Andreas Schäffer

Formation and Fate of Bound Residues from Microbial Biomass during 2,4-D Degradation in Soil

During organic contaminant degradation in soil, bound or nonextractable residues (NER) are formed. Part of these residues may be biogenic, because degrading microorganisms assimilate carbon derived from the pollutant and mineralized CO(2) to form cellular components for example, [fatty acids (FA) and amino acids (AA)], which are subsequently stabilized within soil organic matter (SOM). We investigated the formation and fate of FA and AA from biodegradation of (13)C(6)-2,4-D in soil and the incorporation of the (13)C-label into living biomass via (13)CO(2) fixation. After 64 days of incubation, (13)C-AA in SOM indicated that 44% of the initially applied (13)C(6)-2,4-D equivalents had been converted to microbial biomass and finally to biogenic residues. The intermediate maximum of (13)C-FA in SOM indicated a 20% conversion of (13)C(6)-2,4-D to biomass, but (13)C-FA decreased to 50% of that value whereas (13)C-AA in the SOM remained stable. We provide the first evidence that nearly all bound residues from 2,4-D are biogenic, containing natural microbial residues stabilized in SOM. Because of biogenic residue formation, the potential risk of bound residues from readily metabolized xenobiotics in soils is highly overestimated. Hence, the formation of biogenic residues must be considered in general when performing mass balances of pollutant biodegradation in soils.

Isomer-Specific Degradation of Branched and Linear 4-Nonylphenol Isomers in an Oxic Soil

Using (14)C- and (13)C-ring-labeling, degradation of five p-nonylphenol (4-NP) isomers including four branched (4-NP(38), 4-NP(65), 4-NP(111), and 4-NP(112)) and one linear (4-NP(1)) isomers in a rice paddy soil was studied under oxic conditions. Degradation followed an availability-adjusted first-order kinetics with the decreasing order of half-life 4-NP(111) (10.3 days) > 4-NP(112) (8.4 days) > 4-NP(65) (5.8 days) > 4-NP(38) (2.1 days) > 4-NP(1) (1.4 days), which is in agreement with the order of their reported estrogenicities. One metabolite of 4-NP(111) with less polarity than the parent compound occurred rapidly and remained stable in the soil. At the end of incubation (58 days), bound residues of 4-NP(111) amounted to 54% of the initially applied radioactivity and resided almost exclusively in the humin fraction of soil organic matter, in which chemically humin-bound residues increased over incubation. Our results indicate an increase of specific estrogenicity of the remaining 4-NPs in soil as a result of the isomer-specific degradation and therefore underline the importance of understanding the individual fate (including degradation, metabolism, and bound-residue formation) of isomers for risk assessment of 4-NPs in soil. 4-NP(1) should not be used as a representative of 4-NPs for studies on their environmental behavior.

Slow Biotransformation of Carbon Nanotubes by Horseradish Peroxidase

Due to steady increase in use and mass production carbon nanotubes (CNTs) will inevitably end up in the environment. Because of their chemical nature CNTs are expected to be recalcitrant and biotransform only at very slow rates. Degradation of CNTs within days has recently been reported, but excluding one study, conclusions relied solely on qualitative results. We incubated 13 different types of CNTs and subjected them to enzymatic oxidation with horseradish peroxidase and concluded that the analytical methods commonly employed for studying degradation of CNTs did not have the sensitivity to unequivocally demonstrate degradation of these materials. To obtain unambiguous results with regard to the biotransformability of CNTs in the horseradish peroxidase system we incubated: (a) (14)C-labeled multiwalled CNTs, homologous to Baytubes CNTs; and (b) (13)C-depleted single-walled CNTs, used in previous studies. Our results show that (14)C-CO2 evolved linearly at a rate of about 0.02‰ per day, and at the end of the 30-day incubations the CO2 evolved amounted to about 0.5‰ of both initial substrates, the (14)C-labeled multiwalled and (13)C-depleted single-walled CNTs. These results clearly show that CNT material is oxidized in the horseradish peroxidase system but with half-lives of about 80 years and not a few days as has been reported before. Adequately addressing biotransformation rates of CNTs is key toward a better understanding of the fate of these materials in the environment.

