Acridine was shown to be positive in the SOS chromotest with Escherichia coli K12 and a yeast-based reporter-gene assay in which DNA damage induces the expression of green fluorescent protein (GFP) via the RAD54 promoter [45]. It was not mutagenic in the Ames assay [12], [46] but produced clastogenic effects detected with the yeast DEL assay without metabolic activation [47]. Carbazole was demonstrated to be clastogenic by Jha et al [48] but was not mutagenic in the Ames assay [49] or carcinogenic in mice [43].…”

“…The biological effects so far cover, e.g. toxicity to Daphnia and green algae, mutagenicity in the Ames assay, embryotoxicity to embryos of the zebrafish ( Danio rerio ) or effects mediated by the aryl hydrocarbon and retinoid receptor, respectively [12], [13], [14], [15], [16], [17], [18].…”

“…Furthermore, it is well-accepted that a substance’s potential to induce micronuclei represents an important toxicological effect with potential adverse effects on the population level [21], [27], [28]. The substances tested were chosen to match the set of compounds identified earlier by the project framework KORA (retention and degradation processes to reduce contaminations in groundwater and soil) [12], [29] and comprised indole, 1-benzothiophene, benzofuran, 2-methyl benzofuran, 2,3-dimethyl benzofuran, quinoline, 6-methyl quinoline, carbazole, dibenzothiophene, dibenzofuran, acridine, and xanthene. To account for the dissipation of the compounds from the test medium, changes in chemical concentration during incubation were measured by means of gas chromatography-mass spectrometry (GC-MS) analyses [13].…”