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Unité de Phytopharmacie et Médiateurs Chimiques, Institut National de la Recherche Agronomique, Route de Saint-Cyr, F-78026 Versailles Cedex, France
* Corresponding author (mougin{at}versailles.inra.fr)
Received for publication January 27, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: NP, nonylphenol, mixture of isomers • 4NP, 4-n-nonylphenol • SA, sludge from Ambares • SMF, spore multiplication factor • SP, sludge from Plaisir
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Among organic chemicals, wastewater entering treatment plants contains nonylphenols (NP, a mixture of branched isomers), important compounds in the production of many commercial and industrial chemicals. Nonylphenol is used to produce nonylphenol polyethoxylates, which are nonionic surfactants widely used in domestic, agricultural, and industrial applications.
Nonylphenol is subsequently discharged into surface waters through the microbial biodegradation of these polyethoxylates in sewage treatments (Ahel et al., 1994a), and potential aquatic risks have been extensively studied (for reviews see Sumpter, 1998; Servos, 1999; Nilsson, 2000; Vos et al., 2000). In this context, it is important to make a distinction between 4-n-NP (a main compound present in the mixture of isomers) and NP (the mixture of isomers) because they have some distinct physicochemical properties. However, studies concerning the fate and toxic effects of nonylphenol compounds refer in most cases to 4-n-NP alone. Both in vitro and in vivo studies reported that this isomer is a potent endocrine disrupter, modulating steroidogenesis and activity of hormone-metabolizing enzymes, and inducing feminization. It binds onto cellular estrogenic receptors, thereby regulating the expression of estrogen-responsive genes (Machala and Vondracek, 1998), and can cause proliferation of breast cancer cells in women (Soto et al., 1991).
However, most NP formed during wastewater treatment is associated with sludge, amounting to 1 gram or more per kilogram of dry sludge (Ahel and Giger, 1985; Ahel et al., 1994a,b; APE Research Council, 2002). Nonylphenol is released onto terrestrial environments through sludge application on agricultural land. Nevertheless, only little work has been done on the fate of NP in soils (Topp and Starratt, 2000; Hesselsoe et al., 2001, Dubroca et al., 2003), and an assessment of the potential risk has rarely been conducted (APE Research Council, 2002). Yet, some studies revealed that NP can modify the structure of microbial communities of lake sediments (Jontofsohn et al., 2002). In the laboratory, production of carbon dioxide from soil is inhibited by concentrations of NP higher than 100 mg kg-1 (APE Research Council, 2002), which proved to be toxic to filamentous fungi and yeasts as the result of uncoupled respiration (Karley et al., 1997). Finally, it has been established that endocrine disrupters can interfere with flavonoid signaling during plant–bacterial symbiosis (Fox et al., 2001). To our knowledge, experiments have seldom been performed taking into account NP already present in the sludge and including the amendment of soil with the contaminated sludge. Using this experimental approach, Gejlsbjerg et al. (2001) were unable to demonstrate a negative effect of NP on bacterial denitrification, nitrification, or aerobic respiration in soils.
The objective of the present study was to assess the effect of NP on soil fungal populations as a result of contaminated sludge application. Adverse effects on the soil ecosystem depend on the bioavailability, degradation, and toxicity of the contaminant. The fate of NP in soil has been previously published (Dubroca et al., 2003). We examine here (i) the potential concentration of NP to which soil organisms may be exposed and (ii) the relevant toxicity by studying endpoints related to fungal growth (biomass production) and reproduction (sporulation and germination of spores).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Soil and Sludge Characteristics
The soil used in this study was a silt loam with 25.5% sand, 55.0% silt, and 19.5% clay. Its organic matter content was 1.65%. The soil pH was 8.1 and the cation exchange capacity was 10.2 cmol kg-1. The soil, collected in the 10- to 20-cm layer in a field, was sieved (2 mm) and used immediately.
Two types of sludge exhibiting different characteristics were applied on soils (Table 1) . The sludge from Ambares (SA) was formed following the treatment of both urban (90000 equivalent inhabitants) and industrial wastewaters, whereas that from Plaisir (SP) collected mostly urban wastewater (42000 equivalent inhabitants).
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All other characteristics of the sludge have been determined using normalized methods (Association Française de Normalisation, 2000).
