Toxicity of the antimicrobial oxytetracycline to soil organisms in a multi-species-soil system (MS.3) and influence of manure co-addition.

The effects of oxytetracycline (OTC) on soil organisms have been studied using a multi-species-soil system (MS.3). Oxytetracycline concentrations of 0.01 mg/kg, 1 mg/kg and 100 mg/kg soil were added to the 20 cm top arable soil layer, with and without horse/cow manure (0.15 g organic N/kg soil) co-addition. No mortality was observed for Eisenia foetida S. but significant effects on soil microbial enzymatic activities (phosphatase, dehydrogenase) were observed. The effects on soil microorganism were observed earlier but then recovered in systems with manure co-addition. More important, OTC related plant inhibition was observed in the manured but not in the non-manured systems. Oxytetracycline reached 0.19 and 1.85 mg/l in the leachate of the soil spiked with 1 and 100 mg OTC/kg, respectively and 0.05 and 1.14 mg/l for the same OTC concentrations in the manured systems. The results confirm that manure can modify both the fate and the effects of OTC and that the multi-species-soil systems can reproduce the conditions for a realistic effect estimation of veterinary medicines.

Effects of sulfachlorpyridazine in MS.3-arable land: a multispecies soil system for assessing the environmental fate and effects of veterinary medicines.

A multispecies soil system (MS.3) has been used to evaluate the ecological effects of veterinary pharmaceuticals in soil as a result of routine agricultural practices. Different experimental conditions were tested and the variation of the different parameters was evaluated for a final design. A protocol for the MS.3-arable land is presented here. Emergence of seedlings, plant elongation and biomass, earthworm mortality, and soil microbial enzymatic activities have been selected as toxicological endpoints for soil organisms. Toxicity tests were conducted with the leachate on aquatic organisms (in vitro fish cell lines, daphnids, and algae). The system was used for assessing the effects of the antimicrobial sulfachlorpyridazine that was tested in triplicate at concentrations of 0.01, 1, and 100 mg/kg. The chemical was mixed uniformly with a 20-cm depth soil column to resemble the distribution of manure within arable soil. Reversible and nonreversible effects on soil enzymatic activities were observed at 1 and 100 mg/kg, respectively. Earthworms were not affected. Significant reduction of plant elongation and biomass was observed at the highest concentration. Degradation and leaching contributed to the dissipation of sulfachlorpyridazine from the soil column. The undiluted leachate was highly toxic to Daphnia magna. The parent chemical was assumed responsible for the leachate toxicity although the role of mobile metabolites could not be excluded fully. No significant effects were observed for green algae Chlorella vulgaris and for the rainbow trout established cell lines RTG-2 (rainbow trout gonads) and RTL-WI (rainbow trout liver). The MS.3 system offers a cost-effective experimental approach to measure simultaneously fate and effects of chemicals on a realistic soil system under controlled laboratory conditions. The advantages of using MS.3-effect endpoints are discussed.

In vitro toxicity of antimicrobials on RTG-2 and RTL-W1 fish cell lines.

This study proposed a battery of endpoints based on in vitro fish cell lines. Two fish cell lines and four toxicity endpoints are considered. The tetracycline (TC), oxytetracycline (OTC), sulfachlorpyridazine (SCP), and septrin(®) (ST) were selected as model antimicrobials and chlorpyrifos as positive control. EROD was induced by septrin(®) (formulation containing sulfamethoxazole and trimethoprim) at concentrations higher than 20mg/L, but inhibited by tetracycline, oxytetracycline and chlorpyrifos. Dose dependent inhibition responses were observed for ß-galactosidase (ß-Gal) activity in cells exposed to septrin(®), tetracycline or oxytetracycline. The EROD EC50s ranged between 2.29×10(-2)mg/L (chlorpyrifos) and 167.63mg/L (tetracycline). The ß-Gal EC50s ranged between 22.1 (septrin(®)) and 84.59mg/L (tetracycline). Data suggest that in vitro testing using a battery of endpoints can be a cost-effective solution for screening the toxicity of antimicrobials on fish. The absence of in vitro effects at concentrations well above those expected/measured in the environment may replace the need for conducting acute lethality tests on fish.