A fluorescence-based bioassay for aquatic macrophytes and its suitability for effect analysis of non-photosystem II inhibitors

Background, Goals and Scope During the last years the miniaturization of toxicity test systems for rapid and parallel measurements of large quantities of samples has often been discussed. For unicellular algae as well as for aquatic macrophytes, fluorescence-based miniaturized test systems have been introduced to analyze photosystem II (PSII) inhibitors. Nevertheless, high-throughput screening should also guarantee the effect detection of a broad range of toxicants in order to ensure routinely applicable, high-throughput measuring device experiments which can cover a broad range of toxicants and modes of action others than PSII inhibition. Thus, the aim of this study was to establish a fast and reproducible measuring system for non-PSII inhibitors for aquatic macrophyte species to overcome major limitations for use. Methods A newly developed imaging pulse-amplitude-modulated chlorophyll fluorometer (I-PAM) was applied as an effect detector in short-term bioassays with the aquatic macrophyte species Lemna minor. This multiwell-plate based measuring device enabled the incubation and measurement of up to 24 samples in parallel. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, polycyclic aromatic hydrocarbons (PAHs) and pharmaceuticals and personal care products (PPCPs), which are often detected in the aquatic environment. The I-PAM was used (i) to establish and validate the sensitivity of the test system to the three non-PSII inhibitors, (ii) to compare the test systems with standardized and established biotests for aquatic macrophytes, and (iii) to define necessary time scales in aquatic macrophyte testing. For validation of the fluorescence-based assay, the standard growth test with L. minor (ISO/DIS 20079) was performed in parallel for each chemical. Results The results revealed that fluorescence-based measurements with the I-PAM allow rapid and parallel analysis of large amounts of aquatic macrophyte samples. The I-PAM enabled the recording of concentration-effect-curves with L. minor samples on a 24-well plate with single measurements. Fluorescence-based concentration-effect-curves could be detected for all three chemicals after only 1 h of incubation. After 4–5 h incubation time, the maximum inhibition of fluorescence showed an 80–100% effect for the chemicals tested. The EC50 after 24 h incubation were estimated to be 0.06 mg/L, 0.84 mg/L and 1.69 mg/L for paraquatdichloride, alizarine and triclosan, respectively. Discussion The results obtained with the I-PAM after 24 h for the herbicide paraquat-dichloride and the polycyclic aromatic hydrocarbon alizarine were in good accordance with median effective concentrations (EC50s) obtained by the standardized growth test for L. minor after 7 d incubation (0.09 mg/L and 0.79 mg/L for paraquat-dichloride and alizarine, respectively). Those results were in accordance with literature findings for the two chemicals. In contrast, fluorescence-based EC50 of the antimicrobial agent triclosan proved to be two orders of magnitude greater when compared to the standard growth test with 7 d incubation time (0.026 mg/L) as well as with literature findings. Conclusion Typically, aquatic macrophyte testing is very time consuming and relies on laborious experimental set-ups. The I-PAM measuring device enabled fast effect screening for the three chemicals tested. While established test systems for aquatic macrophytes need incubation times of ≥ 7 d, the I-PAM can detect inhibitory effects much earlier (24 h), even if inhibition of chemicals is not specifically associated with PSII. Thus, the fluorescence-based bioassay with the I-PAM offers a promising approach for the miniaturization and high-throughput testing of chemicals with aquatic macrophytes. For the chemical triclosan, however, the short-term effect prediction with the I-PAM has been shown to be less sensitive than with long-term bioassays, which might be due to physicochemical substance properties such as lipophilicity. Recommendations and Perspectives The results of this study show that the I-PAM represents a promising tool for decreasing the incubation times of aquatic macrophyte toxicity testing to about 24 h as a supplement to existing test batteries. The applicability of this I-PAM bioassay on emergent and submerged aquatic macrophyte species should be investigated in further studies. Regarding considerations that physicochemical properties of the tested substances might play an important role in microplate bioassays, the I-PAM bioassay should either be accompanied by evaluating physicochemical properties modeled from structural information prior to an experimental investigation, or by intensified chemical analyses to identify and determine nominal concentrations of the toxicants tested. The chemicals paraquat-dichloride, alizarine and triclosan were chosen as representatives for the toxicant groups of non-PSII herbicides, PAHs and PPCPs which are often detected in the aquatic environment. Nevertheless, in order to ensure a routinely applicable measuring device, experiments with a broader range of toxicants and samples of surface and/or waste waters are necessary. ESS-Submission Editor: Dr. Markus Hecker (MHecker@Entrix.com)     

Relationship Between Crown Condition and Tree Diameter Growth in Southern Swedish Oaks

