Pollution-induced Community Tolerance Research Articles

PO 43− dependence of the tolerance of autotrophic and heterotrophic biofilm communities to copper and diuron

Pollution-induced community tolerance (PICT) concept is based on the assumption that the toxicant exerts selection pressure on the biological communities when exposure reaches a critical level for a sufficient period of time and therefore sensitive species are eliminated. However, induced tolerance of microbial biofilm communities cannot be attributed solely to the presence of toxicants in rivers but also to various environmental factors, such as amount of nutrients. An experimental study was undertaken to highlight the potential impact of a phosphorus gradient on the sensitivity of periphytic microbial community to Cu and diuron. Biofilms were exposed to real-world levels of chronic environmental contamination of toxicants with a phosphorus gradient. Biofilm sensitivity to Cu and diuron was assessed by performing short-term inhibition tests based on photosynthetic efficiency to target photoautotrophs, extracellular enzyme activity (β-glucosidase and leucine-aminopeptidase) and substrate-induced respiration activity to target heterotrophs. The impact of P-gradient associated to pollution was evaluated by measuring pesticide concentrations in biofilms, biomass parameters (chl a, AFDW), bacterial cell density, photosynthetic efficiency and community structure (using 18S and 16S rDNA gene analysis to target eukaryotes and DGGE and HPLC pigment analysis to target bacteria and photoautotrophs). The obtained results show that depending on the studied toxicant and the used structural or functional parameter, the effect of the phosphorus gradient was variable. This highlights the importance of using a range of parameters that target all the biological communities in the biofilm. The PICT method can be regarded as a good tool for assessing anthropogenic environmental contamination, but it is necessary to dissociate the real impact of toxicants from environmental factors.

The periphyton as a multimetric bioindicator for assessing the impact of land use on rivers: an overview of the Ardières-Morcille experimental watershed (France)

Developing new biological indicators for monitoring toxic substances is a major environmental challenge. Intensive agricultural areas are generally pesticide-dependent and generate water pollution due to transfer of pesticide residues through spray-drift, run-off and leaching. The ecological effects of these pollutants in aquatic ecosystems are broad-ranging owing to the variety of substances present (herbicides, fungicides, insecticides, etc.). Biofilms (or periphyton) are considered to be early warning systems for contamination detection and their ability to reveal effects of pollutants led researchers to propose a variety of methods to detect and assess the impact of pesticides. The present article sought to provide new insights into the ecological significance of biofilm microbial communities and to discuss their bioindication potential for water quality and land use by reporting on 4 years of research performed on the French Ardieres-Morcille experimental watershed (AMEW). Various biological indicators have been applied during several surveys on AMEW, allowing the characterisation of (i) the structure and diversity of biofilm communities [community level finger printing (CLFP) such as PCR–DGGE and pigment classes], (ii) functions associated with biofilm [community level physiological profiles (CLPP) such as extracellular enzymes, pesticides biodegradation or carbon sources biodegradation] and (iii) biofilm tolerance assessment (pollution-induced community tolerance, PICT) of the main contaminant in the AMEW (copper and diuron). Approaches based on CLFPs and PICT were consistent with each other and indicated the upstream–downstream impact due to the increasing land use by vineyards and the adaptation of algal and bacterial communities to the pollution gradient. CLPPs gave a contrasted bioindication because some parameters (most of the tested extracellular enzymes activities) did not detect a pollution gradient. Such CLPPs, CLFPs and PICT methods applied to biofilm could constitute the basis for a relevant in situ assessment both for chemical effects and aquatic ecosystem resilience.

Development of pollution-induced community tolerance is linked to structural and functional resilience of a soil bacterial community following a five-year field exposure to copper

