An In Vivo Whole-Transcriptomic Approach to Assess Developmental and Reproductive Impairments Caused by Flumequine in Daphnia magna.

Among veterinary antibiotics, flumequine (FLU) is still widely used in aquaculture due to its efficacy and cost-effectiveness. Although it was synthesized more than 50 years ago, a complete toxicological framework of possible side effects on non-target species is still far from being achieved. The aim of this research was to investigate the FLU molecular mechanisms in Daphnia magna, a planktonic crustacean recognized as a model species for ecotoxicological studies. Two different FLU concentrations (2.0 mg L-1 and 0.2 mg L-1) were assayed in general accordance with OECD Guideline 211, with some proper adaptations. Exposure to FLU (2.0 mg L-1) caused alteration of phenotypic traits, with a significant reduction in survival rate, body growth, and reproduction. The lower concentration (0.2 mg L-1) did not affect phenotypic traits but modulated gene expression, an effect which was even more evident under the higher exposure level. Indeed, in daphnids exposed to 2.0 mg L-1 FLU, several genes related with growth, development, structural components, and antioxidant response were significantly modulated. To the best of our knowledge, this is the first work showing the impact of FLU on the transcriptome of D. magna.

Delayed toxicity of three fluoroquinolones and their mixtures after neonatal or embryonic exposure, in Daphnia magna.

Fluoroquinolones (FQs) are antibacterial drugs, used both in human and veterinary medicine, that are currently considered as emerging micropollutants. This study investigated the delayed toxic effects of enrofloxacin (ENR), flumequine (FLU), levofloxacin (LEV) and their binary mixtures in D. magna. For this purpose, a 10-day follow-up in pure medium was added to the standard D. magna immobilization test. During this follow-up, phenotypic alterations were evidenced, which were related to scarce or zeroed egg production and early mortality. Consequently, the EC50 s recalculated at the end of the follow-up were always remarkably lower than those obtained after the 48 h immobilization test: ENR 3.13 vs. 16.72 mg L-1; FLU 7.18 vs. 25.35 mg L-1; LEV 15.11 vs. > 40 mg L-1. To analyse the possible interactions within the binary mixtures, the method of nonlinear additive isoboles was applied. The three compounds showed invariably to follow the principle of concentration addition. Furthermore, as previous experiments showed toxicity of FLU and ENR after embryonic exposure of D. magna at a concentration of 2 mg L-1, an additional two embryonic tests were conducted with identical design: one with 2 mg L-1 LEV and the other with a ternary mixture containing 0.66 mg L-1 of each of the three FQs. The embryos were exposed for three days in vitro to the drug solutions and were then reconducted to pure medium for 21 days observation. Both the tests ended-up with only non-significant effects on growth and reproduction, confirming the lower toxicity of LEV, when compared to ENR and FLU, and the absence of any evident synergistic interaction among the three FQs. Overall, these studies have shown two relevant features related to the toxicity of the three FQs: (1) they give rise to delayed toxic effects in D. magna that are undetectable by the standard immobilization test; (2) their interaction in mixtures follow the principle of Concentration Addition. Both these indications concern the Environmental Risk Assessment of FQs and may be of interest to regulatory authorities.

Nano-immobilized flumequine with preserved antibacterial efficacy.

Flumequine was nano-immobilized by self-assembly on iron oxide nanoparticles, called surface active maghemite nanoparticles (SAMNs). The binding process was studied and the resulting core-shell nanocarrier (SAMN@FLU) was structurally characterized evidencing a firmly immobilized organic canopy on which the fluorine atom of the antibiotic was exposed to the solvent. The antibiotic efficacy of the SAMN@FLU nanocarrier was tested on a fish pathogenic bacterium (Aeromonas veronii), a flumequine sensitive strain, in comparison to soluble flumequine and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were assessed. Noteworthy, the MIC and MBC of soluble and nanoparticle bound drug were superimposable. Moreover, the interactions between SAMN@FLU nanocarrrier and microorganism were studied by transmission electron microscopy evidencing the ability of the complex to disrupt the bacterial wall. Finally, a preliminary in vivo test was provided using Daphnia magna as animal model. SAMN@FLU was able to protect the crustacean from the fatal consequences of a bacterial infection and showed no sign of toxicity. Thus, in contrast with the strength of the interaction, nano-immobilized FLU displayed a fully preserved antimicrobial activity suggesting the crucial role of fluorine in the drug mechanism of action. Besides the importance for potential applications in aquaculture, the present study contributes to the nascent field of nanoantibiotics.

