[Gastrointestinal symptoms may reflect complicated falciparum malaria]. / Gastrointestinale Symptome als Ausdruck einer komplizierten Malaria tropica.

HISTORY: A 42-year-old female presented with a two-day history of vomiting, diarrhea, fever and chills. Two weeks before she had returned to Germany from a Safari in Tanzania. She had disregarded the recommendation to take antimalarial chemoprophylaxis. CLINICAL FINDINGS AND DIAGNOSIS: The thin blood film showed Plasmodium falciparum-parasitized erythrocytes, and Plasmodium falciparum malaria was diagnosed. The full blood count showed thrombocytopenia and ultrasound imaging revealed splenomegaly. Initially the criteria for complicated malaria were not fulfilled. THERAPY AND COURSE: We started oral therapy with atovaquone/proguanil. The patient vomited the tablets twice. Therefore therapy was switched to intravenous artesunate. Subsequently, parasitemia dropped from 2.8 to 1.0 % within 22 hours. After 3 days of artesunate i. v., treatment could then be completed with oral atovaquone/proguanil, and the symptoms resolved. CONCLUSIONS: Patients with malaria and persistent vomiting should be treated intravenously and monitored closely, as severe gastrointestinal symptoms may reflect impending organ failure. We therefore propose including persistent vomiting in the list of criteria for complicated malaria.

Enhancing insights: exploring the information content of calorespirometric ratio in dynamic soil microbial growth processes through calorimetry.

Catalytic activity of microbial communities maintains the services and functions of soils. Microbial communities require energy and carbon for microbial growth, which they obtain by transforming organic matter (OM), oxidizing a fraction of it and transferring the electrons to various terminal acceptors. Quantifying the relations between matter and energy fluxes is possible when key parameters such as reaction enthalpy (∆rH), energy use efficiency (related to enthalpy) (EUE), carbon use efficiency (CUE), calorespirometric ratio (CR), carbon dioxide evolution rate (CER), and the apparent specific growth rate (µapp) are known. However, the determination of these parameters suffers from unsatisfying accuracy at the technical (sample size, instrument sensitivity), experimental (sample aeration) and data processing levels thus affecting the precise quantification of relationships between carbon and energy fluxes. To address these questions under controlled conditions, we analyzed microbial turnover processes in a model soil amended using a readily metabolizable substrate (glucose) and three commercial isothermal microcalorimeters (MC-Cal/100P, TAM Air and TAM III) with different sample sizes meaning varying volume-related thermal detection limits (LODv) (0.05-1mW L-1). We conducted aeration experiments (aerated and un-aerated calorimetric ampoules) to investigate the influence of oxygen limitation and thermal perturbation on the measurement signal. We monitored the CER by measuring the additional heat caused by CO2 absorption using a NaOH solution acting as a CO2 trap. The range of errors associated with the calorimetrically derived µapp, EUE, and CR was determined and compared with the requirements for quantifying CUE and the degree of anaerobicity (ηA). Calorimetrically derived µapp and EUE were independent of the instrument used. However, instruments with a low LODv yielded the most accurate results. Opening and closing the ampoules for oxygen and CO2 exchange did not significantly affect metabolic heats. However, regular opening during calorimetrically derived CER measurements caused significant measuring errors due to strong thermal perturbation of the measurement signal. Comparisons between experimentally determined CR, CUE,ηA, and modeling indicate that the evaluation of CR should be performed with caution.

scMAR-Seq: a novel workflow for targeted single-cell genomics of microorganisms using radioactive labeling.

IMPORTANCE: A central question in microbial ecology is which member of a community performs a particular metabolism. Several sophisticated isotope labeling techniques are available for analyzing the metabolic function of populations and individual cells in a community. However, these methods are generally either insufficiently sensitive or throughput-limited and thus have limited applicability for the study of complex environmental samples. Here, we present a novel approach that combines highly sensitive radioisotope tracking, microfluidics, high-throughput sorting, and single-cell genomics to simultaneously detect and identify individual microbial cells based solely on their in situ metabolic activity, without prior information on community structure.

Hypertonic stress induced changes of Pseudomonas fluorescens adhesion towards soil minerals studied by AFM.

