Assessment of the leaching potential of pesticides and their metabolites is an important part of the authorization procedure for pesticides in Europe. To protect groundwater quality, it must be demonstrated that concentrations of active substances in the upper groundwater do not exceed 0.1 μg/L before a pesticide can be approved for use. For the purpose of exposure assessment, this concentration limit is imposed on the water leaching downward at 1 m depth in the soil profile. For a given substance and application pattern, this leaching concentration can vary in space by several orders of magnitude, due to variation in site conditions, most importantly soil properties and climate. Spatially distributed leaching modelling (SDLM) is a methodology for exposure assessment over large spatial extents, dealing with this spatial variability in a comprehensive way. It involves performing simulations for many parametrizations representative for a spatial region and can be used to generate maps or calculate spatio-temporal percentiles of leaching concentrations. While such tools are already used in exposure assessment at national level in several EU member states, no generally accepted SDLM tool is available at the European level. In 2020, a working group of Society of Environmental Toxicology and Chemistry (SETAC) was formed with the purpose to develop a harmonized framework for SDLM across Europe (EU27 + UK). A first version of an SDLM—referred to as GeoPEARL-EU—was built around the pesticide leaching model PEARL, a field-scale model of pesticide fate in the soil-plant system. PEARL mechanistically simulates pesticide behaviour in a 1D soil column based on explicit descriptions of transport in the liquid and gas phases, sorption to the solid phase, degradation, volatilisation, and plant-uptake. Soil moisture content and fluxes are provided by the SWAP hydrological model. PEARL is used in regulatory exposure assessment for groundwater and soil. Furthermore, a spatially distributed tool based on PEARL (GeoPEARL) is used for exposure assessment in the Netherlands. To apply PEARL to Europe, pan-European gridded datasets were collected for several variables, including soil texture, pH, soil organic carbon, weather, irrigation patterns and crop area. These datasets were used to develop a set of parametrizations covering the variability of climate and soil conditions in Europe. To this end, all 1x1 km grid cells for the EU27 + UK were partitioned into approximately 10,000 clusters using k-means clustering, based on several soil- and climate-related variables relevant for leaching vulnerability. Subsequently, a representative grid cell was selected for each cluster, which was used to obtain the data required to parameterize PEARL from the spatial data sets. Pedotransfer functions were used to derive soil hydraulic parameters. We will present results from GeoPEARL-EU for several test cases with specific attention to the effect of the spatial aggregation approach on the model predictions. Moreover, we discuss how the tool could be used in the tiered approach of the regulatory exposure assessment for groundwater in the EU.

The European Commission asked EFSA to undertake a 'repair action' of the FOCUS surface water report after the EFSA Pesticide Steering Network had been consulted. The main request was to introduce into all FOCUS surface water scenarios (both run-off and drainage) a 20-year assessment period instead of the current 12- or 16-month assessment period. Because of the 20-year assessment period, the way application dates are defined needed to be reviewed, reconsidering the functionality of the pesticide application timing currently used. Guidance on how substance parameters should be handled when correlated with soil properties has been provided. Foliar wash-off calculated in MACRO and Pesticide Root Zone Model was aligned and the appropriateness of including rotational crop aspects was discussed. Processing time and how to use the results of the exposure assessment were considered.

EFSA performed a public consultation of the draft scientific report of EFSA on the “repair action” of the FOCUS surface water scenarios from 24 September to 5 November 2018. EFSA was asked the European Commission to prepare a public consultation on the draft scientific report. The EFSA scientific report provides an update of the current FOCUS surface water report currently used for surface water exposure assessments of substances to aquatic organisms when performing risk assessments according to Regulation (EC) No 1107/2009 of the European Parliament and the Council. This report presents statistics on the comments received and answers to them. These comments were considered when finalising the EFSA scientific report.

