Background, aim, and scope Soil remediation with ethylenediamine tetraacetic acid (EDTA) leaching is capable of removing only part of the total metal concentration in the soil, mostly the labile, bioavailable metal species (metal bioavailability stripping). However, reintroduction of remediated soil in the environment exposes the soil to various environmental factors, which could potentially shift nonlabile residual metals back to labile bioavailable forms. We studied the effect of autochthonous earthworm species as model biotic environmental factor on the fractionation and bioavailability of Cu residual in soil after remediation. Materials and methods We used soil from a 50-year-old vineyard regularly managed and treated with CuSO₄•5H₂O (Bordeaux mixture) as fungicide. Soil containing 400 mg kg⁻¹ of Cu was leached with total 15 mmol kg⁻¹ EDTA. Remediated and nonremediated soil was processed by fully clitellated adult specimens of Lumbricus terrestris L., a prevailing autochthonous soil earthworm species. Cu fractionation, phytoavailability, and oral-bioavailability in processed and nonprocessed soil were determined using six-step sequential extraction, extraction with diethylenediamine pentaacetic acid, and in vitro physiologically based extraction test, respectively. Results EDTA leaching removed 41% of the pseudototal Cu, mostly from the soil Fe- and Mn-oxides, carbonates, and organic matter. A 2.7-fold decrease in Cu phytoavailability and a 4.4- and 2.8-fold decrease in Cu oral-bioavailability in the stomach and small intestine fractions, respectively, were achieved after remediation. In nonremediated soil, earthworms increased the share of nonlabile Cu in residual soil fraction, while in remediated soil they increased the share of Cu bound to carbonates. A statistically significant 1.1- and 1.7-fold increase in Cu phytoavailability and intestinal oral-bioavailability, respectively, was observed in earthworm processed remediated soil. Discussion Cu occurs in various soil “pools” of different solubilities with different chemical characteristics and consequently different functions. By removing the labile part of the metals from the soil during remediation, we disrupt the chemical equilibrium; the nonlabile residual metals left in soil after remediation might become more labile in time in tendency to re-establish that equilibrium. Earthworms alter the physical and chemical properties of soil affecting consequently the fractionation of metals. The increase in earthworm's gut pH due to the excretion of ammonia and/or calcium carbonate into the intestine could lead to the transbounding of metals into the carbonate fraction. However, their activity in remediated soil increased Cu phytoavailability and intestinal oral-bioavailability, and it would, therefore, be improper to generalize the influence of earthworms on metal availability in soil. Conclusions The results presented here show that residual Cu in remediated soil is affected by environmental factors such as earthworms, which should be considered in evaluating the effect of Cu polluted soil remediation. Recommendations and perspectives Information on the behavior of residual metals in soil after its remediation is surprisingly scarce. The development of new effective remediation techniques should imply also the evaluation of postremediation effects on remediated soil. The results presented in this work indicate a possible tool for assessing the effect of biotic environmental factors on residual metals left in soil after its remediation.

Background, aim, and scope Aquatic microcontaminants (MCs) comprise diverse chemical classes, such as pesticides, biocides, pharmaceuticals, consumer products, and industrial chemicals. For water pollution control and the evaluation of water protection measures, it is crucial to screen for MCs. However, the selection and prioritization of which MCs to screen for is rather difficult and complex. Existing methods usually are strongly limited because of a lack of screening regulations or unavailability of required data. Method and models Here, we present a simple exposure-based methodology that provides a systematic overview of a broad range of MCs according to their potential to occur in the water phase of surface waters. The method requires input of publicly available data only. Missing data are estimated with quantitative structure-property relationships. The presented substance categorization methodology is based on the chemicals' distribution behavior between different environmental media, degradation data, and input dynamics. Results Seven different exposure categories are distinguished based on different compound properties and input dynamics. Ranking the defined exposure categories based on a chemical's potential to occur in the water phase of surface waters, exposure categories I and II contain chemicals with a very high potential, categories III and IV contain chemicals with a high potential, and categories V and VI contain chemicals with a moderate to low potential. Chemicals in category VII are not evaluated because of a lack of data. We illustrate and evaluate the methodology on the example of MCs in Swiss surface waters. Furthermore, a categorized list containing potentially water-relevant chemicals is provided. Discussion Chemicals of categories I and III continuously enter surface waters and are thus likely to show relatively steady concentrations. Therefore, they are best suited for water monitoring programs requiring a relatively low sampling effort. Chemicals in categories II and IV have complex input dynamics. They are consequently more difficult to monitor. However, they should be considered if an overall picture is needed that includes contaminants from diffuse sources. Conclusions The presented methodology supports compound selection for (a) water quality guidance, (b) monitoring programs, and (c) further research on the chemical's ecotoxicology. The results from the developed categorization procedure are supported by data on consumption and observed concentrations in Swiss surface waters. The presented methodology is a tool to preselect potential hazardous substances based on exposure-based criteria for policy guidance and monitoring programs and a first important step for a detailed risk assessment for potential microcontaminants.