We studied correlation between crown conditions and tree-ring widths in 260 trees of pedunculate oak (Quercus robur L.) growing on 33 sites in southern Sweden. The tree-ring increment over 1998-2002 was highest in trees with healthy crowns, intermediate in trees with moderately declined crowns, and lowest in trees with heavily declining crowns. The time period with significant correlation between crown status and tree-ring increment varied between 10 years (given autocorrelation in tree-ring chronologies preserved) and 4 years (autocorrelation removed). In pairwise comparisons of three crown classes, differences in tree-ring increment between trees with healthy crowns and trees with heavily declining crowns were the most pronounced, Fisher LSD P value staying below 0.05 over 13 years (autocorrelation preserved ) or 4 years (autocorrelation removed). Over two 5-year periods (1993-1997 vs. 1998-2002) the cumulative increment increased significantly for trees with healthy crowns, did not change in trees with moderately declining crowns, and significantly decreased in trees with heavily declining crowns. For trees with healthy crowns, this dynamics may represent growth recovery after 1992 drought. Instead, oaks with defoliation above 60% appear to reach a threshold in their ability to recover growth. At sites on nutrient-poor soils cumulative increments over 1998-2002 differed significantly among trees with different crown condition and no differences were observed at sites on nutrient-rich soils. Analyses and interpretation of the oak growth trends as recovered from tree-ring chronologies may be improved by controlling for the crown status of the trees sampled, e.g., by using sampling strategy that would represent the average crown and growth conditions of the sites.     

Reducing external costs of nitrogen pollution by relocation of pig production between regions in the European Union

This paper tests the hypothesis that relocation of pig production within the EU27 can reduce the external costs of nitrogen (N) pollution. The external cost of pollution by ammonia and nitrate from agriculture in the European Union (EU27) in 2008 was estimated at 61–215 billion € (0.5 to 1.8% of the GDP). Per capita it ranged from more than 1000 € in north-west EU27 to 50 € in Romania. The average contribution of pig production was 15%. Using provincial data (224 NUTS2 regions in EU27), the potential reduction of external N cost by relocation of pig production was estimated at 14 billion € (10% of the total). Regions most eligible for decreasing the pig stock were in western Germany, Flemish region, Denmark, the Netherlands and Bretagne, while Romania is most eligible for increasing pig production. Relocating 20 million pigs (13% of the total EU stock) decreased average external costs per capita from 900 to 785 € in the 13 NUTS2 regions where pigs were removed and increased from 69 to 107 € in 11 regions receiving pigs. A second alternative configuration of pig production was targeted at reducing exceedance of critical N deposition and closing regional nutrient cycles. This configuration relocates pigs within Germany and France, for example from Bretagne to Northern France and from Weser-Ems to Oberbayern. However, total external cost increases due to an increase of health impacts, unless when combined with implementation of best N management practices. Relocation of the pig industry in the EU27 will meet many socio-economic barriers and realisation requires new policy incentives.     

Evaluation of aerosol sources at European high altitude background sites with trajectory statistical methods

This study has investigated the influence of synoptic weather patterns and long-range transport episodes on the concentrations of several compounds related to different aerosol sources (EC, OC, SO₄^2?, Ca²⁺, Na⁺, K⁺, ²¹⁰Pb, levoglucosan and dicarboxylic acids) registered in PM10 or PM2.5 aerosol samples collected at three remote background sites in central Europe. Air mass back-trajectories arriving at these sites have been analysed by statistical methods. Firstly, air mass back-trajectories have been grouped into clusters. Each cluster corresponds to specific meteorological scenarios, which were extracted and discussed. Finally, redistributed concentration fields have been computed to identify the main potential source regions of the different key aerosol components. A marked seasonal pattern is observed in the occurrence of the different clusters, with fast westerly and northerly Atlantic flows during winter and weak circulation flows in summer. Spring and fall were characterised by advection of moderate flows from northeastern and eastern Europe. Significant inter-cluster differences were observed for concentrations of receptor aerosol components, with the highest concentrations of EC, OC, SO₄^2?, K⁺ and ²¹⁰Pb associated with local and mesoscale aerosol sources located over central Europe related to enhanced photochemical processes. Emissions produced by fossil fuel and biomass burning processes from the Baltic countries, Byelorussia, western regions of Russia and Kazakhstan in spring and fall also contribute to elevated levels of EC, OC, SO₄^2?, K⁺ and ²¹⁰Pb. In the summer period long-range transport episodes of mineral dust from North-African deserts were also frequently detected, which caused elevated concentrations of coarse Ca²⁺ at sites. The baseline aerosol concentrations in central Europe at the high altitude background sites were registered in winter, with the exception of coarse Na⁺. While the relatively high concentrations of Na⁺ can be explained by sea salt advected from the Atlantic, the low levels of other aerosol components are caused by efficient aerosol scavenging associated to advections of Atlantic air masses, as well as lower emissions of these species over the Atlantic compared to those over the European continent and very limited vertical air mass exchange over the continent.     