Copper (Cu) is accumulating in agricultural soils worldwide creating concern for adverse impacts on soil microbial communities and associated ecosystem services. In order to evaluate the structural and functional resilience of soil microbial communities to increasing Cu levels, we compared a Cu-adapted and a corresponding non-adapted soil microbial community for their abilities to resist experimental Cu pollution. Laboratory soil microcosms were set-up with either High-Cu soil from Cu-amended field plots (63 g Cu m −2) or with Low-Cu control soil from the same five-year field experiment. Laboratory treatments consisted of Cu amendments in the presence or absence of pig manure. Microbial activities (soil respiration, substrate-induced respiration, [ 3H]leucine incorporation), bacterial community structure (terminal restriction fragment length polymorphism, T-RFLP), community-level physiological profiles, and pollution-induced bacterial community tolerance (PICT detected using the [ 3H]leucine incorporation technique) were monitored for 12 weeks. The High-Cu and Low-Cu soil microbial communities initially exhibited almost identical structure and function and could only be distinguished from each other by their differential Cu tolerance. Experimental Cu pollution inhibited microbial activities, affected bacterial community structure, and induced further bacterial community tolerance to Cu. However, Low-Cu and High-Cu soil microbial communities showed essentially identical responses. Manure amendment did not protect against Cu toxicity and slightly increased Cu bioavailability as measured by a Cu-specific whole-cell bacterial biosensor. Our results indicate convergence of bacterial community structure and function in the High-Cu and Low-Cu soils during the five-year field experiment. We conclude that soil bacterial communities can exhibit structural and functional resilience to a five-year Cu exposure by virtue of their ability to develop Cu tolerance without affecting overall community structure. The observed increased Cu tolerance may involve phenotypic adaptation or selection at the micro-diversity level, for example an increased proportion of Cu-resistant strains within each bacterial species, which go undetected by T-RFLP community fingerprinting. Finally, our results indicate that Cu-dissolved organic matter complexes contribute to microbial toxicity in manure-amended soils implying that free Cu may comprise a poor predictor of metal toxicity.

In situ relationships between spatio-temporal variations in diuron concentrations and phototrophic biofilm tolerance in a contaminated river

Microbial biofilms are useful indicators for monitoring the ecological state of rivers. When assessing the effects of toxicants on microbial communities in natural ecosystems, special attention must be paid to the distinction between such effects and those resulting from other environmental variables. The pollution-induced community tolerance (PICT) approach offers the means to partially isolate effects of individual toxicants within a complex ecosystem by studying shifts in community sensitivity. To validate PICT for site-specific risk assessment, we investigated how closely diuron tolerance acquisition by photoautrophic biofilm communities could reflect their previous in situ exposure to this herbicide. To this end, a field survey was conducted for 9 months in a French river contaminated by diuron inputs from adjacent vineyards. Spatio-temporal variations in diuron tolerance capacities of photoautotrophic communities were estimated monthly using short-term photosynthetic bioassays. The biological survey was coupled with intensive physico-chemical monitoring to analyze the environmental factors influencing tolerance induction. We revealed a possible influence of three co-varying environmental variables (nitrates, conductivity and temperature), but statistical analysis clearly demonstrated that the main factor explaining variation in diuron sensitivity was the mean in situ diuron exposure level during biofilm colonization periods. A remarkable exponential correlation between EC 50 values and in situ diuron concentrations ( R 2 = 0.90, p < 0.0001) was recorded, confirming that PICT can be a powerful tool for the environmental monitoring of rivers.

Co-tolerance of phytoplankton communities to photosynthesis II inhibitors

Natural variability in sensitivity and pollution induced community tolerance (PICT) to atrazine, isoproturon and diuron and a mixture of these three herbicides to natural algal assemblages in mesocosms was determined. The specificity of PICT was examined by evaluating co-tolerance pattern for these photosystem-II (PSII) inhibitors. Phytoplankton communities were constantly exposed to equipotent concentrations of atrazine, isoproturon, diuron namely the 30% hazard concentration (HC 30) obtained from species sensitivity distributions and an equitoxic mixture (Σ3 × 1/3 × HC 30 of each herbicide) for five weeks in outdoor mesocosms. Induction of tolerance to the various herbicides was investigated by photosynthetic efficiency measurements of the algal assemblages in short-term laboratory tests. The composition of the algal communities in the various treatments was determined and ordination techniques such as the principal component analysis (PCA) were applied to log-transformed data to compare the seasonal community structure development. Temporal variation in sensitivity of the control algal assemblage to atrazine and isoproturon, but less to diuron was observed. The results further demonstrated that the control communities were in general more sensitive than the treated ones over the whole period tested indicating an enhanced tolerance of pre-exposed phytoplankton in the mesocosms. Co-tolerance was also observed for atrazine pre-exposed algal community to isoproturon, however, not vise versa. A pre-exposure to diuron induced similar tolerance to all three herbicides. A pre-exposure to the mixture treatment also lead to tolerance to isoproturon and diuron, less to atrazine. Overall, the observed co-tolerance pattern indicates that co-tolerance was not comparable between the herbicides with strong similarity in their biochemical mode of action.