Accumulation and Distribution of Multiwalled Carbon Nanotubes in Zebrafish (Danio rerio)

No data on the bioaccumulation and distribution of multiwalled carbon nanotubes (MWCNTs) in aquatic vertebrates is available until now. We quantified uptake and elimination of dispersed radiolabeled MWCNTs ((14)C-MWCNT; 1 mg/L) by zebrafish (Danio rerio) over time. The influences of the feeding regime and presence of dissolved organic carbon (DOC) on accumulation of the nanomaterial were determined. The partitioning of radioactivity to different organs and tissues was measured in all experiments. A bioaccumulation factor of 16 L/kg fish wet weight was derived. MWCNTs quickly associated with the fish, and steady state was reached within 1 day. After transfer to clear medium, MWCNTs were quickly released to the water phase, but on average 5 mg of MWCNTs/kg fish dry weight remained associated with the fish. The nanomaterial mainly accumulated in the gut of all fish. Feeding led to lower internal concentrations due to facilitated elimination via the digestive tract. In the presence of DOC, 10-fold less was taken up by the fish after 48 h of exposure compared to without DOC. Quick adhesion to and detachment from superficial tissues were observed. Remarkably, little fractions of the internalized radioactivity were detected in the blood and muscle tissue of exposed fish. The part accumulated in these fish compartments remained constant during the elimination phase. Hence, biomagnification of MWCNTs in the food chain is possible and should be a subject of further research.

Thin-film photovoltaic cells: long-term metal(loid) leaching at their end-of-life.

The photovoltaic effect of thin-film copper indium gallium selenide cells (CIGS) is conferred by the latter elements. Organic photovoltaic cells (OPV), relying on organic light-absorbing molecules, also contain a variety of metals (e.g., Zn, Al, In, Sn, Ag). The environmental impact of such technologies is largely unknown, in particular when the physical integrity deteriorates upon end-of-life, possibly facilitating cell constituent leaching. This study analyzed long-term inorganic leaching from damaged OPV and CIGS into different model waters. Leachate concentrations were put into perspective by calculating the predicted environmental concentrations (PEC) for several scenarios. Roof-top acidic rain runoff from CIGS was found to be the predominant emission source for metals and metalloids, with Cd released to such extents that PEC (173.4 μg Cd L(-1)) would considerably exceed acute toxicity concentrations for Daphnia magna . Other PEC for CIGS (9.9 mg Mo L(-1) and 9.4 μg Se L(-1)) were in the range of teratogenic effects. In contrast, OPV released little metals with calculated PEC being below even conservative drinking water guidelines. Time-resolved single-particle ICP-MS indicated that some metals (Zn, Mo, Ag) were in nanoparticulate form, raising nanotoxicity concerns. Leaching kinetics called for revision of existing standardized (accelerated) leaching protocols because long-term release was most relevant.

Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation

To date, only a few reports about studies on toxic effects of carbon nanotubes (CNT) are available, and their results are often controversial. Three different cell lines (rainbow trout liver cells (RTL-W1), human adrenocortical carcinoma cells (T47Dluc), and human adrenocarcinoma cells (H295R)) were exposed to multiwalled carbon nanotubes, the antimicrobial agent triclocarban (TCC) as well as the mixture of both substances in a concentration range of 3.13 to 50 mg CNT/L, 31.25 to 500 μg TCC/L, and 3.13 to 50 mg CNT/L + 1% TCC (percentage relative to carbon nanotubes concentration), respectively. Triclocarban is a high-production volume chemical that is widely used as an antimicrobial compound and is known for its toxicity, hydrophobicity, endocrine disruption, bioaccumulation potential, and environmental persistence. Carbon nanotubes are known to interact with hydrophobic organic compounds. Therefore, triclocarban was selected as a model substance to examine mixture toxicity in this study. The influence of multiwalled carbon nanotubes and triclocarban on various toxicological endpoints was specified: neither cytotoxicity nor endocrine disruption could be observed after exposure of the three cell lines to carbon nanotubes, but the nanomaterial caused intracellular generation of reactive oxygen species in all cell types. For TCC on the other hand, cell vitality of 80% could be observed at a concentration of 2.1 mg/L for treated RTL-W1 cells. A decrease of luciferase activity in the ER Calux assay at a triclocarban concentration of 125 μg/L and higher was observed. This effect was less pronounced when multiwalled carbon nanotubes were present in the medium. Taken together, these results demonstrate that multiwalled carbon nanotubes induce the production of reactive oxygen species in RTL-W1, T47Dluc, and H295R cells, reveal no cytotoxicity, and reduce the bioavailability and toxicity of the biocide triclocarban.