Studying bacterial adhesion to mineral surfaces is crucial for understanding soil properties. Recent research suggests that minimal coverage of sand particles with cell fragments significantly reduces soil wettability. Using atomic force microscopy (AFM), we investigated the influence of hypertonic stress on Pseudomonas fluorescens adhesion to four different minerals in water. These findings were compared with theoretical XDLVO predictions. To make adhesion force measurements comparable for irregularly shaped particles, we normalized adhesion forces by the respective cell-mineral contact area. Our study revealed an inverse relationship between wettability and the surface-organic carbon content of the minerals. This relationship was evident in the increased adhesion of cells to minerals with decreasing wettability. This phenomenon was attributed to hydrophobic interactions, which appeared to be predominant in all cell-mineral interaction scenarios alongside with hydrogen bonding. Moreover, while montmorillonite and goethite exhibited stronger adhesion to stressed cells, presumably due to enhanced hydrophobic interactions, kaolinite showed an unexpected trend of weaker adhesion to stressed cells. Surprisingly, the adhesion of quartz remained independent of cell stress level. Discrepancies between measured cell-mineral interactions and those calculated by XDLVO, assuming an idealized sphere-plane geometry, helped us interpret the chemical heterogeneity arising from differently exposed edges and planes of minerals. Our results suggest that bacteria may have a significant impact on soil wettability under changing moisture condition.

Changes in cell surface properties of Pseudomonas fluorescens by adaptation to NaCl induced hypertonic stress.

Determination of the effect of water stress on the surface properties of bacteria is crucial to study bacterial induced soil water repellency. Changes in the environmental conditions may affect several properties of bacteria such as the cell hydrophobicity and morphology. Here, we study the influence of adaptation to hypertonic stress on cell wettability, shape, adhesion, and surface chemical composition of Pseudomonas fluorescens. From this we aim to discover possible relations between the changes in wettability of bacterial films studied by contact angle and single cells studied by atomic and chemical force microscopy (AFM, CFM), which is still lacking. We show that by stress the adhesion forces of the cell surfaces towards hydrophobic functionalized probes increase while they decrease towards hydrophilic functionalized tips. This is consistent with the contact angle results. Further, cell size shrunk and protein content increased upon stress. The results suggest two possible mechanisms: Cell shrinkage is accompanied by the release of outer membrane vesicles by which the protein to lipid ratio increases. The higher protein content increases the rigidity and the number of hydrophobic nano-domains per surface area.

Water Stress-Driven Changes in Bacterial Cell Surface Properties.

Increased drought intensity and frequency exposes soil bacteria to prolonged water stress. While numerous studies reported on behavioral and physiological mechanisms of bacterial adaptation to water stress, changes in bacterial cell surface properties during adaptation are not well researched. We studied adaptive changes in cell surface hydrophobicity (CSH) after exposure to osmotic (NaCl) and matric stress (polyethylene glycol 8000 [PEG 8000]) for six typical soil bacteria (Bacillus subtilis, Arthrobacter chlorophenolicus, Pseudomonas fluorescens, Novosphingobium aromaticivorans, Rhodococcus erythropolis, and Mycobacterium pallens) covering a wide range of cell surface properties. Additional physicochemical parameters (surface chemical composition, surface charge, cell size and stiffness) of B. subtilis and P. fluorescens were analyzed to understand their possible contribution to CSH development. Changes in CSH caused by osmotic and matric stress depend on strain and stress type. CSH of B. subtilis and P. fluorescens increased with stress intensity, R. erythropolis and M. pallens exhibited a generally high but constant contact angle, while the response of A. chlorophenolicus and N. aromaticivorans depended on growth conditions and stress type. Osmotically driven changes in CSH of B. subtilis and P. fluorescens are accompanied by increasing surface N/C ratio, suggesting an increase in protein concentration within the cell wall. Cell envelope proteins thus presumably control bacterial CSH in two ways: (i) by increases in the relative density of surface proteins due to efflux of cytoplasmic water and subsequent cell shrinkage, and (ii) by destabilization of cell wall proteins, resulting in conformational changes which render the surface more hydrophobic. IMPORTANCE Changes in precipitation frequency, intensity, and temporal distribution are projected to result in increased frequency and intensity of droughts and heavy rainfall events. Prolonged droughts can promote the development of soil water repellency (SWR); this impacts the infiltration and distribution of water in the soil profile, exposing soil microorganisms to water stress. Exposure to water stress has recently been reported to result in increased cell surface hydrophobicity. However, the mechanism of this development is poorly understood. This study investigates the changes in the physicochemical properties of bacterial cell surfaces under water stress as a possible mechanism of increased surface hydrophobicity. Our results improve understanding of the microbial response to water stress in terms of surface properties, the variations in stress response depending on cell wall composition, and its contribution to the development of SWR.