Community responses to contaminants: Using basic ecological principles to predict ecotoxicological effects

Community ecotoxicology is defined as the study of the effects of contaminants on patterns of species abundance, diversity, community composition, and species interactions. Recent discoveries that species diversity is positively associated with ecosystem stability, recovery, and services have made a community-level perspective on ecotoxicology more important than ever. Community ecotoxicology must explicitly consider both present and impending global change and shift from a purely descriptive to a more predictive science. Greater consideration of the ecological factors and threshold responses that determine community resistance and resilience should improve our ability to predict how and when communities will respond to, and recover from, xenobiotics. A better understanding of pollution-induced community tolerance, and of the costs of this tolerance, should facilitate identifying contaminant-impacted communities, thus forecasting the ecological consequences of contaminant exposure and determining the restoration effectiveness. Given the vast complexity of community ecotoxicology, simplifying assumptions, such as the possibility that the approximately 100,000 registered chemicals could be reduced to a more manageable number of contaminant classes with similar modes of action, must be identified and validated. In addition to providing a framework for predicting contaminant fate and effects, food-web ecology can help to identify communities that are sensitive to contaminants, contaminants that are particularly insidious to communities, and species that are crucial for transmitting adverse effects across trophic levels. Integration of basic ecological principles into the design and implementation of ecotoxicological research is essential for predicting contaminant effects within the context of rapidly changing, global environmental conditions.

Pollution-induced tolerance of soil bacterial communities in meadow and forest ecosystems polluted with heavy metals

Pollution-induced community tolerance (PICT) allows finding a cause–effect relationship between pollution and adverse changes in a community. In our previous study we found that functional diversity of bacterial communities decreased significantly with increasing metal concentration, in both forest humus and meadow topsoil. Thus, the aim of the present study was to test whether tolerance of soil bacterial communities had increased as an effect of long-term metal pollution. Bacterial tolerance was tested with the use of the Biolog ® ECO plates in soils originating from the most polluted and the least polluted sites from three forest and five meadow transects located near smelters in Avonmouth (England), Clydach (Wales), and Głogów and Olkusz (Poland). We found that tolerance of bacterial communities was significantly increased in polluted meadow soils when compared to control meadow soils. On the contrary, no increase in tolerance was detected in polluted forest humus.

Increased Pollution-Induced Bacterial Community Tolerance to Sulfadiazine in Soil Hotspots Amended with Artificial Root Exudates

Sulfadiazine (SDZ) residues constitute an important pollutant in soils that may increase environmental reservoirs of antibiotic resistance. Our primary aim was to compare the development of pollution-induced community tolerance (PICT) to SDZ concentration levels in bulk soil and nutrient amended soil hotspots. Agricultural soil microcosms were amended with different concentrations of SDZ with or without weekly additions of artificial root exudates corresponding to realistic rhizodeposition rates. Bacterial community tolerance to SDZ residues, as determined by the [3H]leucine incorporation technique, increased progressively with elevated SDZ exposure, and was significantly increased in soil hotspots (LOEC of 1microg kg(-1)). An alternative PICT approach based on single-cell esterase probing by flow cytometry failed to demonstrate SDZ impacts. Bacterial growth rates ([3H]leucine incorporation) were significantly reduced in both bulk soil and hotspots 24 h after amendment with environmentally relevant concentrations of SDZ, while soil respiration remained unaffected even at 100 microg SDZ g(-1). Our study for the first time demonstrates a drastically increased PICT response of a soil bacterial community due to increased carbon substrate amendment per se. Hence, hotspot soil environments such as rhizosphere and manure-soil interfaces may comprise key sites for proliferation of bacteria that are resistant or tolerant to antibiotics.