Background, aim, and scope Concentration monitoring as a basis for risk assessment is a valid approach only if there is an unambiguous relation between concentration and effect. In many cases, no such unambiguous relation exists, since various substances can exert the same effect with differing potencies. If some or all of these substances contributing to a biological effect are unknown, effect-related monitoring becomes indispensable. Endocrine-disrupting substances in water bodies, including the groundwater, are a prominent example of such a case. The aim of the investigations described here was to detect hormonally active substances in the groundwater downstream of obsolete landfills by using the E-screen assay and to possibly assign the biological effect to individual chemical compounds by means of instrumental analyses carried out in parallel. Materials and methods Grab samples of the groundwater were collected downstream from abandoned landfills and prepared by liquid/liquid extraction. The total estrogenic activity in these samples was determined in vitro by applying the E-screen assay. The human breast cancer cells (MCF-7) used in the E-screen proliferate in response to the presence of estrogenically active compounds. Expressed in concentration units of the reference substance 17β-estradiol (E2), the test system allows the quantification of estrogenicity with a limit of detection (LOD) in the range of 0.1 ng/L. Aliquots of the samples were screened using gas chromatography/mass spectrometry (GC/MS) in order to quantify known estrogenically active substances and to identify unknown compounds. Estrogen-positive samples were extracted at different pH values, split into acidic, neutral, and basic fractions and analyzed by GC/MS, searching for individual components that display estrogenic activity. Results and discussion Estrogenic activity exceeding the LOD and the provisional benchmark of 0.5 ng E2/L was found at three out of seven abandoned waste disposal sites tested. The low concentrations of known xenoestrogens such as bisphenol-A, nonylphenols, or phthalic acid esters determined by GC/MS, however, were not sufficient to explain the detected activity. Neither natural nor synthetic hormones have caused the activity because these chemical structures are readily degradable and cannot persist in abandoned landfills for decades. The highest activity in the E-screen assay was found in the acidic fractions. Hydroxy-polychlorinated biphenyls (PCBs), hydroxylated polycyclic aromatic hydrocarbons (PAHs) and hetero-PAHs, as well as alkylphenols could be identified as further compounds with possible hormonal activity. Conclusions Estrogenically active substances may occur in the groundwater below obsolete landfills, especially those that contain PCBs or waste from gasworks. These substances are not part of analytical programs routinely applied to contaminated sites and may therefore escape detection and assessment. Analyses using the E-screen assay and GC/MS in parallel have shown that the total estrogenic activity found in groundwater samples is to be ascribed to a multitude of individual compounds, some of which cannot be quantified due to lack of standard substances or assessed due to lack of a standardized procedure for determination of their estrogenic potency. By comparison with provisional guide values for estradiol (0.5 ng/L) and ethynylestradiol (0.3 ng/L), the damaging potential of the total estrogenic activity in groundwater samples can in fact be assessed, but specific remediation measures are impossible unless the hormonal activity can be attributed to individual chemical substances. Recommendations and outlook On the one hand, further analyses of samples taken from possible pollution sources should be conducted in order to characterize the extent of groundwater pollution with xenoestrogens. On the other hand, the most potent individual compounds should be identified according to their estrogenic potency. To this end, bioassay-directed fractionation and structure elucidation should be carried out with concentrated samples.