Self-assembly of chlorin-e6 on γ-Fe2O3 nanoparticles: Application for larvicidal activity against Aedes aegypti.

Aedes aegypti mosquitos are widespread vectors of several diseases and their control is of primary importance for biological and environmental reasons, and novel safe insecticides are highly desirable. An eco-friendly photosensitizing magnetic nanocarrier with larvicidal effects on Aedes aegypti was proposed. The innovative core-shell hybrid nanomaterial was synthesized by combining peculiar magnetic nanoparticles (called Surface Active Maghemite Nanoparticles - SAMNs, the core) and chlorin-e6 as photosensitizer (constituting the shell) via self-assembly in water. The hybrid nanomaterial (SAMN@chlorin) was extensively characterized and tested for the photocidal activity on larvae of Aedes aegypti. The SAMN@chlorin core-shell nanohybrid did not present any toxic effect in the dark, but, upon light exposure, showed a higher photocidal activity than free chlorin-e6. Moreover, the eco-toxicity of SAMN@chlorin was determined in adults and neonates of Daphnia magna, where delayed toxicity was observed only after prolonged (≥4 h) exposure to intense light, on the green alga Pseudokirchneriella subcapitata and on the duckweed Lemna minor on which no adverse effects were observed. The high colloidal stability, the physico-chemical robustness and the magnetic drivability of the core-shell SAMN@chlorin nanohybrid, accompanied by the high photocidal activity on Aedes aegypti larvae and reduced environmental concerns, can be proposed as a safe alternative to conventional insecticides.

Transgenerational toxicity of flumequine over four generations of Daphnia magna.

In this study, the effects of both continuous and alternate exposure to 2 mg L-1 of flumequine (FLU) on survival, growth and reproduction of Daphnia magna were evaluated over four generations. Mortality was the most evident effect, with an average mortality rate of 23 ±â€¯14% across generations. Individuals destined to succumb were identifiable well in advance through their discolouration and lack of development, and limited or zero reproductive capacity. Inhibition of reproduction in surviving mothers varied across the four generations (14.3 ±â€¯17%) without an apparent correlation with the duration of exposure over generations. Significant reproductive inhibition was observed in the generation that followed three non-exposed generations (the fourth generation), pointing to a transgenerational toxicity of FLU. In another experiment, in vitro exposure of 72 D. magna embryos to 2 mg L-1 FLU caused 14% mortality (versus 7% in the control). Among the 62 individuals that hatched alive, six showed birth defects and only one was able to survive the next few days. The other, apparently healthy newborns were randomly assigned to two groups and submitted to a reproduction test, either in the absence or in the presence of 2 mg L-1 FLU. A high mortality rate and/or strongly significantly inhibited reproduction were detected in both groups. As with previously run analogous tests with enrofloxacin, the multigenerational and embryonic tests showed a clear disruption to this crustacean population which would not be evidenced by the standard official acute and chronic tests. This indicates the necessity of taking a different and more comprehensive approach to the evaluation of substances having an inherent ability to interact with genetic material.

Synergistic toxicity of some sulfonamide mixtures on Daphnia magna.

In livestock farming, sulfonamides (SAs) are used prophylactically and simultaneously in large numbers of animals. Therefore, traces of these compounds, alone or in combination, have been repeatedly detected in the environment. Synergistic interactions among chemicals in such mixtures represent an area of concern for the regulatory authorities. In this study, the acute toxic effects of binary and ternary mixtures of SAs were evaluated in Daphnia magna, in order to verify whether, based on their individual toxicity, they jointly exert a larger effect than would be predicted by individual actions alone. First, following the Concentration Addition (CA) principle, some preliminary observations were made by testing a number of drug combinations with an expected 50% effect. Then, mixtures more recognised for their synergistic effect (four binary and two ternary) were assayed in a range of reducing concentrations. The data acquired were processed using CompuSyn software, which integrates the different shape of the curves obtained in calculating the Combination Index (CI) for the evaluation of synergistic effects. For binary mixtures, synergy was also evaluated using the curvilinear isobologram method for heterodynamic drugs. Results indicate that most of the selected mixtures exhibit a synergistic effect using the CI methodology. For binary mixtures, these findings were also confirmed by isobologram analysis. Detected synergies indicate that the CA is not always precautionary as a reference model for the evaluation of the aquatic toxicity of SAs mixtures.