Microcosm test for pesticide fate assessment in planted water filters: 13C,15N-labeled glyphosate as an example.

Planted filters are often used to remove pesticides from runoff water. However, the detailed fate of pesticides in the planted filters still remains elusive. This hampers an accurate assessment of environmental risks of the pesticides related to their fate and thereby development of proper mitigation strategies. In addition, a test system for the chemical fate analysis including plants and in particular for planted filters is not well established yet. Therefore, we developed a microcosm test to simulate the fate of pesticide in planted filters, and applied 2-13C,15N-glyphosate as a model pesticide. The fate of 2-13C,15N-glyphosate in the planted microcosms over 31 day-incubation period was balanced and compared with that in the unplanted microcosms. The mass balance of 2-13C,15N-glyphosate turnover included 13C mineralization, degradation products, and the 13C and 15N incorporation into the rhizosphere microbial biomass and plants. We observed high removal of glyphosate (> 88%) from the water mainly due to adsorption on gravel in both microcosms. More glyphosate was degraded in the planted microcosms with 4.1% of 13C being mineralized, 1.5% of 13C and 3.8% of 15N being incorporated into microbial biomass. In the unplanted microcosms, 1.1% of 13C from 2-13C,15N-glyphosate was mineralized, and only 0.2% of 13C and 0.1% of 15N were assimilated into microbial biomass. The total recovery of 13C and 15N was 81% and 85% in planted microcosms, and 91% and 93% in unplanted counterparts, respectively. The microcosm test was thus proven to be feasible for mass balance assessments of the fate of non-volatile chemicals in planted filters. The results of such studies could help better manage and design planted filters for pesticide removal.

Technologies and perspectives for achieving carbon neutrality.

Global development has been heavily reliant on the overexploitation of natural resources since the Industrial Revolution. With the extensive use of fossil fuels, deforestation, and other forms of land-use change, anthropogenic activities have contributed to the ever-increasing concentrations of greenhouse gases (GHGs) in the atmosphere, causing global climate change. In response to the worsening global climate change, achieving carbon neutrality by 2050 is the most pressing task on the planet. To this end, it is of utmost importance and a significant challenge to reform the current production systems to reduce GHG emissions and promote the capture of CO2 from the atmosphere. Herein, we review innovative technologies that offer solutions achieving carbon (C) neutrality and sustainable development, including those for renewable energy production, food system transformation, waste valorization, C sink conservation, and C-negative manufacturing. The wealth of knowledge disseminated in this review could inspire the global community and drive the further development of innovative technologies to mitigate climate change and sustainably support human activities.

Superabsorbent polymer as a supplement substrate of constructed wetland to retain pesticides from agricultural runoff.