INTRODUCTION: In this work, we propose an innovative application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a methodological approach for the chemical characterisation of black crusts on stone monuments, associated with traditional micro-morphological (optical and scanning electron microscopy) and infrared spectroscopic techniques (FTIR). METHODS: This new approach was tested on black crusts developing on two marble substrates, one, the columns of the San Cosimato cloister in Rome, and the other, a sculpture representing an angel, located in Pessano con Bornago, a small industrial town near Milan. DISCUSSION: The main aim of this study was to develop and test the reliability of the LA-ICP-MS analytical method on black crusts and to explore the idea that trace element concentrations in black crusts can be applied to investigate their origin and the relations between concentrations of polluting elements in black crusts and environmental conditions. CONCLUSION: The results obtained by applying traditional techniques find considerable support in the innovative method used here, which could determine the concentrations of a large number of trace elements (including heavy metals) in the black crusts examined, and thus could also be used as a reliable indicator of environmental pollution.

Background, aim, and scope This study developed an integrated approach to identify pollutant sources of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) of workers based on their blood contents. Materials and methods We first measured blood PCDD/F contents of sinter plant workers and residents living near the plant. By comparing those blood indicatory PCDD/Fs found for residents with those for sinter plant workers, exposure-related blood indicatory PCDD/Fs were identified for each selected worker. We then measured PCDD/F concentrations of four different sinter plant workplaces and three different ambient environments of the background. By comparing those airborne indicatory PCDD/Fs found for ambient environments with those for sinter plant workplaces, exposure-related airborne indicatory PCDD/Fs for each workplace were obtained. Finally, by matching exposure-related blood indicatory PCDD/Fs with exposure-related airborne indicatory PCDD/Fs, all suspected pollutant sources were identified for each selected worker. Results Poor Pearson correlations were found between workers' blood contents and their corresponding PCDD/F exposures. Significant differences were found in the top three blood indicatory PCDD/Fs among the selected workers. By matching exposure-related blood indicatory PCDD/Fs with exposure-related airborne indicatory PCDD/Fs, two to three suspected pollutant sources were identified for each selected worker. Discussion The poor Pearson correlation found between workers' airborne PCDD/Fs exposures and their blood contents was because workers' blood PCDD/Fs contents were contributed not only by workers' occupational exposures, but also by other exposure sources and exposure routes. The difference in blood indicatory PCDD/Fs among the selected workers were obviously due to the intrinsic differences in their time/activity patterns in the involved workplaces. While workers used a dust respirator to perform their jobs, gas phase exposure-related airborne indicatory PCDD/Fs played an important role on identifying suspected pollutant sources. But if a dust respirator was not used, the gas + particle phase exposure-related airborne indicatory PCDD/Fs would become the key factor for identifying suspected pollutant sources. Conclusions The developed integrated approach could identify all suspected pollutant sources effectively for selected workers based on their blood contents. The identified pollutant sources were theoretically plausible since they could be verified by examining workers' time/activity patterns, their status in using dust respirators, and the concentrations of PCDD/Fs found in the selected workplace atmospheres. Recommendations and perspectives The developed technique can be used to identify possible pollutant sources not only for workers but also for many other exposure groups associated with various emission sources and exposure routes in the future.