Bioavailability of Nonylphenols
Amounts of NP bioavailable for soil fungi were determined using the protocol described by Gaillardon and Dur (1995). Soil and soil–sludge samples (10 g equivalent dry matter) were placed in 5-cm-diameter Petri dishes to give a 3- to 4-mm-thick layer. Aqueous solutions of unlabeled NP and labeled (4.0 kBq) 4-n-NP were applied to the surface of the soil alone by pipette to ensure 80% of the moisture-holding capacity of the soil, and a final concentration of 40 mg kg-1. Sludge samples were spiked with the mixture to allow the same final NP concentration in the soil. After adding the chemical, the solvent (acetone) was left to evaporate for 30 min. The chemicals were sorbed to the sludge for 24 h at 4°C under nitrogen atmosphere before the sludge was mixed with the soil. Soil to sludge ratio was 95:5 on a dry weight basis, considering only the first centimeters of the soil contaminated with sludge and not the whole plowed layer. All the dishes were placed in the dark at 4°C to avoid biotransformation.
Concentrations of NP in the soil solution were determined 4 h after treatment (soil alone) or mixing (with sludge). Two superposed 42.5-mm-diameter GF/A glass microfiber filters (Whatman, Maidstone, UK) were laid on the soil surface and a slight pressure was applied for 10 s to favor wetting of the filters. The upper filter was then recovered. The volume of soil solution and the dissolved radioactivity retained in the filter were determined by weighing and liquid scintillation counting.
Adsorption Isotherms
Adsorption isotherms were obtained using the batch equilibrium method. Aliquots of 25 mL of 0.05, 0.10, 0.15, 0.20, 0.30, and 0.40 mg L-1 water solutions of NP (unlabeled NP and labeled 4NP) supplemented with 10-2 M CaCl2 were added to 5 g of soil or soil–sludge mixture in centrifuge glass tubes. Equilibration was achieved by stirring for 24 h at 20°C. Five-milliliter aliquots of the supernatants were removed by centrifugation at 4220 x g for 30 min and immediately analyzed. Concentrations of free NP in the supernatant (the equilibrium concentrations, Ce) were determined by liquid scintillation counting. Control blanks were run in parallel to measure tube-wall adsorption of NP. Adsorption data were fitted with the Freundlich equation, x/m = KCe(1/n), where x/m is the amount of NP adsorbed in mg kg-1, and Ce is the equilibrium solution concentration of NP in mg L-1. The constant K is a measure of the magnitude of adsorption, or adsorption capacity of the sorbent.
Fungi
The fungal strains Fusarium oxysporum, F. solani (Martius) Saccardo, and Mucor racemosus Fresenius used in this study were all isolated from soil samples. The white-rot fungi Phanerochaete chrysosporium Burdsall and Trametes versicolor (L.: Fr.) Pilat were obtained from the American Type Culture Collection (ATCC; referenced as 24725 and 32745, respectively). They were maintained at 4°C on agar plates.
Fungal Liquid Cultures and Preparation of Suspensions of Spores
Fungal strains used to produce suspensions of spores were grown on a culture medium already published, containing glycerol (P. chrysosporium; Mougin et al., 1994) or maltose (all other strains; Lesage-Meesen et al., 1996) and ammonium tartrate as carbon and nitrogen sources. A mycelial mat on agar plugs (10-mm diameter) was inoculated into 10 mL of the culture medium in a 150-mL Erlenmeyer flask. Cultivation was performed statically in the dark at 25°C. After 8 to 12 d of growth, the spores were harvested by shaking the cultures with glass beads, counted, and conditioned as liquid suspensions containing 2.5 ± 0.5 x 106 spores mL-1 for inoculation. Spore counting was achieved by placing aliquots (200 µL) of the medium into a Thomas counting chamber.
Short-Term Toxicity Assessment
Culture media were supplemented with NP ranging from 10-3 to 10-7 M immediately after inoculation (acetonic solutions, 50 µL per Erlenmeyer flask). Effect of NP on spore germination was determined after a 24-h exposure by calculating the germinating rate. Fungal biomass was separated from the medium after 2, 4, and 8 d of culture and dried overnight at 105°C for dry weight determination. Effect of NP on sporulation was estimated by harvesting the spores formed from the cultures and inoculating them in liquid media not supplemented with NP. The germinating rate was also calculated after 24 h.
