Cinzia Civitareale

Toxicity of several important agricultural antibiotics to Artemia

The possible effects of antibiotic drug contamination in marine and brackish systems were evaluated using a new methodological approach. Five drugs, namely aminosidine (A), bacitracin (B), erythromycin (E), flumequine (F) and lincomycin (L), were subjected to toxicity tests using nauplii and cysts of Artemia as a model of drug contamination from intensive farming. Tests on nauplii were performed by the ArToxKit-M test (Persoone and Van Haecke, 1981), test on cysts by our experimental protocol (Migliore et al., 1993a, b). The lethal effect on nauplii were evaluated between 24 and 120 h: the sequence of decreasing toxicity was B > F > L > A > E. A high toxicity of B was recorded. In addition, B and F were tested on cysts. B significantly lowered hatching; this depended on the interference of B with normal development. F did not depress hatching, but it deeply altered nauplii pigmentation. The interest in assessing the possible environmental risks due to drugs used in intensive farming and the need for suitable standards to improve sea-water quality is discussed.

Effect of sulphadimethoxine contamination on barley (Hordeum distichum L, Poaceae, Liliopsida)

Animal wastes from intensive farming are generally collected for field fertilisation. They may contain drugs that can become soil pollutants. To evaluate the possible effects of such contamination in terrestrial systems, sulphadimethoxine has been subjected to laboratory tests (in vitro, synthetic medium, and soil) using seeds of barley (Hordeum distichum L.). The drug suppressed normal post-germinative development and growth of roots and leaves in both test conditions; this effect was dependent on the bioaccumulation rate, which was higher on synthetic medium than in soil. Bioaccumulation was higher in roots than foliage and this was markedly evident in soil and, in particular, in soils with a low humus content. The environmental risk of sludge application on soils and the possible contamination of food chains are discussed.

Antibiotics of zootechnical use: Effects of acute high and low dose contamination on Daphnia magna Straus

To gather data on the effects of antibiotic pollution in freshwater systems and to determine methodological approaches, aminosidine (A), bacitracin (B), erythromycin (E) and lincomycin (L) were subjected to acute high dose toxicily tests (AHDTT) using Daphnia magna and following the EEC protocol (EEC Commission 84/449, 1984); the lethal effect evaluated at 24, 48 and 72 h determined the sequence of decreasing toxicity to be B > E > A > L. Furthermore, the four antibiotics were tested in acute low dose totoxiciiy teats (ALDTT) using Daplmia magna by studying the alteration of phototactic behaviour. This test showed that L and B depressed phototaxis, while A increased the light induced migration rate. E did not alter the phototactic behaviour.

Effects of sulphadimethoxine on cosmopolitan weeds (Amaranthus retroflexus L., Plantago major L. and Rumex acetosella L.).

Animal wastes from intensive farming are generally collected for field fertilisation. They may contain drugs that can become soil pollutants. The effect of such contamination on terrestrial biota has been demonstrated in laboratory tests on different plant species, using a common antimicrobial, sulphadimethoxine. In the near future, the monitoring of antimicrobial contamination in arable lands and their crops will be of importance for the protection of natural ecosystems and consumers. A possible tool for this monitoring is the use of weeds that can constitute a ‘mesh’ from which antimicrobial contamination can be detected. In laboratory tests, some direct effects of sulphadimethoxine contamination were demonstrated on normal development and growth of three cosmopolitan weed species, Amaranthus retroflexus L., Plantago major L. and Rumex acetosella L. These effects depended on the very high accumulation rates in plants (thousand μg g−1). P. major accumulated the highest amount of drug followed by A. retroflexus and R. acetosella. These data further highlight the environmental risk of sludge application on soils and the possible contamination of food nets; but also give a potential tool for the monitoring of antimicrobial soil contamination.

Simultaneous analysis of 17α-estradiol and 17β-estradiol in bovine serum by liquid chromatography–tandem mass spectrometry

A new LC-MS/MS method for the separation, identification and quantification of residues of 17alpha-estradiol (17alpha-E2) and 17beta-estradiol (17beta-E2) in bovine serum is reported. Deuterium-labelled 17beta-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M-H](-), were generated at m/z 271 and 274 for 17alpha/17beta-E2 and 17beta-E2-d(3), respectively. The decision limits obtained (CCalpha, i.e., critical concentration alpha) were 0.06 ng/mL and 0.03 ng/mL, respectively for 17alpha-E2 and 17beta-E2. Detection capability (CCbeta, i.e., critical concentration beta) values were 0.08 ng/mL and 0.04 ng/mL, respectively, for 17alpha-E2 and 17beta-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.

Determination of the banned growth promoter moenomycin A in feed stuffs by liquid chromatography coupled to electrospray ion trap mass spectrometry

Flavomycin complex is an antibiotic banned in the European Union as an additive in feed stuffs. As a consequence, the monitoring programmes for official control within the Community require analysis of feeds for possible illegal use of flavomycin. A method for unambiguous identification and quantification of moenomycin A, the main pharmacologically active component of flamomycin complex, in several feeds by liquid chromatography coupled to electrospray ion trap mass spectrometry (LC/ESI-MS/MS) is herein described for the first time. The method was developed to be used as a confirmative analytical tool for the network of Italian official control laboratories; both the singly and doubly charged molecular ions were observed as precursor ions, from which four product ions were selected for both quantitative analysis and unambiguous identification of moenomycin A. The method was in-house validated for feeds in the concentration range 0.50-30.0 microg/g, according to the Regulation 882/2004/EC requirements. Mean recoveries ranging between 83.9-94.2% and relative standard deviations <23% account for method trueness and repeatability, respectively. Moreover, other analytical performance parameters, i.e. method specificity, ruggedness, the linearity of detector response, the limit of quantification (LOQ), the limit of detection (LOD), and measurement uncertainty were evaluated and reported. The ion trap LC/ESI-MS/MS method is highly selective and reliable; high drug recovery, good reproducibility and an LOQ down to 0.10 microg/g guarantee its applicability for confirmatory purposes in the official control activity in Italy.

Sphingomonas turrisvirgatae sp. nov., an agar-degrading species isolated from freshwater

A yellow pigmented and agar-pitting colony was isolated from a water sample obtained from a drainage ditch within a disused system of constructed wetlands. The strain was purified and named MCT13T. This rod-shaped, Gram-negative, oxidase- and catalase-positive, aerobic, non-spore-forming, and non-motile strain formed round colonies and grew optimally at pH 7.5±0.2, at 28-30 °C on LB agar, with 0-0.5 % NaCl. The 16S rRNA gene sequence analysis placed the MCT13T isolate within the Sphingomonas (sensu stricto) cluster. The DNA G+C content was 65.3 %. The only observed ubiquinone was Q10. The major fatty acids included C17 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major polyamine was spermidine. The 16S rRNA gene phylogenetic analysis performed on the whole sequence, showed the closest relative of MCT13T to be Sphingomonas koreensis (98.52 %); however, there are several genotypic and phenotypic differences between the novel isolate and the type strain JSS26T of S. koreensis. On the basis of these results, strain MCT13T represents a novel species in the genus Sphingomonas, for which the name Sphingomonas turrisvirgatae sp. nov. is proposed. The type strain is MCT13T (=DSM 105457T=BAC RE RSCIC 7T).