Elsevier

Chemosphere

Volume 89, Issue 1, September 2012, Pages 1-9
Chemosphere

The effect of triclosan on microbial community structure in three soils

https://doi.org/10.1016/j.chemosphere.2012.04.002Get rights and content

Abstract

The application of sewage sludge to land can expose soils to a range of associated chemical toxicants. In this paper we explore the effects of the broad spectrum anti-microbial compound triclosan on the phenotypic composition of the microbial communities of three soils of contrasting texture (loamy sand, sandy loam and clay) using phospholipid fatty-acid (PLFA) analysis. Each soil type was dosed and subsequently re-dosed 6 weeks later with triclosan at five nominal concentrations in microcosms (10, 100, 500, 1000 mg kg−1 and a zero-dose control). PLFA profiles were analysed using multivariate statistics focussing on changes in the soil phenotypic community structure. Additionally, ratios of fungal:bacterial PLFA indicators and cyclo:mono-unsaturated PLFAs (a common stress indicator) were calculated. It was hypothesised that triclosan addition would alter the community structure in each soil with a particular effect on the fungal:bacterial ratio, since bacteria are likely to be more susceptible to triclosan than fungi. It was also hypothesised that the PLFA response to re-dosing would be suppressed due to acclimation. Although the microbial community structure changed over the course of the experiment, the response was complex. Soil type and time emerged as the most important explanatory factors. Principal component analysis was used to detect phenotypic responses to different doses of triclosan in each soil. As expected, there was a significant increase in the fungal:bacterial ratio with triclosan dose especially in treatments with the highest nominal concentrations. Furthermore, the PLFA response to re-dosing was negligible in all soils confirming the acclimation hypothesis.

Highlights

► The effects triclosan on microbial community structure was explored using PLFAs. ► Community structure changes were complex. ► Soil type and time were the most important explanatory factors. ► There was a significant increase in the fungal:bacterial ratio with triclosan dose.

Introduction

Soil microbial communities control nutrient cycling and soil organic matter decomposition (e.g. Wang et al., 2008, Harris, 2009). The microbial community structure of agricultural soils can be influenced by several factors including soil texture (Bach et al., 2010), moisture content (Williams and Rice, 2007), pH (Rousk et al., 2010), organic matter content (Grandy et al., 2009) and temperature (Norris et al., 2002). In addition, microbial communities can be affected by agricultural management practices such as tillage (Helgason et al., 2010) and can be exposed to a wide range of potential toxicants either directly through the application of pesticides, or indirectly through the addition of contaminated manure (Blackwell et al., 2009) or sewage sludge (Heidler et al., 2006, Kannan et al., 2007).

One contaminant which has received recent attention in the literature is triclosan (e.g. Capdevielle et al., 2008, Reiss et al., 2009), an anti-microbial compound used in personal-care products. Triclosan is frequently detected in sewage-sludge as a result of partitioning during the waste-water treatment process (Bester, 2003) and can become a terrestrial contaminant through the sludge to land route. Aquatic studies have shown that triclosan can have a detrimental effect on algae at relatively low concentrations (Orvos et al., 2002, Wilson et al., 2003), and inhibited bacterial metabolism has also been observed (DeLorenzo et al., 2008). In soils, triclosan has been shown to increase dehydrogenase activity (Ying et al., 2007), and to affect microbial respiration rates (Waller and Kookana, 2009, Butler et al., 2011a). Triclosan concentrations as low as 4 mg kg−1 have been observed to affect microbial populations and inhibit the degradation of other xenobiotic compounds (Svenningsen et al., 2011). Increased persistence in soil may lead to an increased risk of leaching, although the leaching risk is mitigated by a high affinity for soil solids (Gustafson, 1995). In a previous study (Butler et al., 2011a), we showed that very high doses of triclosan relative to typical environmental exposure (e.g. 20–55 mg kg−1 in sewage sludge, Heidler and Halden, 2007 and 23.6–66.6 μg kg−1 in soil, Lozano et al., 2010), altered soil functional behaviour by way of short-term respiration inhibition and depression of the microbial biomass. Soil response to subsequent re-dosing suggested microbial acclimation, with evidence that triclosan was being utilised as a substrate.

