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      Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

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          Abstract

          Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC 50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians.

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          Most cited references26

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          Assessment of the environmental fate and effects of ivermectin in aquatic mesocosms.

          Pharmaceuticals in the environment have been subject to increasing public concern and scientific investigation over the past years. More than 100 active pharmaceutical ingredients have been detected in surface waters worldwide at the ng to microg L(-1) range. At these low levels it is commonly assumed that only chronic and/or mixture toxic effects will be discernible in aquatic ecosystems and that there are orders of magnitude between exposure and effect concentrations. Assessment of potential ecosystem risk of pharmaceuticals are recommended but rarely performed in mesocosms, so for most risk assessments the final tier to reduce extrapolation uncertainty is missing. This paper describes the fate and effects of the anthelmintic drug ivermectin for a 265-day period following treatment (nominal concentration levels of 0, 30, 100, 1000 ng L(-1) (or parts per trillion (ppt)) in fifteen 12,000 L outdoor aquatic mesocosms. Although it is established that ivermectin is highly toxic towards invertebrates, it has been believed that ivermectin does not present notable risks to aquatic systems due to the rapid dissipation of the compound and binding to the sediment. Hence, fate and exchange of ivermectin between water and sediment were evaluated in this study. The ivermectin DT(50aqueous) in water was found to be 3-5 days, but concentrations increased and appeared to be stabile in the sediment at 20-30 ng kg(-1) with no assessable DT(50sed). Acute effects (first week) following ivermectin exposure were identified and cladocerans were particularly sensitive (nom. 100 ppt). Chronic responses ( 229 days) were identified for more sediment-active organisms (e.g. Chydoriae and Ephemeroptera) (nom. 1000 ppt). This is the first study to demonstrate the potential environmental risk of ivermectin at or below the predicted environmental concentration using a standardized test methodology (mesocosm) with minimal extrapolation uncertainty.
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            Development of a reconstituted water medium and preliminary validation of the frog embryo teratogenesis assay--Xenopus (FETAX).

            A reconstituted water medium was developed for use in the Frog Embryo Teratogenesis Assay--Xenopus (FETAX). FETAX solution was then tested on three compounds with known mammalian teratogenicity (ethanol, caffeine, and 5-fluorouracil) as was a non-teratogen (saccharin). The results obtained were then compared with results from tests on these compounds in two other media that had previously been used in the assay. Saccharin was not teratogenic. Ethanol and caffeine were weak and moderate teratogens, respectively. 5-fluorouracil was a strong teratogen. The results compare favorably with those obtained in mammalian studies. The amount of growth inhibition in embryos in the 96 h tests was positively correlated with the degree of teratogenicity of the compound. Final validation of FETAX will allow it to be used to screen and rank compounds for further testing and as a tool for studying the basic mechanisms of teratogenesis.
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              Veterinary medicines in the environment.

              The impact of veterinary medicines on the environment will depend on a number of factors including physicochemical properties, amount used and method of administration, treatment type and dose, animal husbandry practices, manure storage and handling practices, metabolism within the animal, and degradation rates in manure and slurry. Once released to the environment, other factors such as soil type, climate, and ecotoxicity also determine the environmental impact of the compound. The importance of individual routes into the environment for different types of veterinary medicines varies according to the type of treatment and livestock category. Treatments used in aquaculture have a high potential to reach the aquatic environment. The main routes of entry to the terrestrial environment are from the use of veterinary medicines in intensively reared livestock, via the application of slurry and manure to land, and by the use of veterinary medicines in pasture-reared animals where pharmaceutical residues are excreted directly into the environment. Veterinary medicines applied to land via spreading of slurry may also enter the aquatic environment indirectly via surface runoff or leaching to groundwater. It is likely that topical treatments have greater potential to be released to the environment than treatments administered orally or by injection. Inputs from the manufacturing process, companion animal treatments, and disposal are likely to be minimal in comparison. Monitoring studies demonstrate that veterinary medicines do enter the environment, with sheep dip chemicals, antibiotics, sealice treatments, and anthelmintics being measured in soils, groundwater, surface waters, sediment, or biota. Maximum concentrations vary across chemical classes, with very high concentrations being reported for the sheep dip chemicals. The degree to which veterinary medicines may adsorb to particulates varies widely. Partition coefficients (K(d)) range from low (0.61 L kg(-1)) to high (6000 L kg(-1)). The variation in partitioning for many of the compounds in different soils was significant (up to a factor of 30), but these differences could be not be explained by normalization to the organic carbon content of the soils. Thus, to arrive at a realistic assessment of the availability of veterinary medicines for transport through the soil and uptake into soil organisms, the K(oc) (which is used in many of the exposure models) may not be an appropriate measure. Transport of particle-associated substances from soil to surface waters has also been demonstrated. Veterinary medicines can persist in soils for days to years, and half-lives are influenced by a range of factors including temperature, pH, and the presence of manure. The persistence of major groups of veterinary medicines in soil, manure, slurry, and water varies across and within classes. Ecotoxicity data were available for a wide range of veterinary medicines. The acute and chronic effects of avermectins and sheep dip chemicals on aquatic organisms are well documented, and these substances are known to be toxic to many organisms at low concentrations (ng L(-1) to microg L(-1)). Concerns have also been raised about the possibility of indirect effects of these substances on predatory species (e.g., birds and bats). Data for other groups indicate that toxicity values are generally in the mg L(-1) range. For the antibiotics, toxicity is greater for certain species of algae and marine bacteria. Generally, toxicity values for antibacterial agents were significantly higher than reported environmental concentrations. However, because of a lack of appropriate toxicity data, it is difficult to assess the environmental significance of these observations with regard to subtle long-term effects.

                Author and article information

                Journal
                ScientificWorldJournal
                ScientificWorldJournal
                TSWJ
                The Scientific World Journal
                The Scientific World Journal
                1537-744X
                2012
                1 May 2012
                : 2012
                : 605804
                Affiliations
                Laboratory for Ecotoxicology, Department of Environment, Spanish National Institute for Agricultural and Food Research and Technology (INIA), Carretera de la Coruña, Km 7.5, 28040 Madrid, Spain
                Author notes
                *Federica Martini: martini@ 123456inia.es

                Academic Editor: Peter Maček

                Article
                10.1100/2012/605804
                3354565
                22629159
                1f263bf8-fc0a-4bb7-810f-eb2baaf7e5dd
                Copyright © 2012 Federica Martini et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 28 October 2011
                : 22 December 2011
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