Reduction of Baltic Sea Nutrient Inputs and Allocation of Abatement Costs Within the Baltic Sea Catchment

The Baltic Sea Action Plan (BSAP) requires tools to simulate effects and costs of various nutrient abatement strategies. Hierarchically connected databases and models of the entire catchment have been created to allow decision makers to view scenarios via the decision support system NEST. Increased intensity in agriculture in transient countries would result in increased nutrient loads to the Baltic Sea, particularly from Poland, the Baltic States, and Russia. Nutrient retentions are high, which means that the nutrient reduction goals of 135 000 tons N and 15 000 tons P, as formulated in the BSAP from 2007, correspond to a reduction in nutrient loadings to watersheds by 675 000 tons N and 158 000 tons P. A cost-minimization model was used to allocate nutrient reductions to measures and countries where the costs for reducing loads are low. The minimum annual cost to meet BSAP basin targets is estimated to 4.7 billion €.     

A review of plant–pharmaceutical interactions: from uptake and effects in crop plants to phytoremediation in constructed wetlands

Pharmaceuticals are commonly found both in the aquatic and the agricultural environments as a consequence of the human activities and associated discharge of wastewater effluents to the environment. The utilization of treated effluent for crop irrigation, along with land application of manure and biosolids, accelerates the introduction of these compounds into arable lands and crops. Despite the low concentrations of pharmaceuticals usually found, the continuous introduction into the environment from different pathways makes them ‘pseudo-persistent’. Several reviews have been published regarding the potential impact of veterinary and human pharmaceuticals on arable land. However, plant uptake as well as phytotoxicity data are scarcely studied. Simultaneously, phytoremediation as a tool for pharmaceutical removal from soils, sediments and water is starting to be researched, with promising results. This review gives an in-depth overview of the phytotoxicity of pharmaceuticals, their uptake and their removal by plants. The aim of the current work was to map the present knowledge concerning pharmaceutical interactions with plants in terms of uptake and the use of plant-based systems for phytoremediation purposes.     

Technical Aspects of Lime/Limestone Scrubbers for Coal-Fired Power Plants

This is the conclusion of a 2-part article dealing with the technical aspects of lime/limestone scrubbers for coal-fired power plants, it covers instrumentation, participate removal and sludge disposal. Part I (June JAPCA) covered process chemistry and scrubber systems     

Mobile passive samplers: concept for a novel mode of exposure

Integrative passive sampling with devices such as semipermeable membrane devices generally relies on rigs for month-long static exposures in water. We evaluate here whether mobile exposures of passive samplers can provide reliable estimates of dissolved contaminant concentrations. Mobile exposures were obtained by towing samplers fastened to the end of a benthic trawl net. Significant and reproducible absorption of polycyclic aromatic hydrocarbons during 5 h-long deployments was made possible by high sampling rates resulting from high water turbulences during towing at 1.2-1.5 knots. Sampling rates (72-215 L d⁻¹) estimated from the dissipation of performance reference compounds were supported by in situ calibration with samplers exposed for a 30 days in the vicinity of the test site. Higher fluoranthene and pyrene absorption in samplers exposed to the trawling-induced sediment plume could be attributed to desorption from re-suspended sediments. This mode of exposure has the potential to be used in monitoring programmes.     

Long-term effects of clear-cutting and selective cutting on soil methane fluxes in a temperate spruce forest in southern Germany

Based on multi-year measurements of CH₄ exchange in sub-daily resolution we show that clear-cutting of a forest in Southern Germany increased soil temperature and moisture and decreased CH₄ uptake. CH₄ uptake in the first year after clear-cutting (−4.5 ± 0.2 μg C m⁻² h⁻¹) was three times lower than during the pre-harvest period (−14.2 ± 1.3 μg C m⁻² h⁻¹). In contrast, selective cutting did not significantly reduce CH₄ uptake. Annual mean uptake rates were −1.18 kg C ha⁻¹ yr⁻¹ (spruce control), −1.16 kg C ha⁻¹ yr⁻¹ (selective cut site) and −0.44 kg C ha⁻¹ yr⁻¹ (clear-cut site), respectively. Substantial seasonal and inter-annual variations in CH₄ fluxes were observed as a result of significant variability of weather conditions, demonstrating the need for long-term measurements. Our findings imply that a stepwise selective cutting instead of clear-cutting may contribute to mitigating global warming by maintaining a high CH₄ uptake capacity of the soil.