Phospholipid fatty acids (PLFAs) are present in the membranes of all living cells but are rapidly decomposed by hydrolytic cellular enzymes after cell death. This means that the primary source of PLFAs in soil is living microbial cells. Extracted PLFAs can provide a phenotypic description of the associated soil community, reflecting species composition and abundance (Findlay, 2004). This method has been used extensively to track both spatial (Ritz et al., 2004, Mele and Crowley, 2008) and temporal (e.g. Hamel et al., 2006, Moore-Kucera and Dick, 2008) changes in community structure, and has also been used to determine the effects of pollutants on soil communities, including metals (e.g. Sverdrup et al., 2006, Tischer et al., 2008, Farrell et al., 2010) and organic toxicants (e.g. Elsgaard et al., 2001, Bartling et al., 2009, Zhang et al., 2010).

Changes in community structure can potentially affect the associated function of the soil ecosystem. For example, fungi can secrete compounds capable of binding soil particles into aggregates which provide physical protection for soil organic matter (van Groenigen et al., 2007) and, thus, enhance carbon sequestration (Six et al., 2006). Certain bacteria (e.g. Actinobacteria) are critical in the decomposition of organic matter and the release of inorganic nutrients and in humus formation (Steger et al., 2007). Others are exclusively responsible for nitrification (e.g. Purkhold et al., 2000). Fungi and bacteria have different C/N ratios (Bååth and Anderson, 2003), which is important in controlling the magnitude and direction of nitrogen mineralisation or immobilisation during litter decomposition. There are also important differences between these microbial groups in terms of their observed reaction to different stressors, such as metals (Rajapaksha et al., 2004), substrate starvation (Demoling et al., 2008) and soil pH (Rousk et al., 2010).

In this paper, we augment the results reported by Butler et al. (2011a) by evaluating the effect of triclosan addition on the composition of soil microbial community structure using PLFA profiling. We hypothesise that triclosan will have different toxic effects on different components of the microbial community, resulting in an altered phenotypic structure after dosing. Although triclosan is a broad spectrum anti-microbial compound, it is likely to have a more potent effect on bacteria than on fungi, due to its mode of action in targeting the FabI gene, which controls bacterial fatty acid synthesis. Fungi use a different fatty acid synthesis pathway which is not controlled by this gene (Carr et al., 2011), resulting in a relative increase in fungi as a result of dosing with high triclosan concentrations. In addition, we hypothesise that the microbial community can build up some resistance and resilience to triclosan dosing resulting in a less significant phenotypic response on re-dosing, in line with the respiration–inhibition response reported by Butler et al. (2011a). Note that the study design is not appropriate for deriving ecotoxicological effect end points such as lowest or no observable effect levels (LOELs or NOELs).

Section snippets

Soil microcosm preparation and sampling

A 70-d incubation experiment was conducted in soil microcosms. Methods employed in soil selection, soil sampling and soil preparation, microcosm set-up and dosing are described in full by Butler et al. (2011a). Briefly, bulk soil samples (approximately 20 kg) were collected from the upper 20 cm of three different agricultural soils (a sandy loam, clay, and loamy sand) at Silsoe Farm in Bedfordshire, United Kingdom. All sites have been under a similar arable rotation dominated by winter wheat and

Full factorial analysis

As a starting point, PCA was performed on all soils at all sampling times and at all nominal triclosan concentrations. However, third order interactions were observed in this analysis, making interpretation difficult. In particular, soil type made a major contribution to observed variability and masked other more subtle variations such as the effect of time and triclosan dose (see ANOVA Table 1). Time after dosing explained the next greatest proportion of the variance, with triclosan dose also

Discussion

Several factors may be responsible for altering the soil phenotypic community structure observed here. The most significant factor in this study was soil type. This could be driven by a number of factors including pH (Bååth and Anderson, 2003), texture and organic carbon content (Patra et al., 2008) which will influence the bioavailability and toxicity of triclosan (e.g. Orvos et al., 2002, Price et al., 2010) as well as affecting microbial community structure directly. The influence of soil

Conclusions

The PLFA analysis presented here suggests that the respiration inhibition observed in the same experiment by Butler et al. (2011a) was associated with parallel changes in microbial community structure. PCA projections showed that there was a temporal shift in the microbial community structure of all three soils, which may have been the result of pre-dosing disturbance. The PLFA data support the observation of damped respiration inhibition to re-dosing, corroborating the interpretation of

Acknowledgements

We are grateful to Dr Mark Pawlett for guidance in the analysis of PLFAs and to Jane Hubble for assistance with the analytical equipment. We are also grateful to the Engineering and Physical Sciences Research Council (EPSRC) and Unilever for funding.

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