Long-Term Toxicity Assessment
Suspensions of spores were used to inoculate Erlenmeyer flasks containing 10 mL of liquid media supplemented with 5.0 x 10-6 M NP, or solutions of the chemical diluted by 2 at each inoculation to reach 3.1 x 10-7 M at the end of the experiment. The cultures were then allowed to grow and sporulate in the dark at 25°C for 10 d. Spores were harvested as described above and the experiments were still repeated four times. Fungal biomass was measured on a dry weight basis. Germination and sporulation were expressed as a spore multiplication factor (SMF), which reflects the ratio between the amounts of spores produced by a culture versus the amounts of spores used for inoculation.
Laccase Induction
Cultivation was performed as described above for 4 d. After this time, ethanolic solutions of NP or 4NP were added to the cultures to give concentrations ranging from 10-7 to 0.5 x 10-3 M (100 µL solution per Erlenmeyer flask). Aliquots were assayed for laccase activity after a further 3-d incubation.
Laccase Activity Measurements
Laccase production was assessed by measuring enzymatic oxidation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm (
= 3.6 x 104 cm-1 M-1) according to Wolfenden & Wilson (1982). The reaction mixture contained 20 µL extracellular fluid and 980 µL 1 mM ABTS in 0.1 M KH2PO4–citric acid buffer (pH 3.0) at 30°C. The buffer solution was saturated with air by bubbling before the experiment. One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 µmol ABTS in 1 min.
Experimental Design
Each experiment was performed in triplicate. Results are expressed as means ± SD. The IC50 values were calculated by nonlinear regression analysis. In several experiments, a Student's t test was performed to determine significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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These results incited us to measure NP concentration in soil solutions for nonamended and amended soils. Values obtained were 1.93 ± 0.23 mg L-1 for the soil alone, 1.46 ± 0.73 mg L-1 for the soil–SA mixture, 0.083 ± 0.004 mg L-1 for the soil–SP mixture. Mean values corresponded to concentrations of 8.8 x 10-6 M NP for the nonamended-soil solution, 6.6 x 10-6 M (after SA application: A limit), and 3.8 x 10-7 M (after SP application: P limit) NPs in soil solutions (Table 2) .
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1) (Table 2). The K values for the soil alone and the soil amended with SA were similar, suggesting no great change of adsorption of NP due to SA application. By contrast, SP application led to an increased adsorption of NP, with a 20-fold enhancement of the K constant.
Short-Term Toxicity of Nonylphenol on Fungi
We examined the dose–response relationship between NP concentration (ranging from 10-3 to 10-7 M) and the development of fungi in liquid cultures. This wide range included values determined above in the soil solution, but also higher ones intended to magnify potential effects. Only four fungal strains, namely P. chrysosporium, F. oxysporum, F. solani, and M. racemosus produced spores in our culture conditions, whereas T. versicolor was unable to sporulate. This strain was inoculated, as mycelial mats grown on agar plugs.
We first calculated the concentration decreasing the germination of spores by 50% after 24 h (IC50) compared with untreated controls (Table 3) . Our results showed that the "mother" spores obtained from P. chrysosporium were very sensitive to NP, with an IC50 of 7.5 x 10-6 M on germination rate. This value is similar to the A limit. The "mother" spores obtained from the three other strains were affected to a lesser extent.
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In addition, high doses of NP (greater than 10-4 M) induced morphological defects in F. solani hyphae. In summary, NP treatment resulted in a swelling of hyphae, associated with the loss of hyphal apical dominance, and increased branching (data not shown).
Finally, we studied the ability of "daughter" spores produced by fungi grown in the presence of NP to germinate in media free of the chemical. Their sensitivity appeared somewhat increased regarding those of "mother" spores, as calculated IC50 values were 2- to 80-fold lower (Table 3).
In addition, the suspensions of "daughter" spores obtained from the two strains of Fusarium grown in the presence of high concentrations of NP included numerous small spores (microspores) that were not able to germinate within the 24-h period taken into account (data not shown).
The IC50 values influencing fungal growth and reproduction are in most cases higher than the A limit, the highest value measured for the amount of NP bioavailable in soil solutions.