Surface water runoff can export pesticides from agricultural fields into adjacent aquatic ecosystems, where they may pose adverse effects to organisms. Constructed wetlands (CWs) are widely used to treat agricultural runoff contaminated by pesticides, but the removal of hydrophilic pesticides is usually low. In this study, we suggest superabsorbent polymer (SAP), a cross-linked hydrophilic polymer, as a supplement to substrates of CWs and tested the hypothesis that SAP results in an enhanced removal of hydrophilic pesticides. Therefore, batch experiments were conducted to study the retention capacity of water-saturated SAP (w-SAP) for several hydrophilic pesticides. Retention of the pesticides on w-SAP was related to the ionization state and water solubility of the pesticides. The retention of neutral pesticides, imidacloprid, metalaxyl and propiconazole, was about 20% higher than that measured for anionic pesticides, bentazone, glyphosate and MCPA. The retention of the pesticides by w-SAP mainly resulted from their distribution in the gel-water phase of w-SAP, while less water soluble pesticides might have also been adsorbed on the molecular backbone of SAP. Furthermore, we tested the efficacy of w-SAP for treatment of runoff water contaminated by pesticides in lab-scale horizontal subsurface flow CWs. SAP in CWs improved the removal of the pesticides, including the recalcitrant ones. The removal enhancement was owing to the increase of hydraulic retention time and improvement of biodegradation. The removal of the pesticides in SAP containing CWs was > 93% for MCPA, glyphosate, and propiconazole, 62 - 99% for imidacloprid, 50 - 84% for metalaxyl, and 38 - 73% for bentazone. In the control gravel CWs, the removal was > 98% for glyphosate, generally > 83% for MCPA and propiconazole, 46 - 98% for imidacloprid, 32 - 97% for metalaxyl, and 9 - 96% for bentazone.

Retention and distribution of pesticides in planted filter microcosms designed for treatment of agricultural surface runoff.

Pesticides in agricultural surface water runoff cause a major threat to freshwater systems. Installation of filter systems or constructed wetlands in areas of preferential run-off is a possible measure for pesticides abatement. To develop such systems, combinations of filter materials suitable for retention of both hydrophilic and hydrophobic organic pesticides were tested for pesticide removal in planted microcosms. The retention of six pesticides frequently detected in surface waters (bentazone, MCPA, metalaxyl, propiconazole, pencycuron, and imidacloprid) was evaluated in unplanted and planted pot experiments with novel bed material mixtures consisting of pumice, vermiculite, water super-absorbent polymer (SAP) for retention of ionic and water soluble pesticides, and synthetic hydrophobic wool for adsorption of hydrophobic pesticides. The novel materials were compared to soil with high organic matter content. The highest retention of the pesticides was observed in the soil, with a considerable translocation of pesticides into the plants, and low leaching potential, in particular for the hydrophobic compounds. However, due to the high retention of pesticides in soil, environmental risks related to their long term mobilization cannot be excluded. Mixtures of pumice and vermiculite with SAP resulted in high retention of i) water and ii) both hydrophilic and hydrophobic pesticides but with much lower leaching potential compared to the mineral systems without SAP. Mixtures of such materials may provide near natural treatment options in riparian strips and also for treatment of rainwater runoff without the need for water containment systems.

Serial Measurements of Refractive Index, Glucose and Protein to Assess Gastric Liquid Nutrient Transport-A Proof-of-Principal Study.

Delayed gastric emptying contributes to complications as aspiration or malnutrition. Among patients suffering from acute neurological diseases, motility disorders are prevalent but poorly understood. Thus, methods to measure gastric emptying are required to allow for appropriate adaptions of individual enteral nutrition algorithms. For enterally fed patients repetitive concentration measurements of gastric content have been proposed to assess gastric emptying. This approach can be used to calculate the gastric residual volume (GRV) and transport of nutrition formula (NF), but it has not yet been implemented in clinical routine. The aim of this study was to investigate whether refractometry or other likewise straightforward analytical approaches produce the best results under in vitro conditions mimicking the gastric milieu. We measured NF in different known concentrations, either diluted in water or in simulated gastric fluid (SGF), with each of the following methods: refractometer, handheld glucose meter, and Bradford protein assay. Then, in enterally fed patients suffering from acute neurological disease, we calculated GRVs and nutrition transport and tested possible associations with clinical parameters. In water dilution experiments, NF concentrations could be assessed with the readout parameters of all three methods. Refractometry yielded the most precise results over the broadest range of concentrations and was biased least by the presence of SGF (detection range for Fresubin original fibre, given as volume concentration/normalized error of regression slope after incubation with water or SGF: 0-100 vs. 0-100%/0.5 vs. 3.9%; glucose-measurement: 5-100 vs. 25-100%/7.9 vs. 6.1%; Bradford-assay: 0-100 vs. 0-100%/7.8 vs. 15.7%). Out of 28 enterally fed patients, we calculated significant slower nutrition transport in patients with higher blood glucose (Rho -0.391; p = 0.039) and in patients who received high-dose sufentanil (Rho -0.514; p = 0.005). Also, the calculated nutrition transport could distinguish patients with and without feeding intolerance (Median 6 vs. 17 ml/h; Mann-Whitney test: p = 0.002). The results of our study prove that serial refractometry is a suitable and cost-effective method to assess gastric emptying and to enhance research on gastrointestinal complications of stroke.