The surface reactivity of iron oxide nanoparticles as a potential hazard for aquatic environments: A study on Daphnia magna adults and embryos.

Nano-ecotoxicology is extensively debated and nanomaterial surface reactivity is an emerging topic. Iron oxide nanoparticles are widely applied, with organic or inorganic coatings for stabilizing their suspensions. Surface active maghemite nanoparticles (SAMNs) are the unique example of naked iron oxide displaying high colloidal and structural stability in water and chemical reactivity. The colloidal behavior of SAMNs was studied as a function of the medium salinity and protocols of acute and chronic toxicity on Daphnia magna were consequently adapted. SAMN distribution into the crustacean, intake/depletion rates and swimming performances were evaluated. No sign of toxicity was detected in two model organisms from the first trophic level (P. subcapitata and L. minor). In D. magna, acute EC50 values of SAMN was assessed, while no sub-lethal effects were observed and the accumulation of SAMNs in the gut appeared as the sole cause of mortality. Fast depuration and absence of delayed effects indicated no retention of SAMNs within the organism. In spite of negligible toxicity on D. magna adults, SAMN surface reactivity was responsible of membrane bursting and lethality on embryos. The present study offers a contribution to the nascent knowledge concerning the impact of nanoparticle surface reactivity on biological interfaces.

Increasing toxicity of enrofloxacin over four generations of Daphnia magna.

The effects of both continuous and alternate exposure to 2mgL(-1) of enrofloxacin (EFX) on survival, growth and reproduction were evaluated over four generations of Daphnia magna. Mortality increased, reaching 100% in most groups by the end of the third generation. Growth inhibition was detected in only one group of the fourth generation. Reproduction inhibition was >50% in all groups and, in second and third generations, groups transferred to pure medium showed a greater inhibition of reproduction than those exposed to EFX. To verify whether the effects observed in these groups could be explained by the perinatal exposure to the antibacterial, a reproduction test with daphnids obtained from in vitro exposed D. magna embryos was also carried out. Perinatal exposure to EFX seemed to act as an 'all-or-nothing' toxicity effect as 31.4% of embryos died, but the surviving daphnids did not show any inhibition of reproduction activity. However, the embryonic mortality may at least partially justify the inhibition of reproduction observed in exposed groups along the multigenerational test. Concluding, the multigenerational test with D. magna did show disruption to a population that cannot be evidenced by the official tests. The increasing deterioration across generations might be inferred as the consequence of heritable alterations. Whilst the concentration tested was higher than those usually detected in the natural environment, the increasing toxicity of EFX across generations and the possible additive toxicity of fluoroquinolone mixtures, prevent harm to crustacean populations by effects in the real context from being completely ruled out.

Effects of enrofloxacin, ciprofloxacin, and trimethoprim on two generations of Daphnia magna.

Multigenerational tests on Daphnia magna were performed exposing two subsequent generation to enrofloxacin (EFX) and its metabolite ciprofloxacin (CPX), and to trimethoprim (TMP). Mortality rate of 100% and 50% was detected in F0 at concentrations of ≥ 13 mgL(-1) (EFX) and 50 mgL(-1) (TMP), respectively. In F1 with respect to F0, both for growth and reproduction, a worsening trend of the response with EFX, a similar response with CPX and an attenuating trend with TMP was observed. Furthermore, the lowest EC20 for reproduction inhibition (1.3 mgL(-1)) was calculated for F1 exposed to EFX. However, other experimentations, longer and more complex, are necessary in order to confirm that EFX is more hazardous to daphnids than CPX and TMP. EC50 measured for the three assayed antibacterials were in the 6.5-37 mgL(-1) range therefore environmental unrealistic, except in case of exceptional contaminations that may occur in relation to poorly controlled wastewaters from pharmaceutical factories or excessive use of prophylactic treatments in aquaculture.

The sensitivity of Daphnia magna and Daphnia curvirostris to 10 veterinary antibacterials and to some of their binary mixtures.

