J Soils Sediments (2010) 10:556–567 DOI 10.1007/s11368-009-0167-9
SOILS, SEC 4 • ECOTOXICOLOGY • RESEARCH ARTICLE
Effects of nonylphenol on a soil community using microcosms Xavier Domene & Sónia Chelinho & José Paulo Sousa
Received: 6 October 2009 / Accepted: 9 December 2009 / Published online: 12 January 2010 # Springer-Verlag 2010
Abstract Purpose Nonylphenol polyethoxylates (NPEOs) are a group of surfactants known to be toxic and able to mimic estrogen compounds and thus interfere with the action of an animal’s endogenous hormones. NPEOs are easily biodegraded in the environment, but the last end product, nonylphenol (NP), is the most toxic and recalcitrant form and hence can have a longer half-life in the environment. Despite the fact that most NP is finally degraded, a small fraction may remain in soil for longer periods. In soils, the application of sewage sludge is the main source of NPEOs. The aim of this study is to provide data on the effects of NP on a simplified soil invertebrate community since only a few studies using single-species bioassays are available for terrestrial ecosystems in comparison with aquatic ecosystems. Materials and methods In our study, we assessed the effect of increasing NP concentrations (0, 10, 30, 90, and 270 mg NP kg–1) in soil microcosms containing a simplified soil community consisting of natural microorganisms, a primary producer (an oat seedling of Avena sativa), several consumers (the isopod Porcellionides sexfasciatus, the enchytraeid Enchytraeus crypticus, and the collembolans Folsomia candida, Ceratophysella (Hypogastrura) denticulata, and
Proisotoma minuta), and a predator species (the mite Hypoaspis aculeifer). The effects on the different taxa of the different NP concentrations were assessed over three sampling dates (28, 56, and 112 days) using the principal response curves method. Results and discussion The soil community did not change significantly at concentrations below 90 mg NP kg–1, which was selected as the nonobserved effect concentration (NOEC). The highest concentration (270 mg NP kg–1) changed the community significantly after 28 and 56 days, but this effect disappeared after 112 days, in accordance with the known rapid biodegradation of this compound in soil. Conclusions Taking into account the usual NP concentrations in soils with repeated applications of sludge, the environmental risk of NP to soils seems to be limited because the derived NOEC was clearly above the usual concentrations in soil reported in the literature. However, the use of highly polluted sludges or accidental spillages, together with the possible pollution exportation by runoff to aquatic ecosystems, which are highly sensitive to NP pollution, recommend the careful monitoring of this chemical in the environment. Keywords 4-Nonylphenol . Ecotoxicity . Invertebrate community . Microcosms
Responsible editor: Jörg Römbke X. Domene (*) Center for Ecological Research and Forestry Applications (CREAF), Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain e-mail:
[email protected] X. Domene : S. Chelinho : J. P. Sousa IMAR-CMA, Department of Life Sciences, University of Coimbra, 3004-517 Coimbra, Portugal
1 Introduction Nonylphenol polyethoxylates (NPEOs) are a large class of nonionic surfactants known to be toxic, endocrinedisrupting contaminants widely used in industrial, agricultural, commercial, and household applications such as detergents, emulsifiers, wetting and dispersing agents, antistatic agents, demulsifiers, and solubilizers (Staples et al. 2004; Soares et al. 2008).