Fate of pendimethalin in soil and characterization of non-extractable residues (NER).

One important route of degradation of herbicide pendimethalin in soil leads to formation of non-extractable residues (NER). To investigate NER nature (irreversibly, chemically bound, including possible biogenic NER, or strongly sorbed and entrapped) residues of 14C-labelled pendimethalin in soil were investigated after conventional extraction with organic solvents by silylation. After 400 days of incubation, 32.0% of applied radioactivity (AR) was transformed into NER, 39.9% AR remained extractable. Mineralization reached 26.2% AR. Additionally, 14C-pendimethalin was incubated in soil amended with compost for 217 days to investigate the influence of organic amendments on NER formation. NER amounted to 37.8% AR, with 57.9% AR remaining extractable. Mineralization was negligible (1.4% AR). For all sampling times only low amounts of radioactivity were entrapped (<5% AR) in soil without compost amendment. Pendimethalin was present only in trace amounts (ca. 0.4% AR), other released residues consisted of undefined fractions (sum ≈2% AR). In soil amended with compost, silylation overall resulted in release of higher amounts of radioactivity (19% AR). Addition of compost led to an increase in potential entrapment and sorption sites for pendimethalin, forming higher amounts of strongly sorbed, entrapped residues. Furthermore, potential release of non-extractable pendimethalin residues was investigated by incubation of solvent-extracted soil (without compost amendment) mixed with fresh soil for additional 3 months. NER were partly mineralized (7% AR) and 20% became extractable with organic solvents. However, no pendimethalin or any known metabolites were found. It can be concluded that no parent pendimethalin was found and NER of pendimethalin in soil are mainly formed by covalent binding to organic matrix with only low potential of remobilization under natural conditions.

Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing.

Constructed wetlands (CWs) are effective ecological remediation technologies for various contaminated water bodies. Here, we queried for benzene-degrading microbes in a horizontal subsurface flow CW with reducing conditions in the pore water and fed with benzene-contaminated groundwater. For identification of relevant microbes, we employed in situ microcosms (BACTRAPs, which are made from granulated activated carbon) coupled with 13C-stable isotope probing and Illumina sequencing of 16S rRNA amplicons. A significant incorporation of 13C was detected in RNA isolated from BACTRAPs loaded with 13C-benzene and exposed in the CW for 28 days. A shorter incubation time did not result in detectable 13C incorporation. After 28 days, members from four genera, namely Dechloromonas, Hydrogenophaga, and Zoogloea from the Betaproteobacteria and Arcobacter from the Epsilonproteobacteria were significantly labeled with 13C and were abundant in the bacterial community on the BACTRAPs. Sequences affiliated to Geobacter were also numerous on the BACTRAPs but apparently those microbes did not metabolize benzene as no 13C label incorporation was detected. Instead, they may have metabolized plant-derived organic compounds while using the BACTRAPs as electron sink. In representative wetland samples, sequences affiliated with Dechloromonas, Zoogloea, and Hydrogenophaga were present at relative proportions of up to a few percent. Sequences affiliated with Arcobacter were present at < 0.01% in wetland samples. In conclusion, we identified microbes of likely significance for benzene degradation in a CW used for remediation of contaminated water.

Quantitative assessment of microbial necromass contribution to soil organic matter.