Groundwater monitoring is recommended as a higher-tier option in the regulatory groundwater assessment of plant protection products in the European Union. However, to date little guidance has been provided on study designs. SETAC EMAG-Pest GW, a group of regulatory, academic, and industry scientists, was created in 2015 to establish scientific recommendations for conducting such studies. This report provides the SETAC EMAG-Pest GW group’s recommendations on study designs and study procedures. Because of the need to assess the vulnerability to leaching in both site selection and in extrapolating study results, information on how to assess the vulnerability to leaching is a major topic in this report. In the development of groundwater study designs, which groundwater needs to be protected and to what level are key aspects. In the European Union, a groundwater quality standard of 0.1 µg/L applies to active substances and relevant metabolites, but the groundwater to which this standard is applied varies among the Member States. Also, the definition of the concentration may consider temporal or spatial variability (e.g. a single sample or an average concentration over a period of time or geographic area). The SETAC EMAG-Pest GW group does not endorse any specific exposure assessment option. However, 7 different exposure assessment options that consider only the location of the groundwater to which the ground water quality standard is applied were selected to illustrate the impact of the exposure assessment option on the study design. Monitoring can be performed on many different geographical scales. In-field and edge-of-field monitoring focus on residues from applications to a single field, while catchment and aquifer monitoring focus on residues in groundwater over a larger area. The timing of applications in monitoring studies can vary. In a prospective study, an application is made and the movement and degradation of the residues is followed. In a retrospective study, residues from previous applications are monitored. Some studies are both retrospective and prospective—residues from previous applications are monitored and a new application is made and the residues are followed. In addition to the exposure assessment option, study designs must consider the objectives of the study, the properties of the active substance and its metabolites, and the site characteristics. Usually, the objective is to determine whether a substance can move into the groundwater specified in the exposure assessment option as well as the magnitude of residues in groundwater. The objective may also include determining degradation rates in soil as a function of depth, persistence and movement of residues in groundwater, efficacy of mitigation measures, or confirmation of more detailed studies on a wider range of sites. Sampling schedules should consider the expected time required for an active substance to move through the soil into groundwater, as well as expected persistence in both soil and groundwater. Movement and persistence can be affected by both site characteristics and properties of the active substance and its metabolites. The need to tailor study designs to objectives, exposure assessment options, compound properties and site characteristics complicates the development of standardised study designs. Therefore, this report includes a number of example designs. Other key points that must be addressed by study designs are the vulnerability of the chosen sites compared to the vulnerability of all use areas supported by the study, the product use before and during the study, and the connectivity of the sampled groundwater to treated fields. Demonstrating connectivity (a quality criterion in the EU assessment of monitoring sites to exclude false negative measurements) is more challenging for catchment or aquifer monitoring compared to shallow wells installed as part of in-field or edge-of-field studies. This report includes an extensive discussion on assessing vulnerability of monitoring sites. This includes information on different approaches to vulnerability assessment and mapping as well as for setting monitoring sites into context. Lists of available methods and data sources available at the European level are also included. In addition to information on study design and estimating vulnerability, this report includes information on a number of other topics: avoiding contamination during sampling and/or analysis, avoiding influencing residue movement as a result of purging during sampling, and proper study documentation (Good Laboratory Practices and/or quality criteria). Procedures that are discussed include site selection (new or existing wells), installation of monitoring wells, sample collection, and analysis of samples. The report also provides information on causes of outliers (abnormally high concentrations not the result of normal leaching through soil), the use of public monitoring data, information on further hydrological characterisation (such as use of tracers, groundwater age dating, and geophysical methods), and information that should be included in reports providing results of groundwater studies. Groundwater monitoring is recommended as a higher-tier option in the regulatory groundwater assessment of crop protection products in the European Union. However, to date little guidance has been provided on the study designs. The SETAC EMAG-Pest GW group (a mixture of regulatory, academic, and industry scientists) was created in 2015 to establish scientific recommendations for conducting such studies. This report provides recommendations for study designs and study procedures made by the Society of Environmental Toxicology and Chemistry (SETAC) Environmental Monitoring Advisory Group on Pesticides (EMAG-Pest). Because of the need to assess the vulnerability to leaching in both site selection and extrapolating study results, information on assessing vulnerability to leaching is also a major topic in this report. The design of groundwater monitoring studies must consider to which groundwater the groundwater quality standard is applicable and the associated spatial and temporal aspects of its application, the objective of the study, the properties of the active substance and its metabolites, and site characteristics. This limits the applicability of standardised study designs. The effect of the choice of groundwater to which the water quality guideline is applied on study design is illustrated and examples of actual study designs are presented.