Effects of sulfamethoxazole on soil microbial communities after adding substrate

The effect of the antibiotic sulfamethoxazole (SMX) on soil bacteria was studied using two methods (leucine incorporation and Biolog plates) of estimating pollution-induced community tolerance (PICT). SMX was added to an agricultural soil in a microcosm setup. The addition of different substrates (manure and alfalfa), and a non-amended soil, were also studied over 5 weeks. PICT measurements were validated by comparison with other measurements. Community structure was assessed using phospholipid fatty acid (PLFA) analysis and community-level physiological profiling (CLPP), and bacterial growth was estimated using leucine incorporation. Increased PICT was found at SMX concentrations of 20 and 500 mg SMX kg −1 soil in samples containing manure and alfalfa, and at 500 mg SMX kg −1 soil in non-amended soil (only concentration tested) using leucine incorporation. No effect was seen at 1 mg SMX kg −1 soil. It was not necessary to add any substrate to increase the microbial activity in order to detect the effects of a bacteriostatic toxicant such as SMX when using measures based on bacterial growth. Direct inhibition of bacterial growth 2 days after SMX addition was correlated to PICT. No major changes in PICT due to SMX addition were found when using Biolog plates. However, there was a tendency towards increased PICT at the higher SMX concentrations in the manure-amended soil. Thus, different methods of detecting PICT have different sensitivities in detecting the toxic effects of SMX. The effects of substrate amendment were reflected by changes in the microbial community, estimated using both PLFA and CLPP. SMX was found to have a clear effect at the two highest levels of SMX in the manure- and alfalfa-amended soils, with an increase in fungal and a decrease in bacterial PLFAs. Little difference in the PLFA composition was found in the non-amended soil. CLPP was only affected at the highest SMX concentration. Although different variables showed different sensitivities to the effects of SMX, the results were consistent with an initial decrease in bacterial growth rates of sensitive species, which eventually transformed into more tolerant species, altering the community composition.

Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton

This study analyzes psbA gene sequences, predicted D1 protein sequences, species relative abundance, and pollution-induced community tolerance in marine periphyton communities exposed to the antifouling compound Irgarol 1051. The mechanism of action of Irgarol is the inhibition of photosynthetic electron transport at photosystem II by binding to the D1 protein. The metagenome of the communities was used to produce clone libraries containing fragments of the psbA gene encoding the D1 protein. Community tolerance was quantified with a short-term test for the inhibition of photosynthesis. The communities were established in a continuous flow of natural seawater through microcosms with or without added Irgarol. The selection pressure from Irgarol resulted in an altered species composition and an inducted community tolerance to Irgarol. Moreover, there was a very high diversity in the psbA gene sequences in the periphyton, and the composition of psbA and D1 fragments within the communities was dramatically altered by increased Irgarol exposure. Even though tolerance to this type of compound in land plants often depends on a single amino acid substitution (Ser(264)-->Gly) in the D1 protein, this was not the case for marine periphyton species. Instead, the tolerance mechanism likely involves increased degradation of D1. When we compared sequences from low and high Irgarol exposure, differences in nonconserved amino acids were found only in the so-called PEST region of D1, which is involved in regulating its degradation. Our results suggest that environmental contamination with Irgarol has led to selection for high-turnover D1 proteins in marine periphyton communities at the west coast of Sweden.

A retrospective analysis of contamination and periphyton PICT patterns for the antifoulant irgarol 1051, around a small marina on the Swedish west coast

Irgarol is a triazine photosystem II (PSII) inhibitor that has been used in Sweden as an antifouling ingredient since the 1990s. Early microcosm studies indicated that periphyton was sensitive to irgarol at concentrations regularly found in harbours and marinas. However, field studies of irgarol effects on the Swedish west coast in 1994, using the pollution-induced community tolerance (PICT) approach, failed to detect any effects of the toxicant in the field. A PICT study involves sampling of replicate communities in a gradient of contamination, and a comparison of their community tolerance levels, with an increase being an indication that sensitive species have been eliminated and replaced by more tolerant ones. Typically, short-term assays are used to quantify the community tolerance levels. Later PICT studies in the same area over a 10 year period demonstrate that irgarol tolerance levels have increased, although the contamination pattern has been stable. Our results support the hypothesis that that the PICT potential was low initially, due to a small differential sensitivity between the community members, and that a persistent selection pressure was required to favour and enrich irgarol-tolerant species or genotypes.