Soil carbon transformation and sequestration have received significant interest in recent years due to a growing need for quantitating its role in mitigating climate change. Even though our understanding of the nature of soil organic matter has recently been substantially revised, fundamental uncertainty remains about the quantitative importance of microbial necromass as part of persistent organic matter. Addressing this uncertainty has been hampered by the absence of quantitative assessments whether microbial matter makes up the majority of the persistent carbon in soil. Direct quantitation of microbial necromass in soil is very challenging because of an overlapping molecular signature with nonmicrobial organic carbon. Here, we use a comprehensive analysis of existing biomarker amino sugar data published between 1996 and 2018, combined with novel appropriation using an ecological systems approach, elemental carbon-nitrogen stoichiometry, and biomarker scaling, to demonstrate a suit of strategies for quantitating the contribution of microbe-derived carbon to the topsoil organic carbon reservoir in global temperate agricultural, grassland, and forest ecosystems. We show that microbial necromass can make up more than half of soil organic carbon. Hence, we suggest that next-generation field management requires promoting microbial biomass formation and necromass preservation to maintain healthy soils, ecosystems, and climate. Our analyses have important implications for improving current climate and carbon models, and helping develop management practices and policies.

Microbial Turnover of Glyphosate to Biomass: Utilization as Nutrient Source and Formation of AMPA and Biogenic NER in an OECD 308 Test.

Environmental fate assessment of chemicals involves standardized simulation tests with isotope-labeled molecules to balance transformation, mineralization, and formation of nonextractable residues (NER). Methods to predict microbial turnover and biogenic NER have been developed, having limited use when metabolites accumulate, the chemicals are not the only C source, or provide for other macroelements. To improve predictive capability, we extended a recently developed method for microbial growth yield estimation to account for incomplete degradation and multiple-element assimilation and combined it with a dynamic model for fate description in soils and sediments. We evaluated the results against the unique experimental data of 13C3-15N co-labeled glyphosate turnover with AMPA formation in water-sediment systems (OECD 308). Balancing 13C- and 15N- fluxes to biomass showed a pronounced shift of glyphosate transformation from full mineralization to AMPA formation. This may be explained by various hypotheses, for example, the limited substrate turnover inherent to the batch conditions of the test system causing microbial starvation or inhibition by P release. Modeling results indicate initial N overload due to the lower C/N ratio in glyphosate compared to average cell composition leading to subsequent C demand and accumulation of AMPA.

Spatiotemporal disturbance characteristics determine functional stability and collapse risk of simulated microbial ecosystems.

Terrestrial microbial ecosystems are exposed to many types of disturbances varying in their spatial and temporal characteristics. The ability to cope with these disturbances is crucial for maintaining microbial ecosystem functions, especially if disturbances recur regularly. Thus, understanding microbial ecosystem dynamics under recurrent disturbances and identifying drivers of functional stability and thresholds for functional collapse is important. Using a spatially explicit ecological model of bacterial growth, dispersal, and substrate consumption, we simulated spatially heterogeneous recurrent disturbances and investigated the dynamic response of pollutant biodegradation - exemplarily for an important ecosystem function. We found that thresholds for functional collapse are controlled by the combination of disturbance frequency and spatial configuration (spatiotemporal disturbance regime). For rare disturbances, the occurrence of functional collapse is promoted by low spatial disturbance fragmentation. For frequent disturbances, functional collapse is almost inevitable. Moreover, the relevance of bacterial growth and dispersal for functional stability also depends on the spatiotemporal disturbance regime. Under disturbance regimes with moderate severity, microbial properties can strongly affect functional stability and shift the threshold for functional collapse. Similarly, networks facilitating bacterial dispersal can delay functional collapse. Consequently, measures to enhance or sustain bacterial growth/dispersal are promising strategies to prevent functional collapses under moderate disturbance regimes.

Functional Resistance to Recurrent Spatially Heterogeneous Disturbances Is Facilitated by Increased Activity of Surviving Bacteria in a Virtual Ecosystem.

Bacterial degradation of organic compounds is an important ecosystem function with relevance to, e.g., the cycling of elements or the degradation of organic contaminants. It remains an open question, however, to which extent ecosystems are able to maintain such biodegradation function under recurrent disturbances (functional resistance) and how this is related to the bacterial biomass abundance. In this paper, we use a numerical simulation approach to systematically analyze the dynamic response of a microbial population to recurrent disturbances of different spatial distribution. The spatially explicit model considers microbial degradation, growth, dispersal, and spatial networks that facilitate bacterial dispersal mimicking effects of mycelial networks in nature. We find: (i) There is a certain capacity for high resistance of biodegradation performance to recurrent disturbances. (ii) If this resistance capacity is exceeded, spatial zones of different biodegradation performance develop, ranging from no or reduced to even increased performance. (iii) Bacterial biomass and biodegradation dynamics respond inversely to the spatial fragmentation of disturbances: overall biodegradation performance improves with increasing fragmentation, but bacterial biomass declines. (iv) Bacterial dispersal networks can enhance functional resistance against recurrent disturbances, mainly by reactivating zones in the core of disturbed areas, even though this leads to an overall reduction of bacterial biomass.

