Pii: s0045-6535(99)00449-x

E€ects of the antibiotics oxytetracycline and tylosin on soil Angel J. Baguera, John Jensenb,*, Paul Henning Kroghb a Department of Toxicology, Faculty of Veterinary, University of Zaragoza, Miguel Servet, 177 E-50.013 Zaragoza, Spain b Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsùvej 25, P.O. Box 314, DK-8600 Silkeborg, Antibiotics may enter the terrestrial environment when amending soils with manure. A Note of Guidance on ecological risk assessment of veterinary medicines was issued in January 1998. Hardly any information about eco- toxicological e€ects of already existing substances are available. This study has tested the e€ects of two widely used antibiotics, tylosin and oxytetracycline, on three species of soil fauna: Earthworms, springtails and enchytraeids.
Neither of the substances had any e€ect at environmentally relevant concentrations. The lowest observed e€ect con- centration was 3000 mg kgÀ1 and in many cases no e€ect was seen even at the highest test concentration of 5000 mg kgÀ1. Ó 2000 Published by Elsevier Science Ltd. All rights reserved.
Keywords: Antibiotics; Oxytetracycline; Tylosin; Earthworms; Collembola; Enchytraeids This directive outlines the basic requirements for con- ducting an environmental risk assessment of veterinary Veterinary medicinal products such as antibiotics medical products. A detailed evaluation to assess the help to protect the health and ensure the well being of environmental risk of new veterinary medical products is domestic animals. Veterinary medicinal products are li- given in a Technical Guidance Document by the Euro- censed for use by regulatory authorities if they comply pean Agency for the Evaluation of Medicinal products with scienti®c criteria on quality, ecacy and safety. The (EMEA), EMEA/CVMP/055/96 (EMEA, 1997). How- authorities consider safety to the treated animal, to the ever, the environmental risk assessment only concerns consumer, and to the individuals handling the product new products sold after the 1st of January 1998. There is during treatment. In addition to these criteria the envi- currently no European initiative to assess environmental ronmental risk of veterinary medicinal products has re- risk of veterinary medicinal products already on the cently become a matter of increasing public scrutiny and legal requirements. The environmental impact of veter- Very little is known about the ecotoxicological e€ects inary medicines is assessed after di€erent regulations of antibiotics. However, antibiotics are speci®cally de- depending whether the application is therapeutic or non- signed to control bacteria in animals. Obviously this therapeutic. The legislation in the European Union on makes them potentially hazardous to bacteria and other the environmental risk assessment of veterinary medi- micro-organisms in the environment (Warman, 1980; cines is part of the Commission Directive 92/18EEC.
Pursell et al., 1995). For soil fauna and plants hardly any information is available. This paper describes the direct e€ects of antibiotics on soil living fauna. Three soil liv- * Corresponding author. Danish Environmental Research ing invertebrate species (earthworms, springtails and Agency, Vejlesovej 25, DK-8600 Silkeborg, Denmark.
enchytraeids) have been exposed to two widely used 0045-6535/00/$ - see front matter Ó 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 4 4 9 - X A.J. Baguer et al. / Chemosphere 40 (2000) 751±757 antibiotics in controlled laboratory experiments. Oxy- sandy soil, composed by 66.9% coarse sand, 15.8% ®ne tetracycline (OTC), is a broad spectrum antibiotic with a sand, 3.3% coarse silt, 5.3% ®ne silt, 6.2% clay, 2.7% long history in veterinary medicine for the treatment and humus, 1.5% total carbon and a pH-H2O of 5.5, was control of a wide variety of bacterial infections. Tylosin used in the springtail and enchytraeid experiments. A is a macrolide antibiotic that is active mostly against sandy±loamy soil (38.4% coarse sand, 23.6% ®ne sand, Gram-positive bacteria and mycoplasmas. Tylosin is 10.0% coarse silt, 12.3% ®ne silt, 13.0% clay, 2.8% hu- used in pigs, cattle and poultry for the treatment of mus, 1.6% total carbon and a pH-H2O of 6.2) was used conditions caused by sensitive micro-organisms.
in the earthworm tests. In order to eliminate undesired soil fauna, both soils were dried at 80°C and stored at 5°C until use. Thereafter soils were sieved through a Folsomia ®metaria (Linne, 1758) (Collembola: Iso- In order to determine the range of antibiotic con- tomidae) is a euedaphic, nonpigmented, eyeless spring- centrations for use in the ®nal tests, preliminary range- tail that reproduced sexually. A laboratory culture was ®nding tests were performed in each experiment (Lùkke established from ®eld-collected animals that were mass and van Gestel, 1998). Acute mortality tests were carried reared on moistened substrate of plaster of Paris/char- out with F. ®metaria (10 female and 10 male collem- coal. The collembolans were fed dried baker's yeast.