Abstract Following a request from the European Commission, the EFSA Panel on Plant Protection Products and their Residues (PPR Panel) prepared a scientific opinion to provide a comprehensive evaluation of pesticide residues in foods for infants and young children. In its approach to develop this scientific opinion, the EFSA PPR Panel took into account, among the others, (i) the relevant opinions of the Scientific Committee for Food setting a default maximum residue level (MRL) of 0.01 mg/kg for pesticide residues in foods for infants and young children; (ii) the recommendations provided by EFSA Scientific Committee in a guidance on risk assessment of substances present in food intended for infants below 16 weeks of age; (iii) the knowledge on organ/system development in infants and young children. For infants below 16 weeks of age, the EFSA PPR Panel concluded that pesticide residues at the default MRL of 0.01 mg/kg for food for infants and young children are not likely to result in an unacceptable exposure for active substances for which a health‐based guidance value (HBGV) of 0.0026 mg/kg body weight (bw) per day or higher applies. Lower MRLs are recommended for active substances with HBGVs below this value. For infants above 16 weeks of age and young children, the established approach for setting HBGVs is considered appropriate. For infants below 16 weeks of age the approach may not be appropriate and the application of the EFSA guidance on risk assessment of substances present in food intended for infants below 16 weeks of age is recommended. The contribution of conventional food to the total exposure to pesticide residues is much higher than that from foods intended for infants and young children. Because of the increased intake of conventional food by young children, these have the highest exposure to pesticide residues, whereas infants 3–6 months of age generally have lower exposure. The impact of cumulative exposure to pesticide residues on infants and young children is not different from the general population and the EFSA cumulative risk assessment methodology is also applicable to these age groups. Residue definitions established under Regulation (EC) No 396/2005 are in general considered appropriate also for foods for infants and young children. However, based on a tier 1 analysis of the hydrolysis potential of pesticides simulating processing, the particular appropriateness of existing residue definitions for monitoring to cover processed food, both intended for infants and young children as well as conventional food, is questionable.

Following a request from EFSA, the Panelon Plant Protection Products and their Residues developed an opinion on the science to support the potential development of a risk assessment scheme of plant protection products for amphibians and reptiles. The coverage of the risk to amphibians and reptiles by current risk assessments for other vertebrate groups was investigated. Available test methods and exposure models were reviewed with regard to their applicability to amphibians and reptiles. Proposals were made for specific protection goals aiming to protect important ecosystem services and taking into consideration the regulatory framework and existing protection goals for other vertebrates. Uncertainties, knowledge gaps and research needs were highlighted. (c) 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

The EFSA Panelon Plant Protection Products and their Residues reviewed the guidance on how aged sorption studies for pesticides should be conducted, analysed and used in regulatory assessment. The inclusion of aged sorption is a higher tier in the groundwater leaching assessment. The Panelbased its review on a test with three substances taken from a data set provided by the European Crop Protection Association. Particular points of attention were the quality of the data provided, the proposed fitting procedure of aged sorption experiments and the proposed method for combining results obtained from aged sorption studies and lower-tier studies on degradation and adsorption. Aged sorption was a relevant process in all cases studied. The test revealed that the guidance could generally be well applied and resulted in robust and plausible results. The Panelconsiders the guidance suitable for use in the groundwater leaching assessment after the recommendations in this Scientific Opinion have been implemented, with the exception of the use of field data to derive aged sorption parameters. The Panelnoted that the draft guidance could only be used by experienced users because there is no software tool that fully supports the work flow in the guidance document. It is therefore recommended that a user-friendly software tool be developed. Aged sorption lowered the predicted concentration in groundwater. However, because aged sorption experiments may be conducted in different soils than lower-tier degradation and adsorption experiments, it cannot be guaranteed that the higher tier predicts lower concentrations than the lower tier, while lower tiers should be more conservative than higher tiers. To mitigate this problem, the Panelrecommends using all available higher- and lower-tier data in the leaching assessment. The Panelfurther recommends that aged sorption parameters for metabolites be derived only from metabolite-dosed studies. The formation fraction can be derived from parent-dosed degradation studies, provided that the parent and metabolite are fitted with the best-fit model, which is the double first-order in parallel model in the case of aged sorption.