J Soils Sediments (2010) 10:556–567
The biodegradation of nonylphenol ethoxylates leads to the accumulation of the simplest chemical forms of nonylphenol ethoxylates (nonylphenol [NP], nonylphenol monoethoxylate [NP1EO], and nonylphenol diethoxylate [NP2EO]) and nonylphenol carboxy acids (4-nonylphenoxy acetic acid [NP1EC], nonylphenoxy ethoxy acetic acid [NP2EC]) (Ahel et al. 1994b, c; Staples et al. 2001). The accumulation is due to the degradation process, which results in a decrease in the length of the ethoxy chain. This leads to higher hydrophobicity and lower degradation rates (Gejlsbjerg et al. 2001). This is why NP is the nonylphenol with the highest persistence in soil and the main nonylphenol associated with sewage sludge (90%; Soares et al. 2008). In addition, the most prevalent NPEs are also the most toxic intermediates; NPEOs (NP1EO, NP2EO, and especially NP) are more toxic in comparison with NPECs (Staples et al. 2004). Due to the potentially harmful effects of NPEO degradation products, their use and production have been banned in the European Union (European Parliament and Council of the European Union 2009, Directive 2003/53/EC) and they have been replaced by other surfactants (mainly alcohol ethoxylates) which are considered environmentally safer as they degrade more rapidly (Campbell 2002). However, interest in their environmental risk is still high since, despite the prohibition and the decreasing environmental concentrations of this compound, it is still present in environmental samples (Soares et al. 2008). More importantly, widespread use of NPEOs continues in many countries without any legal control (Sjöström et al. 2008). In the environment, nonylphenol (NP) results from the microbial degradation of NPEOs (Roberts et al. 2006; Gabriel et al. 2008). Since NPEOs are synthesized from technical nonylphenol, the NP released during the microbial metabolism of the surfactant is the original technical material. Numerous isomers exist for NP, depending on the structure and the position of the alkyl moiety attached to the phenol ring, but generally more than 90% of the mixture consists of para-substituted NPs (Gabriel et al. 2008). The different isomers also show a variable endocrine-disruption potential depending on the structure of the alkyl side chain (Preuss et al. 2006). The main source of NPEOs in the environment is the discharge of effluents from wastewater treatment plants to aquatic environments (Ahel et al. 1994a). In soils, the application of sewage sludge is the main source of NPEOs (Soares et al. 2008). To a lesser extent, occurrence of NPEOs in soil is also linked to other anthropogenic activities such as landfilling and accidental spillage (Soares et al. 2008). The toxicity of NPEOs and their numerous biodegradation intermediates is higher the shorter the length of the hydrophilic polyethylene glycol (ethoxylate) chain linked to
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the phenol ring. This is why higher lethal and sublethal effects of these chemicals have been shown in the shorterchain NPEOs (Lussier et al. 2000; Staples et al. 2004). Several studies have reported toxic effects on aquatic fauna (Lussier et al. 2000; Staples et al. 2004; Soares et al. 2008), partly due to the fact that NPEOs mimic estrogen compounds, interfering in the action of endogenous hormones by binding to the estrogen receptor and eliciting a biological response resulting in endocrine disruption (Quinn et al. 2006). However, and despite the usual presence of NPEOs in soils, not much is known about their effects on terrestrial organisms in comparison to aquatic organisms, and the available data are restricted to their effects on plants and microorganisms (Gejlsbjerg et al. 2001; Dettenmaier and Doucette 2007; Domene et al. 2009). NPEOs have also been demonstrated to have toxic effects on mammals (de Jager et al. 1999; Ferguson et al. 2000), and the few existing studies have shown noxious effects on soil nematodes (Hood et al. 2000), earthworms, enchytraeids, and collembolans (see Domene et al. 2009 and references therein). In addition, no data regarding the effects on soil invertebrate communities as a whole have been published to date. In a previous study, we addressed the direct effects on soil invertebrates of NPEOs using isolated species with asexual reproduction, concluding that these compounds have low toxicity for soil invertebrates (Domene et al. 2009). In this study, we choose a microcosm approach for the assessment of the effects of NPEOs on soil organisms. Microcosms are enclosed model ecosystems consisting of an assemblage of species from various trophic levels and abiotic media that mimic a real environment. They may be homogeneous systems of sieved soil or intact cores taken from the field. The species may be introduced organisms or natural communities taken from the field (Burrows and Edwards 2002). Despite the fact that, unlike water studies, soil studies can easily be conducted in field conditions, soil microcosms are usually preferred by soil scientists because they allow the replication of experiments at a reasonable cost (Beyers and Odum 1993), the possibility to precisely control environmental conditions and the treatments under investigation (Fraser and Keddy 1997), but also because of their practicality and the rapidity of results (Carpenter 1996). In terms of ecological relevance, microcosms represent a middle point between single-species assays and semifield or field assays (Verhoef 1996; Kampichler et al. 2001; Edwards 2002). The results obtained are only applicable to the small ecosystems reproduced, and their relevance for real ecosystems can only be validated in field conditions (Kampichler et al. 2001). However, and despite the truth of these statements, the use of microcosms is very useful in different contexts, such as preliminary studies or scientific areas that cannot use
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field experiments due to ethical, legal, and environmental concerns or for regulatory purposes (Scott-Fordsmand et al. 2008). In addition, they have been shown to lead to similar conclusions as field studies, as found for soil invertebrates (Jänsch et al. 2006). The main aim of this study was to assess the short- and medium-term effects of NP pollution on a simplified biological community and to present nonobserved effect concentration (NOEC) community values over time. To achieve this goal, a microcosm study was conducted. This approach made it possible to take into account not only the direct effects of NPEOs on each individual species but also the indirect effects due to interaction between these species. In addition, the use of species with sexual reproduction permitted a more accurate assessment of the environmental risks of NPEOs for soils, since it has been suggested that these chemicals are endocrine disruptors.