A unified approach for including non-extractable residues (NER) of chemicals and pesticides in the assessment of persistence.

All chemicals form non-extractable residues (NER) to various extents in environmental media like soil, sediment, plants and animals. NER can be quantified in environmental fate studies using isotope-labeled (such as 14C or 13C) tracer compounds. Previous NER definitions have led to a mismatch of legislation and state of knowledge in research: the residues are assumed to be either irreversibly bound degradation products or at least parts of these residues can be released. In the latter assumption, soils and sediments are a long-term source of slowly released residues. We here present a conceptual experimental and modeling approach to characterize non-extractable residues and provide guidance how they should be considered in the persistence assessment of chemicals and pesticides. Three types of NER can be experimentally discriminated: sequestered and entrapped residues (type I), containing either the parent substance or xenobiotic transformation products or both and having the potential to be released, which has indeed been observed. Type II NER are residues that are covalently bound to organic matter in soils or sediments or to biological tissue in organisms and that are considered being strongly bound with very low remobilization rates like that of humic matter degradation rates. Type III NER comprises biogenic NER (bioNER) after degradation of the xenobiotic chemical and anabolic formation of natural biomolecules like amino acids and phospholipids, and other biomass compounds. We developed the microbial turnover to biomass (MTB) model to predict the formation of bioNER based on the structural properties of chemicals. Further, we proposed an extraction sequence to obtain a matrix containing only NER. Finally, we summarized experimental methods to distinguish the three NER types. Type I NER and type II NER should be considered as potentially remobilizable residues in persistence assessment but the probability of type II release is much lower than that of type I NER, i.e., type II NER in soil are "operationally spoken" irreversibly bound and can be released only in minute amounts and at very slow rates, if at all. The potential of remobilization can be evaluated by chemical, physical and biological methods. BioNER are of no environmental concern and, therefore, can be assessed as such in persistence assessment. The general concept presented is to consider the total amount of NER minus potential bioNER as the amount of xenoNER, type I + II. If a clear differentiation of type I and type II is possible, for the calculation of half-life type I NER are considered as not degraded parent substance or transformation product(s). On the contrary, type II NER may generally be considered as (at least temporarily) removed. Providing proof for type II NER is the most critical issue in NER assessment and requires additional research. If no characterization and additional information on NER are available, it is recommended to assess the total amount as potentially remobilizable. We propose our unified approach of NER characterization and evaluation to be implemented into the persistence and environmental hazard assessment strategies for REACH chemicals and biocides, human and veterinary pharmaceuticals, and pesticides, irrespective of the different regulatory frameworks.

Prediction of the Formation of Biogenic Nonextractable Residues during Degradation of Environmental Chemicals from Biomass Yields.

Degradation tests with radio or stable isotope labeled compounds enable the detection of the formation of nonextractable residues (NER). In PBT and vPvB assessment, remobilisable NER are considered as a potential risk while biogenic NER from incorporation of labeled carbon into microbial biomass are treated as degradation products. Relationships between yield, released CO2 (as indicator of microbial activity and mineralization) and microbial growth can be used to estimate the formation of biogenic NER. We provide a new approach for calculation of potential substrate transformation to microbial biomass (theoretical yield) based on Gibbs free energy and microbially available electrons. We compare estimated theoretical yields of biotechnological substrates and of chemicals of environmental concern with experimentally determined yields for validation of the presented approach. A five-compartment dynamic model is applied to simulate experiments of 13C-labeled 2,4-D and ibuprofen turnover. The results show that bioNER increases with time, and that most bioNER originates from microbial proteins. Simulations with precalculated input data demonstrate that precalculation of yields reduces the number of fit parameters considerably, increases confidence in fitted kinetic data, and reduces the uncertainty of the simulation results.