Aim of this study was to evaluate the suitability of Daphnia curvirostris for the acute toxicity test usually performed on Daphnia magna, and to compare the sensitivity of the two species toward 10 antibacterials [enrofloxacin (EFX), ciprofloxacin(CPX), sulfaguanidine (SGD), sulfadiazine (SDZ), sulfamethazine (SMZ), sulfaquinoxaline (SQO), sulfaclozine (SCZ), sulfamerazine (SMA), sulfadimethoxine (SDM) and trimethoprim (TMP)] and some of their binary mixtures. Furthermore, a tentative prolonged-toxicity test (lasting 13d) was settled up in order to evidence toxic responses with drug concentrations that were uneffective in the classic 48h immobilization test. Results showed that D. curvirostris was more sensitive than D. magna to the majority of compounds (6 out of 10). Lowest 48h EC50s were obtained with EFX (4.3mgL(-1) in D. curvirostris) and SGD (6.2mgL(-1) in D. magna). The toxicity of paired compounds was always concentration-additive or less than concentration-additive. In the prolonged-toxicity test mortality and/or reproduction inhibition were constantly observed. It was concluded that: (1) D. curvirostris could be a suitable model for the evaluation of acute toxicity of antibacterials since its sensitivity was generally greater than that of D. magna; (2) the toxicity of EFX and SGD should be given special attention as the two compounds, in the prolonged test, showed to be active at concentrations of 0.9mgL(-1) and 2.5mgL(-1), respectively; (3) the concentration addition is usually a reasonable worst case estimation of the environmental impact of antibacterial mixtures.

A monitoring of chemical contaminants in waters used for field irrigation and livestock watering in the Veneto region (Italy), using bioassays as a screening tool.

In this study, 50 livestock watering sources (ground water) and 50 field irrigation sources (surface water) from various industrialised areas of the Veneto region were monitored for chemical contaminants. From each site, four water samples (one in each season) were collected during the period from summer 2009 through to spring 2010. Surface water samples and ground water samples were first screened for toxicity using the growth inhibition test on Pseudokirchneriella subcapitata and the immobilisation test on Daphnia magna, respectively. Then, based on the results of these toxicity tests, 28 ground water samples and 26 surface water samples were submitted to chemical analysis for various contaminants (insecticides/acaricides, fungicides, herbicides, metals and anions) by means of UPLC-MS(n) HPLC-MS(n), AAS and IEC. With the exception of one surface water sample where the total pesticides concentration was greater than 4 µg L(-1), positive samples (51.9 %) showed only traces (nanograms per liter) of pesticides. Metals were generally under the detection limit. High concentrations of chlorines (up to 692 mg L(-1)) were found in some ground water samples while some surface water samples showed an excess of nitrites (up to 336 mg L(-1)). Detected levels of contamination were generally too low to justify the toxicity recorded in bioassays, especially in the case of surface water samples, and analytical results painted quite a reassuring picture, while tests on P. subcapitata showed a strong growth inhibition activity. It was concluded that, from an ecotoxicological point of view, surface waters used for field irrigation in the Veneto region cannot be considered safe.

Sublethal effects of trimethoprim on four freshwater organisms.

Sublethal effects of trimethoprim (TMP) were evaluated in four freshwater organisms: Pseudokirchneriella subcapitata and Lemna minor (growth inhibition), Daphnia magna (reproduction and growth inhibition) and Poecilia reticulata (swimming activity inhibition). Cytochrome P4501A induction was also evaluated in P. reticulata. TMP showed varying levels of toxicity in the four test performed, with NOEC for the various endpoints in the range of 3.12-25 mg L(-1). The compound was active on P. reticulata at concentration ≥ 50 mg L(-1) causing inhibition of swimming activity. In the same organism an induction of CYP1A protein, mainly in kidney, gills and intestine, was also detected. L. minor was more sensitive than unicellular algae to TMP, with a NOEC of 12.5 mg L(-1). The lowest NOEC (3.12 mg L(-1)) was obtained in D. magna reproduction test and then a Risk Quotient of <0.03 was calculated by comparing the PNEC (31.2 µg L) and the TMP concentrations usually detected in freshwater (<1 µg L(-1)). However, based on recently reported data, it was concluded that while TMP concentrations normally detected in surface water are below those able to evoke appreciable biological effects in the various aquatic organisms, TMP concentrations in aquaculture and hospital effluents can be one to three orders of magnitude higher. Furthermore, the co-occurrence and additive effects of other antifolic agents should be taken into account for a cautious risk assessment of the drug.

Evaluation of the aquatic toxicity of two veterinary sulfonamides using five test organisms.