bolans were exposed for one week), E. crypticus (10 Every 2±4 weeks the animals were moved to another adult worms were exposed two weeks) and A. caliginosa Petri dish with fresh food and substrate, which stimu- (5 adult earthworms exposed for one week). Five con- lated egg production. A synchronised culture was pro- centrations and a control (two replicates per treatment) duced by collecting approximately 1-week-old eggs, which were allowed to hatch over 3 days (unhatched On the basis of these preliminary range-®nding tests, eggs were removed after this period) (Krogh, 1995).
the test species were exposed to the following OTC Animals 23±26 days old were used in the experiments.
concentration series given as mg/kg soil (dry weight): 0, The sex was discerned by size, as females are bigger than 500, 1000, 2000, 3000 and 5000; The following test concentrations of Tylosin were used: F. ®metaria and Enchytraeus crypticus (Enchytraeidae: Oligochaeta) A. caliginosa, 0, 500, 1000, 2000, 3000 and 5000; were collected from the ®eld and cultured in laboratory E. crypticus, 0, 1000, 2000, 3000, 4000 and 5000. Four conditions in order to get worms of the same size for the replicates per concentration were done.
experiments. Oat ¯akes were used for food and to avoid infections with mites it was boiled. Adult worms, with eggs in the clitellum and approximately the same size, The sublethal toxicity tests with F. ®metaria were The earthworm Aporrectodea caliginosa (Savigny, done following the guideline described by Wiles and 1826) (Annelida: Oligochaeta) were collected in the ®eld Krogh (1998). To each test container 30 g sandy moist from an uncontaminated agricultural soil. Adult animals soil (27 g dry soil and 3 g demineralised water) was (with clitellum) were maintained under laboratory con- added. The antibiotics were dissolved in water and ditions in the same test soil-substrate for one week be- mixed homogeneously into the dry soil one day before fore use (van Gestel et al., 1989). A mixture of test soil the start of the experiments. 10 males and 10 females, and ®nely ground cattle manure (1:1 dry vol.%) subse- 23±26 days old, were added to each test container. 2 mg quently moistened to 50% water content of fresh weight dried baker's yeast was added to the soil surface in each was used as food and applied on the surface of the test container at the start of the experiments and again after soil ad libitum. Adult earthworms were used in the ex- two weeks. The test containers were weighed initially and after two weeks so that water lost by evaporation could be compensated for. Test containers were incu- bated for 21 days at 20°C ‹ 2°C with 12:12 light:dark cycle. After incubation the animals were extracted for 48 Oxytetracycline Dihydrate, C22H24N2O9 2H2O (num.
h in a high-temperature gradient soil fauna extractor of CAS 6153-64-6), and Tylosin Tartrate (num. CAS the MacFadyen type (Petersen, 1978). The extraction 74610-55-2) with a potency of 898 mg tylosin/mg, both lasted 48 h starting at 25°C at the surface of the soil.
by Sigmaâ, were used in the experiments.
Every 12 h, the temperature at the soil surface was in- In order to reach the optimal conditions for the dif- creased by 5°C up to 40°C during the last 12 h. The ferent animal's species two kind of soils were used. A collembolans were collected into containers with a 0.5 A.J. Baguer et al. / Chemosphere 40 (2000) 751±757 cm layer of plaster of Paris/charcoal. The surface of the softly blotted on absorbent paper to remove excess plaster was kept constantly at 3°C during the extraction.
water and weighed. In order to ensure that the replicate The animals were stored at 5°C until automatic counting units for each treatment cover a similar range of initial (Krogh et al., 1998). Measured parameters were survival weights, a Ôranking and blockingÕ procedure was done and reproduction of the collembolan F. ®metaria and the earthworms were assigned the di€erent treat- ments, one in each test container (McIndoe et al., 1999).