Following a request from EFSA, the Panelon Plant Protection Products and their Residues (PPR) developed an opinion on the state of the art of Toxicokinetic/Toxicodynamic (TKTD) models and their use in prospective environmental risk assessment (ERA) for pesticides and aquatic organisms. TKTD models are species- and compound-specific and can be used to predict (sub)lethal effects of pesticides under untested (time-variable) exposure conditions. Three different types of TKTD models are described, viz., (i) the General Unified Threshold models of Survival' (GUTS), (ii) those based on the Dynamic Energy Budget theory (DEBtox models), and (iii) models for primary producers. All these TKTD models follow the principle that the processes influencing internal exposure of an organism, (TK), are separated from the processes that lead to damage and effects/mortality (TD). GUTS models can be used to predict survival rate under untested exposure conditions. DEBtox models explore the effects on growth and reproduction of toxicants over time, even over the entire life cycle. TKTD model for primary producers and pesticides have been developed for algae, Lemna and Myriophyllum. For all TKTD model calibration, both toxicity data on standard test species and/or additional species can be used. For validation, substance and species-specific data sets from independent refined-exposure experiments are required. Based on the current state of the art (e.g. lack of documented and evaluated examples), the DEBtox modelling approach is currently limited to research applications. However, its great potential for future use in prospective ERA for pesticides is recognised. The GUTS model and the Lemna model are considered ready to be used in risk assessment.

Abstract In 2013, EFSA published a comprehensive systematic review of epidemiological studies published from 2006 to 2012 investigating the association between pesticide exposure and many health outcomes. Despite the considerable amount of epidemiological information available, the quality of much of this evidence was rather low and many limitations likely affect the results so firm conclusions cannot be drawn. Studies that do not meet the ‘recognised standards’ mentioned in the Regulation (EU) No 1107/2009 are thus not suited for risk assessment. In this Scientific Opinion, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to assess the methodological limitations of pesticide epidemiology studies and found that poor exposure characterisation primarily defined the major limitation. Frequent use of case–control studies as opposed to prospective studies was considered another limitation. Inadequate definition or deficiencies in health outcomes need to be avoided and reporting of findings could be improved in some cases. The PPR Panel proposed recommendations on how to improve the quality and reliability of pesticide epidemiology studies to overcome these limitations and to facilitate an appropriate use for risk assessment. The Panel recommended the conduct of systematic reviews and meta‐analysis, where appropriate, of pesticide observational studies as useful methodology to understand the potential hazards of pesticides, exposure scenarios and methods for assessing exposure, exposure–response characterisation and risk characterisation. Finally, the PPR Panel proposed a methodological approach to integrate and weight multiple lines of evidence, including epidemiological data, for pesticide risk assessment. Biological plausibility can contribute to establishing causation.

Abstract In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP's informed Integrated Approach for Testing and Assessment (IATA).

8 This EFSA guidance document provides guidance for the exposure assessment of soil organisms to 9 plant protection products (PPPs) and their transformation products according to Regulation EC no 1