Long-Term Toxicity of Nonylphenol on Fungi
We examined the effect of NP on fungi following five cycles of biological events that could affect the size of fungal populations, namely spore germination, biomass formation, and spore production. Three culture conditions were used: controls without NP, assays under constant NP concentration of 5.0 x 10-6 M (below most of the IC50 values calculated above), and assays under decreasing concentrations of NP, thus reaching 3.1 x 10-7 M at the end of the experiment. These decreasing values mimic the natural disappearance of the chemical in the soil due to its transformation. These concentrations are consistent with P and A limits.
The calculated SMF, integrating both the production and germination of spores, varied according to the strains. As a general case, the SMF naturally decreased in control cultures after successive exposure of spores to NP, with a maximal effect between the two first generations (Fig. 1) . Nonylphenol also diversely affected this endpoint. A slight negative effect of NP on P. chrysosporium SMF was significant only at the beginning of the experiments, while a stronger effect was noticed throughout the experiment regarding M. racemosus and F. solani. For these three strains, the effect did not seem related to NP level in the medium. The most intensive effect of NP on fungal SMF was observed on F. oxysporum cultures. The SMF corresponding to this strain dramatically decreased in the absence of the chemical. By contrast, it increased up to 100 times by NP, with an effect increasing with the number of biological cycles. In that case, too, the pollutant level did not modulate the intensity of the effect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The amounts of chemicals bioavailable for soil microorganisms are rarely determined. Our results concerning the amount of NP measured in the soil solution under several adsorbents (soil or soil–sludge mixtures) depend on the physicochemical properties of the sludges. They demonstrate the decrease in bioavailability of NP following the application of the sludge from Plaisir onto the soil. Nevertheless, these amounts remain easily measurable, and our experiments provide minimum and maximum values for the concentrations of NP that can be used in fungal liquid cultures to assess the effect of the chemical in realistic environmental situations.
Experimental data for adsorption of NP to the soil and the soil–sludge mixtures resulted in a good fit to the Freundlich equation. Nonlinear L-shaped isotherms indicated that our nonamended soil has a moderate affinity for NP in the initial stages of adsorption (Giles et al., 1960, 1974). Linear C-type isotherms obtained in the presence of the two sludges suggested a constant partition of NP between the solution and the adsorbent. The calculated adsorption coefficient (K) for NP onto the soil–SP mixture was 20-fold higher than the value obtained with the other adsorbents, evidencing a higher extent of adsorption of NP in SP. These results agree with the lower amount of NP (two orders of magnitude) measured in the soil solution using static measurement. In soils, it has been reported that phenolic compounds of moderate hydrophobicity adsorb onto organic matter. Application of the sludges with similar amounts of organic matter resulted in distinct effects on adsorption, thus suggesting the involvement of other parameters, such as their chemical composition. Taken together, our data provide information for the first time on exposure of soil microorganisms to NP. They show that the bioavailability of NP can be significant when applied to soil following incorporation into sludge. In the absence of measured data, calculated predicted environmental concentrations (PEC) amounted to 2 to 4 mg kg-1 (APE Research Council, 2002).
The level of soil contamination in our study was higher to take into account the worst-case hypothesis, such as massive soil amendment with heavily contaminated sludge. In other terms, our experiments were performed with load of sludge above the limits generally permitted in European countries, which are 10 Mg dry matter ha-1 according to the EU Directive 86/278. In addition, it is noteworthy that concentrations of NP in the sludge were high (above 200 mg kg-1 dry matter). Expectable concentrations of NP in sludge will decrease in the future in Europe. A value limit of 50 mg kg-1 (dry matter) has been proposed in the Working Document intended to revise the directive.
Effect of NP on fungi was then assessed using three strains isolated from soils (namely F. solani, ascomycete; M. racemosus, zygomycete; and F. oxysporum, deuteromycete) and two white-rot basidiomycetes from our collection (P. chrysoporium and T. versicolor) that could also be found in soils. Endpoints take into account reproduction (production and germination of spores) and growth (biomass production), both governing the size of the fungal populations. It is difficult to assess an effect on fungal populations directly grown in soils. For this reason, some experiments were performed using fungal liquid cultures.
