Due to difficulties in tracing engineered nanoparticles (ENPs) in complex media, there are few data on the exposure of soil biota to ENPs. This study used neutron activated cobalt (Co NPs) and silver (Ag NPs) nanoparticles, as well as soluble cobalt and silver salts, to assess the uptake, excretion and biodistribution in the earthworm Eisenia fetida. Concentrations of cobalt in worms after four weeks exposure reached 88% and 69% of the Co ions and Co NPs concentrations in food, respectively, while corresponding values for Ag ions and Ag NPs were 2.3% and 0.4%. Both Ag ions and Ag NPs in earthworms were excreted rapidly, while only 32% of the cobalt accumulated from Co ions and Co NPs were excreted within four months. High accumulation of cobalt was found in blood and in the digestive tract. Metal characterization in the exposure medium was assessed by sequential extraction and ultrafiltration. The Co NPs showed significant dissolution and release of ions, while Ag ions and particularly Ag NPs were more inert.
Studies regarding the environmental impact of engineered nanoparticles (ENPs) are hampered by the lack of tools to localize and quantify ENPs in water, sediments, soils, and organisms. Neutron activation of mineral ENPs offers the possibility of labeling ENPs in a way that avoids surface modification and permits both localization and quantification within a matrix or an organism. Time-course experiments in vivo also may be conducted with small organisms to study metabolism and exposure, two aspects currently lacking in ecotoxicological knowledge about ENPs. The present report explains some of the prerequisites and advantages of neutron activation as a tool for studying ENPs in environmental samples and ecologically relevant organisms, and it demonstrates the suitability of neutron activation for Ag, Co/Co3O4, and CeO2 nanoparticles. In a preliminary experiment with the earthworm Eisenia fetida, the dietary uptake and excretion of a Co nanopowder (average particle size, 4 nm; surface area, 59 m2/g) were studied. Cobalt ENPs were taken up to a high extent during 7 d of exposure (concentration ratios of 0.16-0.20 relative to the ENP concentration in horse manure) and were largely retained within the worms for a period of eight weeks, with less than 20% of absorbed ENPs being excreted. Following dissection of the worms, 60Co was detected in spermatogenic cells, cocoons, and blood using scintillation counting and autoradiography. The experimental opportunities that neutron activation of ENPs offer are discussed.
Eisenia fetida were exposed continuously to (60)Co gamma radiation during two generations (F(0) and F(1)). Adult F(0) reproduction capacity (i.e., number of cocoons produced, hatchability and number of F(1) hatchlings) in controls and at five dose rates (0.18, 1.7, 4, 11 and 43 mGy/h) was measured over a 13-week exposure period. Survival, growth and sexual maturation of F(1) hatchlings were observed for 11 weeks. F(1) adults were exposed for a further 13 weeks to determine their reproduction capacity. There was no radiation-induced effect on the cocoon production rate in either F(0) or F(1). For F(0), hatchability of cocoons produced during the first 4 weeks was reduced to 60% at 43 mGy/h (98% in controls), and none of the cocoons produced at 5-13 weeks hatched. At 11 mGy/h the cocoon hatchability was reduced to 25% at 9-13 weeks. In addition, the number of hatchlings per hatched cocoon was reduced at 11 and 43 mGy/h. Correspondingly, at these dose rates, the total number of F(1) hatchlings per adult F(0) was significantly lower than in the control. This number was also reduced at 4 mGy/h, but the effect was of borderline significance. For adult F(1), the hatchability of cocoons at 11 mGy/h was reduced to 45-69% during the 13-week exposure period. The number of hatchlings (F(2)) per cocoon and the total number of F(2) individuals produced was also reduced. However, and in contrast to the results observed for F(0), hatchability increased with time, suggesting a possible acclimatization or adaptation of the F(1) individuals. In conclusion, chronic irradiation reduced the reproduction capacity of E. fetida, but extensive exposure periods (13 weeks) were needed for these effects to be expressed. The lowest dose rates at which an effect was observed were 4 mGy/h in F(0) and 11 mGy/h in F(1).
Methods for analysing oxidised DNA lesions [formamidopyrimidine glycosylase (Fpg)-sensitive sites] in coelomocytes and spermatogenic cells from the earthworm Eisenia fetida using the Fpg-modified comet assay were established. The DNA integrity (SSBs = strand breaks plus alkali labile sites and Fpg-sensitive sites) in cells from E. fetida continuously exposed to (60)Co gamma-radiation (dose rates 0.18-43 mGy/h) during two subsequent generations (F0 and F1) were measured and related to effects on reproduction end points which have already been reported. The data suggest a slight increase of Fpg-sensitive sites in spermatogenic cells from worms exposed at 11 mGy/h in the F0 generation but not in F1, whereas reduced reproduction had been observed at dose rates at or >4 mGy/h in F0 and at 11 mGy/h in F1. Using acute X-rays (41.9 Gy/h), dose-response relationships were established for SSBs in coelomocytes and spermatogenic cells exposed in vitro. In vivo DNA repair was studied by measuring the decrease in damage (SSBs and Fpg-sensitive sites) in coelomocytes and spermatogenic cells isolated from worms at different times (0-6 h) after acute X-ray exposure (4 Gy). SSBs were repaired in coelomocytes following biphasic kinetics, i.e. with a fast and a slow half-life (t(1/2)) of 36 min (95%) and 6.7 h (5%), respectively. Fpg-sensitive sites were repaired at considerably lower rates (t(1/2) = 4-5 h). In spermatogenic cells, SSB repair during the first hour was observed but a half-life could not be estimated. Repair of Fpg-sensitive sites could not be determined. In general, a reduced repair of Fpg-sensitive sites suggests a higher potential for accumulation of oxidised lesions, compared to SSBs, in earthworms exposed to radiation and other environmental contaminants. This is the first study comparing DNA damage with reproduction in earthworms exposed to ionising radiation.
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