Knowledge about the stabilization of organic matter input to soil is essential for understanding the influence of different agricultural practices on turnover characteristics in agricultural soil systems. In this study, soil samples from a long-term field experiment were separated into silt- and clay-sized particles. In 1967, 14C labeled farmyard manure was applied to three different cropping systems: crop rotation, monoculture and permanent bare fallow. Humic acids (HAs) were extracted from silt- and clay-sized fractions and characterized using photometry, mid-infrared and fluorescence spectroscopy. Remaining 14C was determined in size fractions as well as in their extracted HAs. Yields of carbon and remaining 14C in HAs from silt-sized particles and Corg in clay-sized particles decreased significantly in the order: crop rotation > monoculture ≫ bare fallow. Thus, crop rotation not only had the largest overall C-pool in the experiment, but it also best stabilized the added manure. Mid-infrared spectroscopy could distinguish between HAs from different particle size soil fractions. With spectroscopic methods significant differences between the cropping systems were detectable in fewer cases compared to quantitative results of HAs (yields, 14C, Corg and Nt). The trends, however, pointed towards increased humification of HAs from bare fallow systems compared to crop rotation and monoculture as well as of HAs from clay-sized particles compared to silt-sized particles. Our study clearly shows that the largest differences were observed between bare fallow on one hand and monoculture and crop rotation on the other.

Despite the importance of soil organic matter (SOM), very few long-term data concerning soil organic-C dynamics are available for calibrating and evaluating C models. The long-term (14)C-turnover field experiment, established in 1967 in Fuchsenbigl, Lower Austria, offers the unique opportunity to follow the fate of labeled C under different crop-management systems (bare fallow, spring wheat, crop rotation) over a period of more than 35 y. Compared with the crop-rotation and spring wheat treatments, the decline of total organic C was largest in the bare-fallow treatments, because no significant C input has occurred since 1967. Nonetheless, the decline was not as fast as predicted with the original RothC-26.3-model decomposition rate constants. In this work, we therefore calibrated the Roth-C-26.3 model for the Pannonian climatic region based on the field-experiment results. The main adjustment was in the decomposition rate constant for the humified soil C pool (HUM), which was set to 0.009 instead of 0.02 y(-1) as determined in the original Rothamsted field trial. This resulted in a higher HUM pool in the calibrated model because of a longer turnover period (111 vs. 50 y) The modeled output based on the calibrated model fitted better to measured values than output obtained with the original Roth-C26.3-model parameters. Additionally, the original decomposition rate constant for resistant plant material (RPM) was changed from 0.3 to 0.6 y(-1) to describe the decomposition of (14)C-labeled straw more accurately Application of the calibrated model (modified HUM decomposition rate) to simulate removal of crop residues showed that this can entail a long-term decline of SOM. However, these impacts are strongly dependent on the crop types and on environmental conditions at a given location

The stabilisation of 14C-labelled farmyard manure was investigated under different cropping systems (crop rotation, monoculture, and bare fallow) in a long-term field experiment established in 1967. Solid-state 13C-NMR of bulk soils yielded a gradient of increasing aromatic properties in the order: straw manure–crop rotation < straw manure–monoculture < straw and farmyard manure–bare fallow. The opposite trend was observed for O-alkyl groups. The farmyard manure–bare fallow treatment was used to investigate changes of humic acids (HAs) with time. The FT-IR bands of aromatics, carbonyl groups, and a band of methyls and benzene rings increased over the 36 years of the experiment, whereas 2 amide bands and a band of sulfone and/or ester groups decreased. Fluorescence spectroscopy verified the increase in aromatic properties with age. Consequently, during soil organic matter stabilisation, HAs showed increasing properties of carbonyl and aromatic groups, whereas amidic groups decreased. The dynamic character of HAs, as shown by 14C, was also reflected by distinct spectroscopic changes over the period of investigation.