With regard to the enchytraeid tests, 23.2 g sandy Test containers were incubated for 21 days at 15°C ‹ moist soil (20 g dry soil and 3.2 g demineralized water) 1°C. Food (test soil mixed with cattle manure) was ap- was added to each test container. The antibiotics were plied on the surface of the test soil ad libitum in each dissolved in water and mixed thoroughly into the dry container at the start of the experiments and again after soil one day before the start of the experiments. Then 10 two weeks. The test containers were weighed initially sexually mature enchytraeids, with eggs in the clitellum and evaporated water was replaced after two weeks. Test and approximately the same size, were added to each containers were incubated for 21 days at 15°C ‹ 1°C. At test container. A small amount of boiled oat ¯akes the end of the test the substrates were carefully searched (around 25 mg dry weight) was added and mixed with for surviving earthworms and cocoons. The earthworms the soil in each container just before the worms were were washed, blotted, weighed and counted. Cocoons added to soil. Food supply was added weekly at the soil were counted and maintained in Petri dishes with wet surface to avoid harming the worms. The test vessels ®lter paper and were incubated at 20°C for further nine were weighed initially and once a week the weight loss weeks to allow hatching. For each test, survival, growth, was replenished with the appropriate amount of de- reproduction (number of cocoons produced) and cocoon ionised water. Test containers were covered with plastic viability were measured. As chemicals in line with other and perforated lids and incubated for 21 days at 20°C ‹ stresses may impact the scope for growth of organisms, 2°C in a controlled light dark cycle of 12:12 light:dark that is the energy available for growth and reproduction, cycle. After 21 days, the test substrates were carefully the total biomass produced during the exposure time searched and surviving adults were removed and was used as the most ecological relevant endpoint of counted. The test substrates, including cocoons hatched growth (Holmstrup, personal communication). The juveniles, were incubated under the same test conditions growth of earthworms was calculated as the ®nal bio- for additionally 21 days. After two weeks 25 mg (d.w.) mass (FB), including body growth and the numbers of food was added to each test container. The containers (NC) and mean weight of cocoons (MCW) produced in were weighed once a week and the weight loss was re- each replicate, i.e. FB ˆ FW + (NC ´ MCW). To nor- plenished with the appropriate amount of de-ionised malise the weight data to a uniform weight class all water. At the end of the incubation period the worms weights were divided by the weight of the corresponding were extracted using the wet funnel method (OÕConnor, controls from the same block. Cocoons viability was 1985). The enchytraeids were collected into plastic con- estimated with regard to the number of cocoons pro- tainers and stored at 5°C until counting. In order to duced in each replicate and a hatch rate (HR) was cal- facilitate the counting of the high number of worms after culated as the ratio between the number of hatched the exposure time, the procedure was as follows: ®rst, cocoons (HC) during the nine weeks of incubation and the enchytraeids were transferred to graduate-counting the number of produced cocoons (PC) during the three Petri dishes with a thin layer of water, and their move- ment were decreased by adding 1±2 drops of glycerol.
Afterwards 2±3 drops of Bengalred were added in order to increase the contrast and improve visualisation of the The sublethal toxicity tests with the earthworm A.
Estimation of the no-observed-e€ect concentration caliginosa were carried out following the guideline de- (NOEC) and the lowest-observed-e€ect concentration scribed by Kula and Larink (1998). 800 g dry sandy± (LOEC) was done by comparing the control with each loam soil was weighed into each test container. Four of the concentrations by a DunnettÕs test in an ANOVA replicates for each concentration were used in all tests.
(SAS, 1989). For all tests the level of signi®cance was Solutions of OTC and Tylosin were used to give the 0.05. The validity of the ANOVA has been tested by required antibiotic concentration and percentage water SAS/LAB (SAS, 1992), which check for homogeneity of content in the test soil. The solutions were mixed ho- variances (LeveneÕs test), outliers and normality. Cal- mogeneously into the soil the day before the start of the culation of EC10 and EC50 values for reproduction, experiments. The same volume of de-ionised water (160 growth and cocoon viability was done by using the ICp ml) was added to the controls. At the end of the accli- approach (Norberg-King, 1993). Lethal concentration matisation period (one week), four earthworms were (LCx) values for adults were estimated by use of PRO- collected by hand, carefully washed with tap water, A.J. Baguer et al. / Chemosphere 40 (2000) 751±757 The toxicity of OTC and tylosine to the three tested soil animals was generally very low. The lowest observed signi®cant e€ects were found at 3000 mg kgÀ1 and in many cases no e€ects were observed at the highest test concentration of 5000 mg kgÀ1 (Figs. 1±3). Reproduc- tion was generally a more sensitive endpoint than sur- vival (Tables 1±4). Growth and fertility, expressed as the number of cocoons hatched during a nine weeks post exposure period, were a slightly more sensitive endpoints than survival of earthworms. Estimated EC10 values were found in the range of 134 to more than 5000 mg kgÀ1, whereas all EC50 values were above 2000 mg kgÀ1.
Fig. 3. E€ects of the antibiotics oxytetracycline (rombs) and tylosin (squares) on the reproduction (number of coccoons) of the earthworm A. caliginosa. Data signi®cantly di€erent from controls are marked with an asterisk (*). Error bars ˆ SEM.
Generally large con®dence intervals were found in the Veterinary medicines may be spread to the environ- ment, either directly when using the drugs (minor) or by subsequent excretion from the animals (major). Before entering the environment, the substances may be me- Fig. 1. E€ects of the antibiotics oxytetracycline (rombs) and tylosin (squares) on the reproduction (number of o€spring) of tabolised in the animal. Reactions may consist of oxi- the springtail F. ®metaria. Data signi®cantly di€erent from dation, reduction, hydrolysis or conjugation. The controls are marked with an asterisk (*). Error bars ˆ SEM.
metabolisation changes the physical, chemical and eco- toxicological properties of the substance (see, e.g., Gibson and Skett, 1986). It is, however, shown that metabolites may be reconverted to their parent com- pounds after leaving the animals. Berger et al. (1986) showed that the metabolites chloramphenicol glucor- onide and N-4-acetylated sulphadimidine were convert- ed to the parent compounds chloramphenicol and sulphadimidine in samples of liquid manure.