Pollution‐induced community tolerance as a measure of species interaction in toxicity assessment

1 The assessment of the environmental hazards posed by chemical pollutants typically results from single-species tests that are extrapolated to ecosystems. The aim of the present study was to compare this type of extrapolation for a herbicide with the chronic effects that may be observed at a community level and to evaluate currently applied risk assessment strategies for their ability to predict chemical effects on complex communities. 2 Freshwater periphyton communities, grown in indoor aquaria, were exposed to the pollutant diuron for 3 months. Acute toxic effects of diuron were detected as photosynthesis inhibition using quenching analysis of chla-fluorescence. Chronic effects of the herbicide were observed in terms of changes in biomass and algal class composition as well as pollution-induced community tolerance (PICT). The PICT concept is based on a chemical exerting selection pressure on a community and therefore eliminating sensitive species. As a result, the measured community tolerance increases. 3 Short-term effects of diuron arise from 4–9 µg L−1 as half-maximal effect concentration (EC50). It is further shown that diuron concentrations down to 0·08 µg L−1 caused chronic effects in two independent microcosm studies. The observed threshold concentration of 0·08 µg L−1 still caused changes in biomass and class composition as well as an increased community tolerance. The determined EC50 values increased by a factor of 2–3 in diuron-exposed periphyton communities. This threshold value could not be predicted by advanced extrapolation methods such as species sensitivity distribution or acute-to-chronic effect ratios. 4 Synthesis and applications. The chronic community-level effects of the pollutant diuron were not predictable from single-species tests. However, regulations such as the EC Water Framework Directive or the EC-REACH process (Registrations, Evaluation and Authorisation of Chemicals) rely on this type of information. The management of chemicals in the environment should be based upon higher-tier assessment tools. Species interaction, detectable and quantifiable by the PICT methodology, may serve as a prognostic tool in chemical hazard assessment when extrapolating effects from single-species tests to community level.

No Long-Term Persistence of Bacterial Pollution-Induced Community Tolerance in Tylosin-Polluted Soil

Pollution-induced community tolerance (PICT) of soil bacteria to the antibiotic tylosin was studied over 95 days. Tylosin was added at increasing concentrations, together with different amounts of alfalfa to study the effects of substrate addition on PICT and bacterial growth in soil. The leucine incorporation technique was used to estimate bacterial growth and as a detection method in the PICT concept. Direct inhibition of the bacterial growth rates, resulting in a dose-response curve, was found above 50 mg of tylosin kg(-1) of soil two days after tylosin addition (IC50 value of 960 mg tylosin kg(-1)). After 10 days of exposure to at least 50 mg of tylosin kg(-1), the PICT was observed and correlated to inhibition of bacterial growth by tylosin. A return of the PICT to control levels was found over time, and after 95 days at 1500 mg of tylosin kg(-1), essentially no PICT was found, as compared to the unpolluted control soil. The return of PICT to pre-exposure levels was not totally reflected in the recovery of bacterial growth. Alfalfa addition did not affect the inhibitory effect of tylosin on bacterial growth rates; neither did it alter the PICT. Since tylosin is relatively rapidly degraded in soil, our results indicate that the PICT will return to prepollution levels when the selective pressure of the toxicant is removed and will thus be a useful technique for monitoring remediation measures.

Use of pollution‐induced community tolerance of the bacterial community to detect phenol toxicity in soil

Pollution-induced community tolerance (PICT) was used to study effects of phenol on soil bacteria. Phenol was added to an agricultural soil in a microcosm experiment. The effects were studied for up to four months. Bacterial growth rates were estimated with the leucine incorporation technique. This technique was also used as detection method for PICT. Changes in community structure were studied using the phospholipid fatty acid (PLFA) pattern. Increased phenol PICT of the bacterial community was found at phenol concentrations above 1 micromol/g wet weight soil. Direct inhibiting effect on bacterial growth rates 1 d after adding phenol was correlated to PICT. Phenol toxicity was reflected by changes in the structure of the bacterial community, although PICT appeared more sensitive than the PLFA method. In soil amended with 1 to 10 micromol phenol/g soil, bacterial growth recovered within one week. In the soil amended with the highest phenol concentration (30 micromol/g soil), bacterial growth rate recovered from total inhibition after 27 d, eventually reaching values six times higher than in the control. However, PICT did not change during the four months the experiment was performed. The specificity of PICT was also studied by examining cotolerance to 2-chlorophenol, 2,4-dichlorophenol, 2,3,6-trichlorophenol, Cu, and Zn. Adding phenol induced cotolerance of the bacterial community to the other phenols, although always at a lover level than to phenol. No cotolerance was found to metals in phenol-polluted soil. We conclude that the PICT concept is a valuable tool in determining phenol toxicity to bacterial communities, especially in situations where bacterial growth has recovered. Cotolerance between different phenols can, however, make interpretations of PICT more complicated.

