The veterinary antibiotic oxytetracycline and Cu influence functional diversity of the soil microbial community

Abstract

There are increasing concerns over the effects of veterinary antibiotics and heavy metals in agricultural soils. The widely used veterinary antibiotic oxytetracycline (OTC), Cu and their combination on soil microbial community function were assessed with the Biolog method. The microbial community was extracted from the soil and exposed to a 0.85% sodium chloride solution containing OTC (0, 1, 5, 11, 43, 109 and 217 μM), or Cu (0, 10, 20, 100 and 300 μM), or combination of the two pollutants (OTC 0, 5, 11 μM and Cu 0, 20 μM). Functional diversity, evenness, average well color development (AWCD) and substrate utilization decreased significantly with increasing concentrations of OTC or Cu (p < 0.005). The critical concentrations were 11 μM for OTC and 20 μM for Cu. The combination of OTC and Cu significantly decreased Shannon's diversity, evenness and utilization of carbohydrates and carboxylic acids compared to individual one of the contaminants. The antibiotic OTC and Cu had significant negative effects on soil microbial community function, particularly when both pollutants were present.

Introduction

Veterinary pharmaceuticals are widely used for the therapy of infectious diseases of animals in intensive farming system (Halling-S¢rensen, 2000, Boxall et al., 2003). They are designed to act very effectively at low doses and to be completely excreted from the body after a short time of residence. Only a fraction of the ingested antibiotics is metabolized in the animals, hence a large percentage of the antibiotics are excreted and released into the environment e.g. via manure and sludge used as fertilizer on fields or effluent from aquaculture (Jørgensen and Halling-Sørensen, 2000). Therefore, residual concentrations of pharmaceutical antibiotics can be found in soils, surface and ground water (Hamscher et al., 2002, Kolpin et al., 2002, Simon, 2005). The fate of antibiotics including sorption and fixation, mobility and transport is well documented (Tolls, 2001, Figueroa et al., 2004, Kulshrestha et al., 2004), whereas the knowledge about their ecotoxicity and the effect of antibiotics on soil microbial functioning is scarce (Thiele-Brun, 2003, Boxall et al., 2003).

Under intensive pressure of industrialization in both developing and developed countries, increasing amounts of copper and other heavy metals are entering agricultural fields and contaminate the food-chain. Wastewater irrigation (Luo et al., 2003), compost application, including municipal waste, sewage sludge or their combination (Zheljazkov and Warman, 2003), and the application of agrochemicals containing heavy metals (Besnard et al., 2001) have been reported to contribute to the input of copper and other heavy metals in agricultural soils. Excess copper in soils is toxic to plants and soil organisms. Plant yield reduction and growth retardation (Moreno et al., 1997) and changes in community structure of soil microorganisms and nematodes (Giller et al., 1998, Ellis et al., 2001) have been reported in metal-contaminated soils. However, conflicting findings with respect to microbial responses to metals may arise from differences in bioavailability (McGrath, 1994). Microorganisms respond mainly to the soluble metal fraction and the proportion of total metals present in this fraction may differ with soil type, environmental conditions and time of measurement relative to metal inputs (Dar and Mishra, 1994).

Soil microorganisms play important roles in many ecosystem processes such as biogeochemical cycling of nutrients, soil structural and hydrological properties and energy flow (Doran and Zeiss, 2000, Lesser et al., 2004, Driver et al., 2004, Šantrůčková et al., 2004). Thus, maintenance of the biological activity in the soil is generally regarded as a key feature of sustainable production to ensure ecosystem functions (Swift, 1994), and soil microbial properties are often used as indicators of soil quality. Microbial community function provides a practical and ecologically relevant measure of microbial diversity (Zak et al., 1994). Community functional diversity indicates its potential activity, i.e. the capability of the community to adapt metabolism and/or composition and size to different conditions. Community level physiological profiles (CLPP) (Garland and Mills, 1991) assessed by BIOLOG Microplate^® have been widely used to investigate functional diversity of soil microbial communities (Garland, 1997, Di Giovanni et al., 1999, Mäder et al., 2002, Li et al., 2004). Despite a number of limitations, e.g. focus on bacterial species that are able to respond rapidly to the substrates, changes in community composition during growth (Preston-Mafham et al., 2002), the method can provide insights into the effect of disturbance on microbial communities.

Our interests in oxytetracycline (OTC) and its effect on soil microorganisms was provoked by the evidence that it was widely used, its concentration in fresh feces of Simmental calves treated with 60 mg/kg/d of OTC can be very high (871.7 mg/kg, De Liguoro et al., 2003) and has been shown to reduce nitrification rates and growth of bacteria (Halling-Søensen, 2001, Halling-Søensen et al., 2003). OTC is active in vitro against gram-positive and gram-negative bacteria, but has little effect on fungi (Anderson and Domsch, 1993). It inhibits protein synthesis by disrupting amino acid chain elongation at the 30S subunit of ribosomes (Backhaus and Grimme, 1999). Cu concentrations are high in soils in some regions in China because of the input of agricultural chemicals containing Cu and the use of Cu in animal feed as growth promoter. In these regions the use of antibiotics in animal husbandry is also increasing. Thus, soils in these regions can be contaminated by both pollutants, but little is known about their effect on soil microorganisms.

Objectives of the present study were therefore to elucidate the effects of OTC and Cu and their combined effects on soil community level physiological profiles as an indicator for microbial community function.