The stabilization of soil organic matter (SOM) is an important process in the context of global change and is strongly affected by soil use and management. We investigated the mineralization and stabilization of 14 C‐labeled wheat ( Triticum aestivum L.) straw and 14 C‐labeled farmyard manure under different cropping systems (crop rotation, monoculture, and bare fallow) in a long‐term field experiment established in 1967. Exponential decay equations were fitted to the measured data, and coefficients of the C pools and decay constants were estimated. The different organic amendments and cropping systems resulted in considerably different distributions and half‐lives of three modeled SOM pool compartments. Significantly lower amounts of residual 14 C‐labeled material were found in the bulk soils of the bare fallow systems than the farmyard‐manured crop rotation or monoculture, indicating enhanced organic matter stabilization in the latter systems. In 2004, remaining 14 C‐labeled amendments (as a percentage of the original application in 1967) in bulk soils were 8.7 ± 0.3% for crop rotation, 8.6 ± 0.4% for monoculture, 7.5 ± 0.7% for bare fallow (all farmyard‐manured), and 5.0 ± 0.6% for straw‐manured bare fallow. Humic acids extracted with 1 mol L −1 NaOH responded markedly to the different cropping systems regarding both their extraction yields and the remaining 14 C label. In all cropping systems, the 14 C in the extracted humic acids decreased rapidly with time. Remaining 14 C‐labeled organic residues in this fraction in 2004 (as a percentage of the original application in the fraction) were 0.315 ± 0.002% for crop rotation, 0.31 ± 0.02% for monoculture, and 0.16 ± 0.02% for bare fallow (all farmyard manured). Our study therefore shows that the investigated humic acids are a dynamic SOM pool cycling at decadal and subdecadal rates.

The soil microbial communities of a landfill cover substrate, which was treated with landfill gas (100lCH4m−2d−1) and landfill leachate for 1.5 years, were investigated by phospholipid fatty acid (PLFA), ergosterol and respiratory quinone analyses. The natural 13C depletion of methane was used to assess the activity of methanotrophs and carbon turnover in the soil system. Under methane addition, the soil microbial community was dominated by PLFAs (14:0 and 16:1 isomers) and quinones (ubiquinone-8 and 18-methylene-ubiquinone-8) related to type I methanotrophs, and 18:1 PLFAs contained in type II methanotrophs. While type I methanotrophic PLFAs were 13C depleted, i.e. type I methanotrophs were actively oxidising and assimilating methane, 13C depletion of 18:1 PLFAs was low and inconsistent with their abundance. This, possibly reflects isotopic discrimination, assimilation of carbon derived from type I methanotrophs and a high contribution of non-methanotrophic bacteria to the 18:1 isomers. Landfill leachate irrigation caused the methanotrophic community to shift closer to the soil surface. It also decreased 18:1 PLFAs, while type I methanotrophs were probably stimulated. Gram positive bacteria, but not fungi, were also 13C depleted and consequently involved in the secondary turnover of carbon originating from methanotrophic bacteria. Cy17:0 PLFA was 13C depleted in deep soil layers, indicating anaerobic methane oxidation.

The present paper describes changes in soil organic carbon (SOC) and extractable humic acids (HAs) in a long-term field experiment with different tillage treatments (minimum tillage (MT), reduced tillage (RT) and conventional tillage (CT)). This field experiment is located in the east of Vienna in a Pannonian climate and it was started in 1988. The methodological approach included elemental analyses, FT-IR, 13C NMR and fluorimetric measurements. Both MT and RT revealed significant depth gradients of yields of extractable HAs. In CT no depth gradient was observable, neither for HA yields nor for observed molecular characteristics. This indicated a destruction of the gradient by mixing of the soil in CT. Especially MT showed an increase of aromatic moieties with depth, suggesting an increased humification of HAs in the lower soil layers. Gradients with similar trends were indicated for the carbonylic, the amidic and probably the hydroxylic groups in HAs extracted from MT and RT samples. The data revealed with FT-IR and solid-state 13C NMR spectroscopy were convincing, plausible and meaningful, the highly sensitive fluorescence spectroscopy was limited because of strong quenching by inner filter effects, compromising data reliability. However, the fluorescence results based on a defined HAs concentration (and comparing soils from the same site) were in line with results from the other methods (13C NMR and FT-IR). As a consequence, the influence of tillage treatments can be followed by absence or presence of depth gradients of the according molecular characteristics in extracted HAs.