Background, aim, and scope Organoarsenical-containing animal feeds that promote growth and resistance to parasites are mostly excreted unchanged, ending up in nearby wastewater storage lagoons. Earlier work documented the partial transformation of organoarsenicals, such as, 3-nitro-4-hydroxyphenylarsonic acid (roxarsone) to the more toxic inorganic arsenate [As(V)] and 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA). Unidentified roxarsone metabolites using liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC/ICP-MS) were also inferred from the corresponding As mass balance. Earlier batch experiments in our laboratory suggested the presence of organometallic (Cu) complexes during relevant roxarsone degradation experiments. We hypothesized that organocopper compounds were complexed to roxarsone, mediating its degradation in field-collected swine wastewater samples from storage lagoons. The objective of this study was to investigate the role of organometallic (Cu) complexes during roxarsone degradation under aerobic conditions in swine wastewater suspensions, using electrospray ionization mass spectrometry (ES-MS). Materials and methods Two swine wastewater samples differing in % solids content and total recoverable Cu concentrations were reacted with 500 ppb of roxarsone under aerobic conditions for 16 days. LC/ICP-MS and ES-MS were used for As speciation analyses, and characterization of metal-organoarsenical complexes in swine wastewater subsamples, respectively. Results and discussion An organocopper roxarsone metabolite was found only in the high-Cu wastewater sample, suggesting the role of Cu in roxarsone degradation under aerobic conditions. The organocopper metabolite was not found in the low-Cu wastewater sample, because roxarsone did not undergo degradation under aerobic conditions even after 16 days. Conclusions Aerobic degradation of organoarsenicals (roxarsone) has not been documented before. Preliminary dataset from this study illustrates the direct and/or indirect association of particulate Cu in catalyzing roxarsone degradation under aerobic conditions in samples with high % solids content. Recommendations and perspectives Concerns regarding the degradation of roxarsone in wastewater to the more toxic inorganic As may be partially linked to the presence of particulate Cu. The presence of Cu in wastewater-suspended particle surfaces has never been coupled before to organoarsenicals degradation reactions, thus, further studies are needed to elucidate the related reaction mechanisms and pathways. Water depth-dependent solid particle distribution profiles in wastewater storage lagoons could provide empirical evidence towards the design of effective degradation practices for nitrophenol-containing compounds, such as, organoarsenical-containing antibiotics, or explosive munitions compounds.

Background, aim, and scope Arthropods, with over a million species described, are ubiquitous throughout different environments. Knowledge of their responses to human impact is crucial for understanding and predicting changes in ecosystem structure and functions. Our aim was to investigate the general patterns and to identify sources of variation in changes of the diversity, abundance and fitness of terrestrial arthropods (including Arachnida, Collembola and Insecta) in habitats affected by point polluters. Main features We found 134 suitable studies which were published between 1965 and 2007. These data came from impact zones of 74 polluters in 20 countries with the largest representation from Russia (28 polluters), Poland (12 polluters) and the USA (six polluters). The database allowed calculation of 448 effect sizes (i.e. relative differences between measurements taken from polluted and control sites) on the effects of various point polluters like non-ferrous industries, aluminium plants, cement, magnezite, fertilising and chemical plants, power plants, iron- and steel-producing factories. We used meta-analysis to search for general effects and to compare between polluter types and arthropod groups, and linear regression to describe the latitudinal gradient and to quantify relationships between pollution and arthropod responses. Results The overall effect of pollution on arthropod diversity did not differ from zero. However, species richness of soil arthropods (both living on the soil surface and within the soil) tended to decrease, and species richness of herbivores to increase, near point polluters. Abundance of terrestrial arthropods near point polluters decreased in general. This decrease resulted from strong adverse effects on soil arthropods, especially on decomposers and predators. Densities of herbivores increased, but a number of research biases that we discovered in published data may have led to overestimation of the latter effect. The dome-shaped density pattern along pollution gradients was discovered only in 5% of data sets. Among herbivores, only free-living defoliators and sap-feeders demonstrated higher densities in polluted sites; the effects of pollution on other guilds were not significant. Near the polluters, conifers suffered higher increase in damage from herbivores than deciduous woody plants and herbs. Overall effect of pollution on arthropod performance was negative; in particular, individuals from polluted sites were generally smaller than individuals from control sites. This negative effect weakened with increase in duration of the pollution impact, hinting evolution of pollution resistance in populations inhabiting polluted sites. Stepwise regression analysis demonstrated that pollution-induced changes in both the density and performance of arthropods depended on climate of the locality. Negative effects on soil fauna increased with increase in annual precipitation; positive effects on herbivore population density increased with increases