Short-term (acute) toxicity of NP was assessed first. Generally, the germination rate of fungal spores seemed moderately sensitive to exposure to NP in environmentally sound amounts. Nevertheless, even in the presence of higher amounts of NP, inducing a strong inhibition of germination, the consequences on the size of the resulting populations were slight. In fact, an adverse effect of NP on fungal growth was only detected in young fungal cultures in the 2 d following the treatment with the chemical. This kind of lag phase in growth has already been reported concerning Neurospora cultures exposed to NP with similar IC50 values (Karley et al., 1997). This effect was reduced after 4 d of culture, and totally suppressed after 8 d. These results suggest that nutrient availability remains the main factor governing fungal growth, as soon as the spores germinate. In our experiments, most of the calculated IC50 values for fungal growth (after 2 d of exposure) and spore germination were in the 1 to 10 x 10-6 M range. They compare well with the acute concentrations causing 50% lethality or inhibition in fish, amphibian, invertebrate, mollusk, or algae species (Weinburger et al., 1987; Servos, 1999). They were below the values reported to inhibit bacterial nitrification (Gejlsbjerg et al., 2001).
However, exposure to NP at doses higher than these resulting from soil amendment under good agricultural practice is not without other effects on fungi. An uncoupling effect of NP on respiration has already been described in liquid cultures supplemented with high concentrations of the chemical (Karley et al., 1997). These authors also reported morphological defects in hyphae in Neurospora crassa, consisting of a swelling of hyphae associated with the loss of hyphal apical dominance and increased branching. These abnormalities could be due to disruption of the hyphal free cytosolic Ca2+ gradient, the H+ gradient, and the actin cytoskeleton of the apical cells (Jackson and Heath, 1993; Karley et al., 1997). We observed these abnormalities in F. solani cultures (another ascomycete), as well as the formation of immature microspores. These results must be considered from an ecological point of view, because ascomycetes represent the main populations of soil fungi. In contrast, our results show that the white-rot basidiomycetes studied appeared less sensitive to NP than the other strains.
Possible long-term (chronic effects) were also investigated. We decided to integrate both the production and germination of the spores in a global index named the spore multiplication factor (SMF). The decrease often observed in the nontreated cultures reflects the loss of performance of the fungal strains due to natural nutrients possibly missing in our synthetic culture media. Nevertheless, both an increase of the SMF or its decrease suggest a chemical stress caused by NP. In chronic toxicity tests, no observable effect concentration (NOEC) has been determined as low as 10-5 M in fish and 10-8 M in invertebrates (Servos, 1999). The threshold for chronic toxicity on organisms living in sediments was 10-5 M (APE Research Council, 2002). Our data showed that a constant exposure of some fungal strains to 5.0 x 10-6 M NP modified their sporulation.
How NP can modify the reproduction of fungi remains to be elucidated. It has long been established that inhibition of the germination rate is mainly due to an effect on respiration, a typical mode of action of fungicide compounds (Leroux, 1996). However, little is known on the endogenous factors affecting sporulation, such as sexual hormone signaling. On one hand, several fungi are able to produce or metabolize several steroid hormones, thus affecting fungal growth and development (Brasch, 1997; Rizner et al., 1999; Rizner and ZakeljMavric, 2000). On the other hand, trisporic acid is a sexual hormone of zygomycetes, which triggers the first steps of zygophore formation (Czempinski et al., 1996). It is likely that NP, a well-known endocrine disrupter, may interfere with these sexual hormone pathways. The growth of P. chrysosporium and M. racemosus was not affected by NP in our experimental conditions maintained during five biological cycles.
Nonylphenol has also been shown to induce laccase production. 4-n-Nonylphenol is a more potent inducer than the complete mixture. This effect on extracellular oxidases may be considered as a positive effect, because it leads to a decreased bioavailability of the chemical and to its increased detoxification. This reaction may also lead to the stabilization of the chemical in the soil, thus preventing ground water contamination. As demonstrated earlier (Mougin et al., 2002), our present results confirm that xenobiotics can be potent inducers of extracellular enzymes of the ligninolytic pathway on fungi, even in low concentrations.
The relative tolerance to NP exhibited by several fungal strains could be related to their high efficiency in transforming NP. For example, white-rot basidiomycetes (P. chrysosporium and T. versicolor) are known to secrete extracellular oxidases (peroxidases and/or laccases) that are able to catalyze the rapid polymerization of NP through oxidative coupling (Tsutsumi et al., 2001; Dubroca et al., 2003). The reaction located in the culture medium efficiently reduced the bioavailability and toxic effect of the chemical. In addition, zygomycetes including Cunninghamella and Mucor spp. are known to possess intracellular P450 systems able to efficiently transform pollutants (Mougin, 2002).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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