The aquatic toxicity of sulfaquinoxaline (SQO) and sulfaguanidine (SGD) was evaluated on the following test organisms: Daphnia magna (reproduction test), Pseudokirchneriella subcapitata, Scenedesmus dimorphus, Synecococcus leopoliensis (algal growth inhibition test) and Lemna gibba (duckweed growth inhibition test). Furthermore, the additivity of the two compounds was measured on D. magna (acute immobilisation test) and P. subcapitata (algal growth inhibition test) using the isobologram method. Results show that SQO and SGD are more toxic to green algae and daphnids, respectively, than other veterinary sulfonamides (SAs) and that their mixtures have a less then additive interaction. Taking into account the highest concentrations detected so far in surface waters for SQO (0.112 µg L(-1)) and for SGD (0.145 µg L(-1)) and the lowest NOECs obtained with the five test organisms, divided by an assessment factor of 10, the following PNECs and risk quotients (RQs) were calculated. SQO: PNEC 2 µg L(-1); RQ 0.056. SGD: PNEC 39.5 µg L(-1); RQ 0.004. Consequently, at the concentrations actually detected in the aquatic environment, the two SAs alone should not harm the freshwater organisms. However, it seems advisable, for veterinary mass treatments, the use of other SAs that have a lesser impact on the aquatic environment. Furthermore, considering the high probability of having complex mixtures of different SAs residues in water, each individual contamination should be evaluated by applying to the SAs mixtures the conservative criteria of additivity.

The toxicity of sulfamethazine to Daphnia magna and its additivity to other veterinary sulfonamides and trimethoprim.

Sulfonamides (SAs), the oldest chemotherapeutic agents used for antimicrobial therapy, still play an important role in veterinary mass treatments. Consequently, traces of these compounds, alone or in combinations, have been repeatedly detected in the environment. Sulfamethazine (SMZ) deserves particular attention not only because it is the most used veterinary SA, but also due to its proven effects on fertility in mice and on thyroid hormone homeostasis in rats. In this study, after evaluating the acute toxicity to Daphnia magna of six veterinary SAs and trimethoprim (TMP), the additivity of SMZ to each other compound was tested using the isobologram method. Two reproduction tests on the same biological model were also performed in order to derive LOEC and NOEC of SMZ. The acute EC(50) was in the range 131-270 mgL(-1) for all the compounds tested with the exception of sulfaguanidine (EC(50)=3.86 mgL(-1)). In acute binary tests SMZ showed a complex interaction with sulfaquinoxaline (superadditivity, additivity or subadditivity) at the three different combination ratios tested, simple additivity to TMP and less than additive interaction when paired to the other SAs. LOEC and NOEC of SMZ obtained from reproduction tests were 3.125 and 1.563 mgL(-1), respectively. In conclusion, SMZ should not harm the crustacean population at environmentally realistic concentrations. Its toxicity is comparable to that of other systemic SAs, and their binary interactions are less than additive. The same can not be entirely said for enteric SAs, and considering that these compounds are administered at high doses and mostly excreted in unmetabolised form, further evaluation of their impact to the aquatic environment seems advisable.

Integrated analytical approach in veal calves administered the anabolic androgenic steroids boldenone and boldione: urine and plasma kinetic profile and changes in plasma protein expression.

Surveillance of illegal use of steroids hormones in cattle breeding is a key issue to preserve human health. To this purpose, an integrated approach has been developed for the analysis of plasma and urine from calves treated orally with a single dose of a combination of the androgenic steroids boldenone and boldione. A quantitative estimation of steroid hormones was obtained by LC-APCI-Q-MS/MS analysis of plasma and urine samples obtained at various times up to 36 and 24 h after treatment, respectively. These experiments demonstrated that boldione was never found, while boldenone alpha- and beta-epimers were detected in plasma and urine only within 2 and 24 h after drug administration, respectively. Parallel proteomic analysis of plasma samples was obtained by combined 2-DE, MALDI-TOF-MS and muLC-ESI-IT-MS/MS procedures. A specific protein, poorly represented in normal plasma samples collected before treatment, was found upregulated even 36 h after hormone treatment. Extensive mass mapping experiments proved this component as an N-terminal truncated form of apolipoprotein A1 (ApoA1), a protein involved in cholesterol transport. The expression profile of ApoA1 analysed by Western blot analysis confirmed a significant and time dependent increase of this ApoA1 fragment. Then, provided that further experiments performed with a growth-promoting schedule will confirm these preliminary findings, truncated ApoA1 may be proposed as a candidate biomarker for steroid boldenone and possibly other anabolic androgens misuse in cattle veal calves, when no traces of hormones are detectable in plasma or urine.