in both mean July temperature and annual precipitation. Discussion We detected effects of research methodology on the outcome of published studies. Many of them suffer from research bias—the tendency to collect data on organisms or under conditions in which one has an expectation of detecting significant effects. Pseudoreplicated studies (one polluted site contrasted to one control site) frequently reported larger effects than replicated studies (several polluted sites contrasted with several control sites). These methodological flaws especially influenced herbivory studies; we conclude that increase in herbivory in both heavily and moderately polluted habitats is not as frequent as it was earlier suggested. In contrast, the decrease in abundance of predators is likely to be a widespread phenomenon. Thus, our analysis supports the hypothesis that pollution may favour herbivore populations by creating an enemy-free space. Consistent declines in abundance of soil arthropods in impact zones of different polluters suggest that this group can potentially be used in bioindication of pollution-induced changes in terrestrial ecosystems. Conclusions Main effects of pollution on arthropod communities (decreased abundance of decomposers and predators and increased herbivory) may have negative consequences for structure and services of entire ecosystems. Responses of arthropods to pollution depend on both temperature and precipitation in such a way that ecosystem-wide adverse effects are likely to increase under predicted climate change. Recommendations and perspectives Our analysis confirmed that local severe impacts of industrial enterprises on biota are well-suited to reveal the direction and magnitude of the biotic effects of aerial pollution, as well as to explore the sources of variation in responses of organisms and communities. Although we analysed the effects of point polluters, our conclusions can be applied to predict consequences of pollution impacts on regional and even global scales. We argue that possible interactions between pollution and climate should be accounted for in the analyses of global change impacts on biota.

Background, aim, and scope To stimulate the national economy, a so-called “gold week” comprising May Day and National Day has been put in force by the government, and the first golden-week holiday began on October 1, 1999. Statistical data show that about 15,000 visitors were received each day by Emperor Qin's Terra-Cotta Museum during just such a gold week period. To evaluate the effects of tourism on indoor air, airborne samples were collected by the sedimentation plate method for 5 min during the “Oct. 1” gold week period of 2006, and both composition and changes of airborne bacteria and fungi in indoor/outdoor air in the museums were investigated. Materials and methods Airborne microbes were simultaneously collected by means of gravitational sedimentation on open Petri dishes. Three parallel samples were collected at the same time each day, and samples were subsequently incubated in the lab. Microbiology media were prepared before each experiment by a professional laboratory. Concentrations were calculated and presented as average data of colony-forming units per cubic meter of air (CFU/m³). Results The results show that (1) 13 bacterial genera and eight genera of fungi were identified from indoor and outdoor air at Emperor Qin's Terra-Cotta Museum during “Oct. 1” gold week in 2006. The bacterial groups occupied 61%, the fungi groups occupied 36%, and others occupied 3% of the total number of isolated microorganism genera. (2) As for the comparison of indoor and outdoor samples, the average concentrations of fungi were higher during the afternoon (13:00) than for the morning (09:00). The average concentrations of bacteria in indoor air were higher during the afternoon (13:00) than for the morning (9:00), and in outdoor air, they were lower during the afternoon (13:00) than for the morning (9:00). (3) The average concentrations of five dominant groups of bacteria and three dominant groups of fungi were higher during the afternoon (13:00) than for the morning (9:00) in the indoor air, but the average concentrations of fungi were higher and those of bacteria were lower during the afternoon than for the morning, for outdoor air. (4) As for the comparison of indoor samples, the bacterial daily concentrations and fungal daily concentrations were higher during the afternoon (13:00) than those for the mornings (9:00) over the 10 days. For the comparison of outdoor samples, the bacterial concentration was lower, and the fungal concentrations were higher during the afternoon (13:00) than those for the morning (9:00) over the 10 days. Discussion The results also show that the numbers of airborne bacteria and fungi had a daily character in indoor air and were higher in the afternoon. The airborne microbe concentrations were found to be similar to residential indoor values from other reports; the indoor museum maximum of microbial concentrations was 90 CFU/m³ and did not exceed the Chinese indoor bioaerosol guideline. However, microorganisms may fall on the surface of display items as a result of particle sedimentation and would, as such, be capable of degrading objects by way of their secretions, e.g., enzymes and organic acids. Therefore, the right steps should be taken to prevent any deterioration in the quality of displayed artifacts. Conclusions The results show that museum air was affected by human activity; therefore, it is imperative that the number of visitors be strictly limited and that windows be opened regularly to avoid air pollution. Recommendations and perspectives The data provide a significant scientific basis for indoor air quality control and museum scientific management. It is recommended that the number of visitors be strictly limited.