A 13C natural abundance experiment including GC-c-IRMS analysis of phospholipid fatty acids (PLFAs) was conducted to assess the temporal dynamics of the soil microbial community and carbon incorporation during the mineralization of plant residues under the impact of heavy metals and acid rain. Maize straw was incorporated into (i) control soil, (ii) soil irrigated with acid rain, (iii) soil amended with heavy metal-polluted filter dust and (iv) soil with both, heavy metal and acid rain treatment, over a period of 74 weeks. The mineralization of maize straw carbon was significantly reduced by heavy metal impact. Reduced mineralization rate of the added carbon likely resulted from a reduction of the microbial biomass due to heavy metal stress, while the efficiency of 13C incorporation into microbial PLFAs was hardly affected. Since acid rain did not significantly change soil pH, little impact on soil microorganisms and mineralization rate was found. Temporal dynamics of labelling of microbial PLFAs were different between bacterial and fungal PLFA biomarkers. Utilization of maize straw by bacterial PLFAs peaked immediately after the application (2 weeks), while labelling of the fungal biomarker 18:2ω6,9 was most pronounced 5 weeks after the application. In general, 13C labelling of microbial PLFAs was closely linked to the amounts of maize carbon present in the soil. The distinct higher labelling of microbial PLFAs in the heavy metal-polluted soils 74 weeks after application indicated a large fraction of available maize straw carbon still present in the soil.

Aim of our study was the development of the methodological basis for the characterization of humic fractions of a long-term field experiment. Humic acids (HAs) were extracted from three layers of a nontilled soil using three different extractants (1 M NaOH, 0.1 M Na4P2O7, 1 M Na2CO3), and the humin fraction was enriched. NaOH as extractant for FTIR analysis of humic substances yields higher resolved IR spectra, especially in the important regions of stretching vibrations including aromatic and aliphatic groups and in the fingerprint area including amides, aliphats, and aromats than the other extractants. The NaOH extraction has lower extraction yields as compared to Na4P2O7 and Na2CO3 and represents a different part of the soil organic matter (SOM). This is reflected by lower C : N ratios and higher E4 : E6 and fulvic acid–to–humic acid ratios as compared to the other extractants. The FTIR band areas of HA fraction obtained by NaOH showed an increase of the aromatic and carbonyl groups and a decrease of amide groups with increasing soil depth. Aliphatic groups showed contradicting results: The bands of the stretching vibrations increased, and the band of the bending vibrations decreased. We assume that band interactions in the bending vibrations were responsible for that phenomenon under the assumption of an increase of aliphatic groups with increasing soil depth. The IR bands of the enriched humin fraction showed a decreasing trend in case of both aliphatic bands deriving from stretching vibrations and an increase of aromatic characteristics with depth. Our study led to the conclusion that HA fractions obtained by 1 M NaOH represent a small and dynamic fraction indicated by the measured yields in combination with values of Nt, C : N, E4 : E6 ratios, and ratios of fulvic acids (FA) to HA. The humin fraction has a high contribution to the total organic C and represents a more stabilized fraction of SOM which still shows changes in its aromatic and aliphatic characteristics with soil depth.

We assessed the effects of chronic heavy metal (HM) contamination on soil microbial communities in a newly established forest ecosystem. We hypothesized that HM would affect community function and alter the microbial community structure over time and that the effects are more pronounced in combination with acid rain (AR). These hypotheses were tested in a model forest ecosystem consisting of several tree species (Norway spruce, birch, willow, and poplar) maintained in open top chambers. HMs were added to the topsoil as filter dust from a secondary metal smelter and two types of irrigation water acidity (ambient rain vs. acidified rain) were applied during four vegetation periods. HM contamination strongly impacted the microbial biomass (measured with both fumigation–extraction and quantitative lipid biomarker analyses) and community function (measured as basal respiration and soil hydrolase activities) of the soil microbial communities. The most drastic effect was found in the combined treatment of HM and AR, although soil pH and bioavailable HM contents were comparable to those of treatments with HM alone. Analyses of phospholipid fatty acids (PLFAs) and terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S ribosomal DNA showed that HM treatment affected the structure of bacterial communities during the 4-year experimental period. Very likely, this is due to the still large bioavailable HM contents in the HM contaminated topsoils at the end of the experiment.