How to confirm identified toxicants in effect-directed analysis

Due to the production and use of a multitude of chemicals in modern society, waters, sediments, soils and biota may be contaminated with numerous known and unknown chemicals that may cause adverse effects on ecosystems and human health. Effect-directed analysis (EDA), combining biotesting, fractionation and chemical analysis, helps to identify hazardous compounds in complex environmental mixtures. Confirmation of tentatively identified toxicants will help to avoid artefacts and to establish reliable cause-effect relationships. A tiered approach to confirmation is suggested in the present paper. The first tier focuses on the analytical confirmation of tentatively identified structures. If straightforward confirmation with neat standards for GC-MS or LC-MS is not available, it is suggested that a lines-of-evidence approach is used that combines spectral library information with computer-based structure generation and prediction of retention behaviour in different chromatographic systems using quantitative structure-retention relationships (QSRR). In the second tier, the identified toxicants need to be confirmed as being the cause of the measured effects. Candidate components of toxic fractions may be selected based, for example, on structural alerts. Quantitative effect confirmation is based on joint effect models. Joint effect prediction on the basis of full concentration-response plots and careful selection of the appropriate model are suggested as a means to improve confirmation quality. Confirmation according to the Toxicity Identification Evaluation (TIE) concept of the US EPA and novel tools of hazard identification help to confirm the relevance of identified compounds to populations and communities under realistic exposure conditions. Promising tools include bioavailability-directed extraction and dosing techniques, biomarker approaches and the concept of pollution-induced community tolerance (PICT). [figure: see text]

Lotic biofilm community structure and pesticide tolerance along a contamination gradient in a vineyard area

We investigated the environmental impact of diffuse pesticide pollution on natural river biofilms in the River Morcille, France, during 2 seasons in pristine and contaminated stations. Prokaryotic and eukaryotic community compositions were assessed by denaturing gradient gel electrophoresis, while microalgal community composition was determined by high pressure liquid chromatography pigment analysis. The sensitivity of microphytobenthos to the herbicide Diuron was investigated in the laboratory by short-term photosynthesis inhibition assays. Spatial changes in prokaryotic and eukaryotic species compositions were found in spring and in winter. Community structures were significantly different between contaminated areas and those that were uncontami- nated or less contaminated. Associated changes in biodiversity were not found. Community tolerance towards Diuron (based on EC50 values for photosynthesis) was significantly lower in upstream than in downstream photoautotrophic organisms. Pesticide concentration increased along the downstream gradient. These results strongly suggest contamination-driven changes in biofilm community struc- ture and in the tolerance of the photoautotrophic community, confirming the Pollution-Induced Community Tolerance (PICT) hypothesis.

Long-term Hg pollution induced Hg tolerance in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea)

The aim of our work was to assess the pollution-induced community tolerance (PICT) of isopod gut microbiota and pollution-induced isopod population tolerance (PIPT). Animals collected from a chronically Hg polluted and an unpolluted location were exposed for 14 days to 10 μg Hg/g dry food under laboratory conditions. The lysosomal membrane stability, hepatopancreas epithelium thickness, feeding activity and animal bacterial gut microbiota composition were determined. The results confirm the hypothesis that the response to short-term Hg exposure differs for animals from the Hg polluted and the unpolluted field locations. The animals and their gut microbiota from the Hg polluted location were less affected by Hg in a short-term feeding experiment than those from the unpolluted environment. We discuss the pollution-induced population tolerance of isopods and their gut microbiota as a measure of effects of long-term environmental pollution. The ecological consequences of such phenomena are also discussed.