Background, aim and scope Opposite interests must coexist in coastal areas: the presence of significant cities and urban centres, of touristic and recreational areas, and of extensive shellfish farming. To avoid local pollution caused by treated wastewaters along the Northern Adriatic coast (Friuli Venezia-Giulia and Veneto regions), marine outfall systems have been constructed. In this study, the application of a numerical dispersion model is used to support the traditional monitoring methods in order to link information concerning the hydrodynamic circulation and the microbiological features, to evaluate possible health risks associated with recreational and coastal shellfish farming activities. The study is a preliminary analysis of the environmental impact of wastewater treatment plants (WWTPs) with submarine discharge outfalls. It also could be useful for the water profile definition according to the Directive 2006/7/EC on the quality of bathing water and for the integrated areal analysis (Ostoich et al. 2006), to define the area of influence of each submarine discharge point. Materials and methods Historical data on discharges of the considered WWTPs were recovered and evaluated. Data on discharges' control for Veneto region (WWTPs of Lido and Cavallino) were produced by the WWTPs' manager Veritas Laboratory service, while data for the WWTPs of Friuli Venezia-Giulia region were produced by the regional environmental protection agency in the institutional control activity following official methods. The hydrodynamic model used in this work is the three-dimensional version of the finite element model SHYFEM, developed at ISMAR-CNR (Marine Science Institute of the Italian National Research Council) in Venice (Umgiesser et al. J Mar Syst 51:123-145, 2008). Results and discussion Numerical simulations have been carried out with the 3D version of the finite element model SHYFEM for 3 months during autumn 2007 to evaluate the bacterial pollution dispersion along the coasts of Veneto and Friuli Venezia-Giulia regions, prescribing meteo-marine forcings and concentration values at the points corresponding to the positions of the submarine outfalls. Model results show that during autumn 2007 the discharges of the submarine outfalls of the Venice province seem to have no impact on the surface water quality, while there are some visible effects in the Gulf of Trieste. This reflects the behaviour of the experimental data collected by ARPAV and ARPA FVG and monitoring campaigns both on water and shellfish quality. Further results have been elaborated to identify the area of influence of each discharge point; scenarios were developed with imposed concentrations. The results seem to highlight that the two discharges of the Veneto region are not noticeable, while the discharges of the Gulf of Trieste (in particular the Servola and Barcola ones) are perceptible. Conclusions This study represents a new step towards the study of the microbiological pollution dispersion and impact due to the discharges of the submarine outfalls of the Veneto and Friuli Venezia-Giulia regions (nine considered discharge points). With the 3D version of the finite element model SHYFEM, the information obtained from the hydrodynamic circulation has been linked to the classical methods of analysis, to assess possible risks connected to the microbiological parameter Escherichia coli. Recommendations and perspectives In future studies the time scale for microbiological parameters' decay could be linked to various environmental parameters such as light climate, temperature, and salinity. Interesting information would come from the study of new scenarios with different configurations of the discharge of the pipelines and/or the treatment plants and in particular from the improvements of the 3D version of the SHYFEM model, to take the stratification process into account which occurs during spring-summer, since the Northern Adriatic Sea is a very complex ecosystem, both as physical and ecological processes.