Use of sulfadimethoxine in intensive calf farming: evaluation of transfer to stable manure and soil.

After prophylactic treatment of 50 calves with 62mgkg(-1)day(-1) of sulfadimethoxine (SDM) for five days, the levels of the drug over time were followed in feces, bedding and stable manure, and then in the soil of a manured field and surrounding drainage courses. Analysis were done by HPLC after applying to the different matrices a quick and simple extraction procedure. The half-life of the drug in bedding was very short (24h). In stable manure the degradation rate of the drug slowed down and the calculated half-life was 64 days, with 390microgkg(-1) of SDM still detectable after three months maturation. However, in a five months matured stable manure obtained from other groups of calves subjected to the same prophylactic treatment, levels of SDM were <50muicrokg(-1) (LOD of the analytical method). After field fertilization with this manure, no traces of SDM were found in soil (LOD 10microgkg(-1)) or in the water (LOD 2microgl(-1)) from the surrounding drainage courses. Using the internationally recognised DAPHTOXKIT-Ftrade mark, a SDM toxicity test toward Daphnia magna was performed in the range 10-100mgl(-1). The test gave negative results both after 24 and 48h, confirming that microcrustaceans are less sensitive than other models to the toxicity of antibacterials. However, based on data from other authors, concerning algal toxicity and microbial inhibition, and on the analytical results from the current field study, the calculated worst-case EC50/PEC ratio for SDM both in freshwater and in soil was still >1000.

Field observations in pigs exposed to fumonisin B1 contaminated feed.

Fourteen pigs, fed during the last 5 mo of rearing on fumonisin B1 naturally contaminated feed, were evaluated for hematological and biochemical effects. After slaughtering of the animals, the liver and 1 kidney underwent histopathological examination; the other kidney was tested by HPLC for fumonisin B1 residues. With feed contamination of 2 and 22 mg fumonisin B1/kg, the calculated daily intake of fumonisin B1 was approximately 50 microg/kg bw before the first blood sampling and approximately 500 microg/kg bw before the second blood sampling and slaughter. No hematological changes were observed, while moderate adverse effects on liver function (ALT and cholesterol) and hepatocyte integritywere found. No histopathological alterations nor fumonisin residues were detected (LOD 5 microg/kg) in the kidneys. The levels of contamination had slight but observed effects on swine health. The lack of fumonisin B1 carry-over to edible swine tissues was confirmed and under the studied conditions, no consumer risk from the pork products was expected.

Use of oxytetracycline and tylosin in intensive calf farming: evaluation of transfer to manure and soil.

Antibiotics may enter soils with manure from treated animals. Because of their biological effects, antibiotics are regarded as potential micropollutants. The levels of oxytetracycline and tylosin over time were followed in faeces, bedding and manure, and then in the soil of a manured field and surrounding drainage courses, after oral treatment of calves. Fifty Simmental calves were treated for 5 days with 60 mg/kg/day of oxytetracycline. After 15 days the animals were treated for 5 days with 20 mg/kg/day of tylosin. Tylosin degraded rapidly, and was no longer detected in manure 45 days after cessation of treatment and no trace of the compound was detected in soil or surrounding water (detection limits 10 microg/l). The half-life of oxytetracycline in manure was 30 days and the compound was still detectable in this matrix (820 microg/kg) after 5 months maturation. In the manured soil oxytetracycline was detected at concentrations at least 10 times lower than the European Agency for the Evaluation of Medicinal Products threshold (100 microg/kg) requiring phase II environmental risk assessment. Oxytetracycline was not detected in the water courses (detection limit 1 microg/l). These results demonstrate that the processes occurring between faeces production and application of manure to the soil are very effective in reducing the load of TYL and OTC in the environment. For both drugs a toxicity test was performed using the alga Selenastrum capricornutum. The EC50 was 4.18 mg/l for oxytetracycline and 0.95 mg/l for tylosin. A worst-case hazard assessment for the aquatic environment was performed comparing the ratio between the measured concentrations (LOD) and effect data from previous work (OTC) or from this work (TYL). This showed ratio between toxicity levels (bacteria) (EC50=0.14 mg/l) and measured concentrations (LOD=1 microg/l) for OTC to be 140. The corresponding value for TYL (LOD=10 microg/l) was 95.