Short term exposure to elevated trinitrotoluene concentrations induced structural and functional changes in the soil bacterial community

We investigated the acute impact of trinitrotoluene (TNT) contamination of soil on the aerobic bacterial community composition and function. The contamination of the environment with explosive residues presents a serious problem at sites across the world, with the highly toxic compound TNT being the most widespread explosive contaminant. We investigated the acute impact of trinitrotoluene (TNT) contamination of soil on the aerobic bacterial community composition and function. Soil microcosms were amended with a range of concentrations of TNT for 30 days. A polyphasic approach encompassing culture-independent molecular analysis by DGGE, community-level physiological profiling (CLPP) and cell enumeration revealed that the amendment of soils with TNT resulted in a shift from slower growing k-strategists towards faster growing r-strategists. Pseudomonads became prevalent at high concentrations of TNT. Pollution induced community tolerance (PICT) was observed as TNT concentrations increased. Chemical analyses revealed that TNT was reduced to its amino derivatives, products of reductive microbial transformation. The transformation to amino derivatives decreased at high concentrations of TNT, indicative of inhibition of microbial TNT transformation.

Linking pollution induced community tolerance (PICT) and microbial community structure in chronically metal polluted estuarine sediments

We tested the ability of pollution induced community tolerance (PICT) to detect the effects of chronic metal pollution on estuarine sediment microbial communities, along a gradient spanning two orders of magnitude in metal concentrations. In tandem, we investigated the associated microbial community structure using terminal restriction fragment length polymorphism (T-RFLP). Tolerance of microbes to Cu, measured as IC50 (inhibitory concentration 50%), was strongly correlated with pore water Cu concentration (r(2)=0.842). No strong correlation existed for other metals tested, highlighting the ability of PICT to identify the pollutant causing a toxic effect. There was no correlation between microbial community structure and community tolerance to metals tested, but analysis of community structure did provide some information on reasons for observed PICT response. PICT methodology used here provided a greater strength and consistency of association with pollutant concentration compared to microbial community structure and can be recommended as a sensitive indicator of metal pollution on estuarine sediment microbial communities.

Community-level microalgal toxicity assessment by multiwavelength-excitation PAM fluorometry

In ecotoxicological studies involving community-level investigations, rapid and multiparametric fluorescence-based methods may provide substantial advantages over traditional methods used for structural and functional community analysis. Therefore, multiwavelength-excitation pulse-amplitude modulated (PAM) fluorometry was applied in this study to assess long-term changes in periphyton community structure, short-term effects on periphyton functioning (photosynthesis) and pollution induced community tolerance (PICT). For inter-calibration, periphyton structure was evaluated by chemotaxonomic analysis of accessory pigments and a four-wavelength-excitation PAM fluorometer. Short-term effects of herbicides were evaluated by fluorescence quenching analysis and 14C-incorporation as a proxy of primary production. Subsequently, the method was applied to assess structural and functional changes in periphyton communities after isoproturon exposure for 14 and 26 days, respectively. Results showed good correlation of the PAM fluorescence-based measurements with traditional methods for biofilms in the initial colonisation phase for structural and functional parameters. However, for biofilms older than 9 weeks PAM fluorescence may underestimate biomass. Multiwavelength-excitation PAM fluorometry showed good correlation with marker pigment concentrations indicating that this method provides a reliable estimate of the community structure. PAM fluorometry was able to quantify changes of biomass and follow relative shifts in class composition of biofilms under exposure of isoproturon. Short-term tests based on the quantification of the inhibition of the effective quantum yield revealed a concentration-dependent increase of PICT. The observation of two succession phases of the biofilms after 14 and 26 days of growth, respectively, revealed that sensitivity of biofilms decreased with increasing age and biomass, respectively, but PICT remained a characteristic parameter of exposed communities in a concentration-dependent relationship. In conclusion, multiwavelength-excitation PAM fluorometry has considerable potentials in multispecies and multiparameter assessment of toxic effects on community level in terms of (1) a combined and rapid evaluation of structural and functional parameters in parallel, (2) screening of trends over time, (3) observing effects in replication and (4) being non-destructive. The approach therefore provides a perspective for a better understanding of community-level effects as species interactions in terms of PICT and therefore a higher ecological realism in risk assessment of toxicants.