Background, aim, and scope Soil contamination with heavy metals occurs as a result of both anthropogenic and natural activities. Heavy metals could have long-term hazardous impacts on the health of soil ecosystems and adverse influences on soil biological processes. Soil enzymatic activities are recognized as sensors towards any natural and anthropogenic disturbance occurring in the soil ecosystem. Similarly, microbial biomass carbon (MBC) is also considered as one of the important soil biological activities frequently influenced by heavy metal contamination. The polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) has recently been used to investigate changes in soil microbial community composition in response to environmental stresses. Soil microbial community structure and activities are difficult to elucidate using single monitoring approach; therefore, for a better insight and complete depiction of the soil microbial situation, different approaches need to be used. This study was conducted in a greenhouse for a period of 12 weeks to evaluate the changes in indigenous microbial community structure and activities in the soil amended with different application rates of Cd, Pb, and Cd/Pb mix. In a field environment, soil is contaminated with single or mixed heavy metals; so that, in this research, we used the selected metals in both single and mixed forms at different application rates and investigated their toxic effects on microbial community structure and activities, using soil enzyme assays, plate counting, and advanced molecular DGGE technique. Soil microbial activities, including acid phosphatase (ACP), urease (URE), and MBC, and microbial community structure were studied. Materials and methods A soil sample (0-20 cm) with an unknown history of heavy metal contamination was collected and amended with Cd, Pb, and Cd/Pb mix using the CdSO₄ and Pb(NO₃)₂ solutions at different application rates. The amended soils were incubated in the greenhouse at 25 ± 4°C and 60% water-holding capacity for 12 weeks. During the incubation period, samples were collected from each pot at 0, 2, 9, and 12 weeks for enzyme assays, MBC, numeration of microbes, and DNA extraction. Fumigation-extraction method was used to measure the MBC, while plate counting techniques were used to numerate viable heterotrophic bacteria, fungi, and actinomycetes. Soil DNAs were extracted from the samples and used for DGGE analysis. Results ACP, URE, and MBC activities of microbial community were significantly lower (p < 0.05) in the metal-amended samples than those in the control. The enzyme inhibition extent was obvious between different incubation periods and varied as the incubation proceeded, and the highest rate was detected in the samples after 2 weeks. However, the lowest values of ACP and URE activities (35.6% and 36.6% of the control, respectively) were found in the Cd₃/Pb₃-treated sample after 2 weeks. Similarly, MBC was strongly decreased in both Cd/Pb-amended samples and highest reduction (52.4%) was detected for Cd₃/Pb₃ treatment. The number of bacteria and actinomycetes were significantly decreased in the heavy metal-amended samples compared to the control, while fungal cells were not significantly different (from 2.3% to 23.87%). In this study, the DGGE profile indicated that the high dose of metal amendment caused a greater change in the number of bands. DGGE banding patterns confirmed that the addition of metals had a significant impact on microbial community structure. Discussion In soil ecosystem, heavy metals exhibit toxicological effects on soil microbes which may lead to the decrease of their numbers and activities. This study demonstrated that toxicological effects of heavy metals on soil microbial community structure and activities depend largely on the type and concentration of metal and incubation time. The inhibition extent varied widely among different incubation periods for these enzymes. Furthermore, the rapid inhibition in microbial activities such as ACP, URE, and MBC were observed in the 2 weeks, which should be related to the fact that the microbes were suddenly exposed to heavy metals. The increased inhibition of soil microbial activities is likely to be related to tolerance and adaptation of the microbial community, concentration of pollutants, and mechanisms of heavy metals. The DGGE profile has shown that the structure of the bacterial community changed in amended heavy metal samples. In this research, the microbial community structure was highly affected, consistent with the lower microbial activities in different levels of heavy metals. Furthermore, a great community change in this study, particularly at a high level of contamination, was probably a result of metal toxicity and also unavailability of nutrients because no nutrients were supplied during the whole incubation period. Conclusions The added concentrations of heavy metals have changed the soil microbial community structure and activities. The highest inhibitory effects on soil microbial activities were observed at 2 weeks of incubation. The bacteria were more sensitive than actinomycetes and fungi. The DGGE profile indicated that bacterial community structure was changed in the Cd/Pb-amended samples, particularly at high concentrations. Recommendations and perspectives The investigation of soil microbial community structure and activities together could give more reliable and accurate information about the toxic effects of heavy metals on soil health.