The dominating pathway of environmental release in the terrestrial compartment is by amendment of arable soil with manure or slurry. Very few studies concerning the levels of veterinary medicines in soil after manure amendment have been reported. Warman and Thomas (1981) found chlortetracyclines in soil amended with chicken manure, and Shore et al. (1988) found testos- terone and estrogene in manure from American chick- ens. Van Goll (1993) estimated that if the total amount of growth promoters used in the Netherlands were Fig. 2. E€ects of the antibiotics oxytetracycline (rombs) and spread over all the two million hectares of Dutch arable tylosin (squares) on the reproduction (number of o€spring) of the enchytraeid E. crypticus. Data signi®cantly di€erent from land, an annual average of 130 mg antibiotics and an- controls are marked with an asterisk (*). Error bars ˆ SEM.
tibiotic metabolites per m2 of arable land would be found, corresponding to approximately 0.9 mg/kg of dry A.J. Baguer et al. / Chemosphere 40 (2000) 751±757 Collembola. E€ects of oxytetracycline and tylosin on the survival and reproduction of F. ®metariaa a All concentrations in mg kgÀ1 d.w. Numbers in brackets are 95% con®dence interval.
Enchytraeids. E€ects of oxytetracycline and tylosin on the survival and reproduction of E. crypticusa a All concentrations in mg kgÀ1 d.w. Numbers in brackets are 95% con®dence interval.
Earthworms. E€ects of oxytetracycline and tylosin on the survival and reproduction of A. caliginosaa a All concentrations in mg kgÀ1 d.w. Numbers in brackets are 95% con®dence interval.
Earthworms. E€ects of oxytetracycline and tylosin on the growth of A. caliginosa and the hatchability of produced cocoonsa a All concentrations in mg kgÀ1 d.w. Numbers in brackets are 95% con®dence interval.
soil. Such a uniform distribution is unlikely and local tested the e€ects of the antibiotics chlortetracycline and concentration signi®cantly higher than this must be ex- oxytetracycline on plants when grown in both a nutrient pected. A uniform procedure for estimating the predict solution media and in soils. When grown in a nutrient environmental concentrations (PECs) for veterinary solution of 160 mg/l all plants died and in lower con- medicines is suggested by Spaepen et al. (1997). Empir- centrations both the growth of roots and the dry weight ical estimations predict environmental concentrations in of the shoots were signi®cantly reduced (approximately 60±90%). In the study using soil as growth media a large As soil dwelling organisms potentially will be ex- variation of the sensitivity among plant species was posed to antibiotics it is important to know whether found (Batchelder, 1982). Lanzky and Halling-Sùrensen e€ects on non-target organisms are likely to occur. Very (1997) showed that Chlorella sp. are very sensitive little is known about the possible side e€ects of antibi- (EC10 ˆ 2.03 mg/l and EC50 ˆ 12.5 mg/l) to the antibi- otics to soil fauna and plants. Batchelder (1981, 1982) otics metronidazole. Studies on the e€ect of antibiotics A.J. Baguer et al. / Chemosphere 40 (2000) 751±757 to soil invertebrates have not yet been published. The Holmstrup, M., 1998. Personal communication. National present study shows that the antibiotics oxytetracycline Environmental Research Institute, Department of Terres- and tylosin have a low toxicity to soil dwelling fauna.
Krogh, P.H., 1995. Does a heterogenous distribution of food or 10 values were found from approximately 150 mg kgÀ1. Due to variation in the data this is markedly below pesticide a€ect the outcome of toxicity tests with the NOEC values. A common situation in ecotoxicolo- Colembola. Ecotoxicology and Environmental Safety 30, gy. Many scientists have therefore recommended the use Krogh, P.H., Johansen, K., Holmstrup, M., 1998. Automatic of EC10 values instead of NOEC values in, e.g., the risk counting of collembolans for laboratory experiments. Ap- assessment procedure (e.g., Hoekstra and van Ewijk, Kula, H., Larink, O., 1998. Tests on the earthworms Eisenia Information from the aquatic environment shows fetida and Aporrectodea caliginosa. In: Lùkke, H., van that antibiotics may be toxic for other organisms than Gestel, C.A.M. (Eds.), Handbook of Soil Invertebrate Toxicity Tests. Wiley, Chichester, pp. 95±112.
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