2 Materials and methods 2.1 Soil collection and treatment The soil used in the microcosms was the upper layer (20 cm) of a loamy natural soil collected from an experimental agricultural soil within the campus of the Autonomous University of Barcelona (Cerdanyola del Vallès, Spain). The soil had formerly been used for grain production and had been free of pesticides for at least 5 years. After collection, the soil was air-dried for a week and sieved (5 mm). To remove the natural invertebrate communities that could influence the results, the soil was defaunated by two consecutive freezing–thawing cycles, each consisting in placing soil at −20°C for 4 days followed by a period of 4 days at 20°C. The soil properties are shown in Table 1. The apparently high copper levels found in this soil were inherited from the prior use of copper sulfate in traditional vineyard cultures of this area. This copper level was not expected to affect the results of the bioassays for two reasons. First, in previous studies using this soil, no apparent detrimental effects were found to different soil invertebrate species (Domene et al. 2009). For example,
toxic effects on the collembolan Folsomia fimetaria have been found over 2,400 and 800 mg Cu kg–1 for survival and reproduction, respectively (Pedersen and Van Gestel 2001) in soil spiked with copper. Second, due to aging processes, copper in this soil was expected to be mainly in nonavailable forms (Lock and Janssen 2003). For example, it has been shown that field soils containing up to 2,900 mg Cu kg–1 soil had no toxic effects on the reproduction of this species (Scott-Fordsmand et al. 2000; Pedersen et al. 2000) due to aging processes. Moreover, this copper level is common in many soils in southern Europe (Brun et al. 2001), as it is associated with the use of copper sulfate as a fungicide in viticulture soils since the end of the nineteenth century, thus representing a realistic scenario. 2.2 Nonylphenol spiking procedure Technical-grade 4-nonylphenol of 95% purity (Kao Corporation, Barcelona, Spain) was used in this study, hereafter referred to as NP. Due to its high hydrophobicity, different solutions of NP in acetone (95%, Panreac, Barcelona, Spain) were prepared in order to achieve different soil concentrations (0, 10, 30, 90, and 270 mg of NP kg−1 of soil). For each concentration, the soil was hydrated with a fixed volume of acetone solution containing the appropriate quantity of NP (150 ml of solution per kilogram of soil), a rate which permitted proper spiking of the chemical solution with soil. An equal acetone solution volume was applied to all the test concentrations. Pure acetone was added for the controls. The acetone was then left to evaporate for 24 h in a fume hood. For each concentration prepared, a small portion of the untreated soil (20%) was put aside to be used as microbial inoculums prior to the addition of the acetone solution. Once the acetone had evaporated and just before the beginning of the experiment, this soil portion was mixed with the polluted soil in order to reinoculate it with the original microbial soil community. Just before the construction of the microcosms, the soil batches were hydrated to 25% (corresponding to 45% of their water holding capacity). This moisture corresponded to the maximum water content that allowed this soil to have a crumbly structure, due to its loamy nature.
Table 1 Properties of the soils used in the bioassays pH
EC 25°C dSm−1
Sand %
Silt %
Clay %
C %
N %
C/N
CEC meq(+)/100g
Cd ppm
Cu ppm
Cr ppm
Ni ppm
Pb ppm
Zn ppm
8.3
0.2
36.4
44.9
18.7
2.63
0.18
14.6
13.9
