G. Hamscher, S. Sczesny, H. Nau,|
Department of Food Toxicology, Center of Food Science,
School of Veterinary Medicine,
Bischofsholer Damm 15,
Institute of Soil Technology,
Geological Survey of Lower Saxony,
D-28211 Bremen, Germany.
Drug residues in the environment are of growing interest worldwide. In both human and veterinary medicine a large number of drugs are used. After excretion, these drugs and their metabolites can contaminate the environment. Residues of pharmaceuticals used in human medicine occur in water by passing sewage treatment plants. New investigations show, that more than 40 different drugs can be found in surface waters from the low to the very low µg/L concentration range [for review see references 1 and 2].
Veterinary drugs can enter the environment directly by the use in fish farms, by urine and dung or by liquid manure used as fertilizer (see fig. 1). Important drugs in this field are antibiotics and antiparasitic drugs.
Fig. 1: Anticipated exposure routes of veterinary drugs in the environment 
A recently published study of Germanys Federal Environmental Agency showed, that the degradation rate of tetracycline in liquid manure is approximately 50 % in 5 months . In a screening of 62 pig slurry samples 9 were found positive for tetracycline with amounts of 5 to 24 mg/L.
There is still very little known about the amounts of these veterinary drugs in soil. Therefore, we performed first investigations to evaluate the fate of frequently used drugs such as tetracyclines in soils fertilized with liquid manure in regions with intensive livestock farming. In another approach possible leaching of these compounds into seeping water and ground water was also investigated .
Sampling, sample preparartion and measurement
Soil samples were collected from 12 agricultural fields in Northern Germany in early February 2000, the last fertilization with liquid manure was in September 1999. Samples were collected at depths of 10, 20, 30, 60, and 90 cm below soil surface. Two control samples from fields without slurry fertilization since at least 5 years were also taken from this region. Samples were immediately transported under cooling to the laboratory and stored at 4oC prior analysis.
In another approach the distribution of antibiotics in one long-term soil monitoring area  fertilized with pig slurry was investigated in detail: 8 samples were taken in and beside 4 specially marked areas. The area was fertilized in April, soil sampling was performed in May. Additionely the pig slurry subjected to this area was investigated for tetracylines. In 4 areas "crusty" animal slurry was picked up from the topsoil and in addition soil samples were taken from 0-30 cm.
Water was sampled via pumping from depths of 80 cm and 120 cm in four different areas using 0,5 bar below atmospheric pressure. Two sampling areas were fields belonging to farms housing pigs and cows; the other two places were the control areas mentioned above.
Soil samples were liquid-liquid extracted based on a method for the extraction of tetracyclines from eggs , water samples were pretreated with solid phase extraction. Measurement was obtained by the application of liquid chromatography combined with electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). The mobile phase of the reversed phase C18-LC-system consisted of 0,5% formic acid in water and a linear gradient from 0 to 50% acetontrile. [M+H]+-ions were obtained from all compounds, (ion-)trapped, fragmented and prominent daughter ions registered.
The main results of our study in February are summarized in figure 2. Leaching of the anaylzed compounds into seeping water sampled at a depth of 80-140 cm could not be detected with the methods employed. The results of tetracycline amounts of the detailed investigation of the long-term soil monitoring area and the slurry used for fertilization are given in table 1. The results of the "crusty" slurrys and the amounts of tetracyclines in the soil above are given in table 2. All data are not corrected for recovery (average recoveries were 74,7 % for oxytetracycline, 37,9 % for tetracycline and 69,8 % for chlortetracycline.
Tab. 1: Detailed investigation of a long-term soil monitoring area after fertilization with pig slurry containing 4 mg/L tetracycline and 0,1 mg/L chlortetracycline. 8 samples in- and outside marked areas within the field were sampled in different soil depths.
Fig.2: Investigation of various soil samples with LC-ESI-MS-MS. A und B: Tetracycline in soils 1-14, C: Chlortetracycline in soils 5-14, (average of two replicates). Limit of determination: 5 µg/kg, limit of detection: 1 µg/kg for all compounds.
Tab. 2: Results of the investigation of "crusty" slurrys from 3 different areas.
In a pilot study we detected with sophisticated LC-ESI-MS-MS procedures tetracycline and chlortetracycline in agricultural fields fertilized with animal slurry in concentrations up to 32 µg/kg soil [5, see also fig. 2. a-c]. In that study samples were taken in February, the last application of slurry was approximately 4-5 months ago. These findings showed, that tetracyclines are persistent in the environment and that the detected amounts were in several areas higher than the so called "phase I trigger value" of 10 µg/kg soil recommended by EMEA. If this value is exceeded, additional ecotoxicological tests have to be applied since 1997 for the final registration of a new drug .
Meanwhile we performed a detailed investigation of a long-term soil monitoring area which received slurry in April and soil sampling took place in May. In that case, we also received a pig slurry sample. It could be shown, that the tetracyclines were distributed evenly over the field and that the antibiotics were ploughed in a soil depth of approximately 30 cm. Calculating the predicted environmental concentration (PEC) of tetracyclin in soil following the EMEA guidance  and based on the amount of 4 mg/L measured in liquid manure, amounts of 100 µg/kg in the top 10 cm (or approximately 30 µg/kg in the top 30 cm) should be expected. In our study, the average distribution of tetracycline in the top 30 cm in this field was between 20 and 40 µg/kg which shows, that the worst case scenario for the calculation of PECs is quite realistic for tetracyclines and that these compounds are nearly quantitatively transferred from slurry into soil.
Acute problems with tetracyclines in the environment may arise, when animal slurry is not sufficiently ploughed in the soil. We found an example of an area where liquid manure dried on the topsoil and the amount of chlortetracycline in this "crusty" slurry was as high as 1 mg/kg, which is in the range of the minimal inhibitory concentration for several bacteria, e.g. in plasma.
Leaching of these compounds into seeping or ground water could not be detected with the methods employed. Tetracyclines easily build chelatic complexes with bivalent cations, e.g. with calcium. Currently it is not known, what kind of complexation or adsorption takes place in soil and in seeping water. Therefore, we should not overinterprete our first negative findings in the water samples and work on a further improvement especially of analytical techniques.
Finally we conclude, that our studies show that tetracyclines, which are frequently used worldwide, are not only persistent in animal slurry but also in soil in significant amounts, and that these substances represent an actual environmental problem in intensive livestock farming.
Based on reports in the literature [1-4] and on our investigations  we propose the following:
Further investigation into the fate of tetracyclines in the environment (e.g. degradation rates, local and global distribution, bioavailability).
Further improvement and validation of the employed methods for the analysis of tetracyclines in soil, water and liquid manure.
Development of methods or techniques to accelerate the degradation of tetracyclines in slurry.
Development of analytical methods for other frequently used veterinary drugs including their metabolites (e.g. sulfonamides).
Development of suitable ecotoxicological test methods, especially for antibiotics (acute effects / antibiotic resistance).
Relevant case studies with realistic concentration ranges to perform environmental risk assessment.
Silke Sczesny was supported by a grant of the Wilhelm-Schaumann-Stiftung, Germany. The authors are grateful to the Volkswagenstiftung, Germany, for financial support to build up the laboratory for residue analysis in the Department of Food Toxicology.
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