Predicting the sensitivity of populations from individual exposure to chemicals: The role of ecological interactions

Population responses to chemical stress exposure are influenced by nonchemical, environmental processes such as species interactions. A realistic quantification of chemical toxicity to populations calls for the use of methodologies that integrate these multiple stress effects. The authors used an individual-based model for Daphnia magna as a virtual laboratory to determine the influence of ecological interactions on population sensitivity to chemicals with different modes of action on individuals. In the model, hypothetical chemical toxicity targeted different vital individual-level processes: reproduction, survival, feeding rate, or somatic growth rate. As for species interactions, predatory and competition effects on daphnid populations were implemented following a worst-case approach. The population abundance was simulated at different food levels and exposure scenarios, assuming exposure to chemical stress solely or in combination with either competition or predation. The chemical always targeted one vital endpoint. Equal toxicity-inhibition levels differently affected the population abundance with and without species interactions. In addition, population responses to chemicals were highly sensitive to the environmental stressor (predator or competitor) and to the food level. Results show that population resilience cannot be attributed to chemical stress only. Accounting for the relevant ecological interactions would reduce uncertainties when extrapolating effects of chemicals from individuals to the population level. Validated population models should be used for a more realistic risk assessment of chemicals.

Understanding receptor-mediated effects in rainbow trout: in vitro-in vivo extrapolation using physiologically based toxicokinetic models.

The European REACH regulation requires the use of animal experimentation to assess the risk of industrial chemicals. However, the 3R principle (reduction, replacement, refinement) demands the use of suitable alternative test methods. Many dossiers submitted for the authorization of chemicals have attempted to provide the required data without performing new experiments, relying heavily on in silico methods; in vitro assays were scarcely used. We propose a methodology that uses physiologically based toxicokinetic (PBTK) models to extrapolate in vitro data to the in vivo level. We collected experimental results for in vitro and in vivo ethoxyresorufin-O-deethylase and vitellogenin induction following chemical exposure and compared those results with model predictions. We found that the predictive power of aqueous chemical concentrations was limited; median effect concentrations (EC50s) based on internal concentrations in fish correlated better with in vitro EC50s. Our data show that in vitro assays could offer a substitute for fish studies when combined with PBTK models.

Incorporation mechanisms of a branched nonylphenol isomer in soil-derived organo-clay complexes during a 180-day experiment.

The incorporation process of a defined (13)C- and (14)C-labeled nonylphenol isomer (4-(3,5-dimethylhept-3-yl)phenol) into soil-derived organo-clay complexes was investigated. Isolated organo-clay complexes were separated into humic subfractions. Noninvasive ((13)C-CP/MAS NMR) and invasive methods (sequential chemical degradation, pyrolysis) were applied to obtain detailed information about the mode of incorporation, chemical structure, and change of the incorporation character of nonextractable residues in course of incubation. (13)C-CP/MAS NMR measurements of humic acids revealed an increasing incorporation of phenolic compounds during the experimental time which was referred to residues of the introduced (13)C-labeled NP isomer. Detailed investigations by means of sequential chemical degradation indicated a predominant incorporation of nonextractable NP isomer residues via reversible ester (amide) bonds. In course of time, the amount of releasable compounds decreased, pointing to altering processes which affected the mode of incorporation. BBr3-treatment, RuO4 oxidation, and thermochemolysis released only low portions of nonextractable radioactivity giving evidence of strongly incorporated residues. With the comprehensive application of complementary methods (e.g., humic matter fractionation, (13)C-CP/MAS NMR, sequential chemical degradation) it was possible to provide a comparatively detailed insight into the incorporation behavior of the applied NP isomer.

Microscale In Vitro Assays for the Investigation of Neutral Red Retention and Ethoxyresorufin-O-Deethylase of Biofuels and Fossil Fuels.

Only few information on the potential toxic effectiveness of biofuels are available. Due to increasing worldwide demand for energy and fuels during the past decades, biofuels are considered as a promising alternative for fossil fuels in the transport sector. Hence, more information on their hazard potentials are required to understand the toxicological impact of biofuels on the environment. In the German Cluster of Excellence “Tailor-made Fuels from Biomass” design processes for economical, sustainable and environmentally friendly biofuels are investigated. In an unique and interdisciplinary approach, ecotoxicological methods are applied to gain information on potential adverse environmental effects of biofuels at an early phase of their development. In the present study, three potential biofuels, ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran were tested. Furthermore, we investigated a fossil gasoline fuel, a fossil diesel fuel and an established biodiesel. Two in vitro bioassays, one for assessing cytotoxicity and one for aryl hydrocarbon receptor agonism, so called dioxin-like activity, as measured by Ethoxyresorufin-O-Deethylase, were applied using the permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss). The special properties of these fuel samples required modifications of the test design. Points that had to be addressed were high substance volatility, material compatibility and low solubility. For testing of gasoline, diesel and biodiesel, water accommodated fractions and a passive dosing approach were tested to address the high hydrophobicity and low solubility of these complex mixtures. Further work has to focus on an improvement of the chemical analyses of the fuel samples to allow a better comparison of any effects of fossil fuels and biofuels.