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)     

Factor analysis of corporate environmental responsibility

Under the current situation of environmental pollution and resources shortage, the research of corporate environmental responsibility (CER) is of great importance to promote the sustainable development of society. The article adopts the perspective of the Stakeholder Theory and environmental management to make the empirical analysis of CER. Consequently designed questionnaire investigation is carried out among 30 Shanghai enterprises during 2007 and 2008. Through the correlation analysis, principal component analysis, factor ordering, and variables reappearance of key factors, seven key indexes are identified in the original 35 variables. This article aims to provide reference for enterprise environmental management and forwards the study of Corporate Social Responsibility.     

Hepatic mercury,cadmium, and lead in mink and otter from New York State: monitoring environmental contamination

Many non-linear processes link atmospheric emissions to the bioavailability of metals; consequently, the monitoring of metals in ecosystem components is required to model their ecodynamics. American mink (Neovison vison) and river otter (Lontra canadensis) have the potential to serve as an upper-level-consumer component in monitoring metals bioavailability. However, the relationship of bioaccumulated metals to various environmental factors has not been explored nor have the effects of demographic factors been resolved. To address these limitations, mink and otter, collected throughout New York State during 1998–2002, were analyzed for hepatic concentrations of total mercury (Hg), cadmium (Cd), and lead (Pb). Relationships were investigated between metals concentrations and landscape-level factors (physiographic zone, hydrologic unit, and elevation) and demographic factors (gender and age). Considerable variation in Hg and Cd concentrations was observed relative to both physiographic zone and hydrologic unit for both species. In contrast with Hg, Cd concentration increased predictably with increasing elevation. Mercury concentrations were greater, but for Cd less, in otter than mink. Lead concentrations showed little landscape heterogeneity and were independent of elevation. Age-related bioaccumulation was evident for Hg and Cd, but not for Pb, in both species. Mercury and Cd concentrations were greater in female than male mink; however, Pb concentrations were greater in males than females. Inverse relationships of relative growth (weight/length) to metals concentrations explained gender differences in Hg and Cd in mink. For otter, no gender-related differences in metals concentrations were apparent. The suitability of mink and otter for monitoring programs is discussed.     

Quantification and reduction of erroneous differences between images in remote sensing

The reliability of image data, along with the difficulty of accurately comparing images acquired at different times or from different sensors, is a generic problem in remote sensing. Measurement repeatability errors occur frequently and can substantially reduce the system’s ability to reliably quantify real spectral and spatial change in a target. This paper outlines methodologies for quantifying and reducing erroneous differences between monochrome and multispectral or hyperspectral images. [Each of these image types is acquired by an instrument that collects light photons across a variable range of the electromagnetic spectrum (often referred to as an image band). The hyperspectral image is often referred to as a hyperspectral cube with XY spatial dimensions and many Z bands (spectral demotions)]. In this paper, we specifically discuss the Pixel Block Transform (PBT), the Spectral Averaging Transform (SAT), and the Wavelet Transform (WAVEL). We briefly address sensor fusion. Results indicate that the PBT is a powerful cross-noise and repeatability error reducing tool, applicable to monochrome, multispectral, and hyperspectral images. The SAT is as powerful as the PBT in reducing error but is suitable only for hyperspectral imagery. WAVEL can reduce some of the finer scale noise, but it is not as powerful in reducing cross-noise as PBT or SAT and it requires some trial and error for selecting the appropriate wavelet function. It is important that those involved in developing statistically sound relationships between remotely sensed imagery and other data sources understand the problems and their solutions to prevent wasting time or developing relationships that are statistically insignificant or unstable, or that lead to faulty conclusions.     

The morphogenesis of ephyra in Coronatae (Cnidaria,Scyphozoa)

The morphogenesis of ephyrae of Atorella vanhoeffeni Bigelow, 1909 (Werner, 1967) and of Nausithoe maculata Jarms, 1990 during strobilation is described. We found differences in the developmental pattern of marginal structures and structural changes of longitudinal muscle tubes in particular. During strobilation the polyp’s tetraradiate symmetry is passed to the ephyra of both taxa as a tetraradiate central symmetry that we consider a major plesiomorphic character. The present results also indicate divergent patterns of ephyra development during strobilation that lead to variable marginal symmetries of ephyrae and thus of medusae. Therefore, the marginal symmetry of medusae of N. maculata is octoradiate and of A. vanhoeffeni is hexaradiate. We conclude the latter is stated as derived pattern. These results lead us to maintain both families Nausithoidae and Atorellidae.     

Chemical composition of the slippery epicuticular wax blooms on Macaranga (Euphorbiaceae) ant-plants

Summary. The stems of many Macaranga ant-plants (Euphorbiaceae) are covered by epicuticular wax crystals rendering the surface very slippery for most insects. These wax blooms act as selective barriers protecting the symbiotic ant partners, which are specialized “wax-runners”, against the competition of other ants. Glaucous stems occur almost exclusively among the ant-plants of the genus Macaranga (). We analyzed the cuticular lipids of 16 Macaranga species by GC-MS and investigated the wax crystal morphology using SEM. Presence of crystalline wax blooms was strongly correlated with high concentrations (52%–88%) of triterpenoids. In contrast epicuticular waxes of glossy Macaranga surfaces contained only 0% to 36% of these dominant components. Therefore we conclude that triterpenoids are responsible for the formation of the thread-like Macaranga wax crystals. In all Macaranga ant-plants investigated, the principal components were epitaraxerol and taraxerone accompanied by smaller portions of taraxerol, β-amyrin and friedelin. Only in the case of the non-myrmecophytic M. tanarius did β-amyrin predominate. Moreover, we found that only in M. tanarius, the dense wax crystal lacework is torn into large mosaic-like pieces in the course of secondary stem diameter growth. Both chemical and macroscopic differences may contribute to a reduced slipperiness of M. tanarius stems and appear to be functionally important. The distribution of wax crystals and their composition amongst different sections of the genus suggests that glaucousness is a polyphyletic character within Macaranga. Received 7 October 1999; accepted 3 December 1999     

Controlling enzymatic activity by immobilization on graphene oxide

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells’ viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.     

Efficiency of Reovirus Concentration from Water with Positively Charged Filters

This study examined the efficacy of reovirus concentration from large volumes of water using two positively charged filters: Zeta Plus 1MDS and NanoCeram. The results indicated that an average of 61 and 81% of input reoviruses were effectively recovered, respectively, from recycled water and tap water using NanoCeram filtration.     

Benzene Exposure Assessment at Indoor, Outdoor and Personal Levels. The French Contribution to the Life MACBETH Programme

Many VOC represent hazards to human health through chronic exposure. Recent European and world-wide legislation proposes limit values for ambient concentrations of these compounds. However, very little experimental data exists for true population exposure. In 1996, the European MACBETH initiative set out to measure population exposure to benzene in six European cities. This study details the French contribution to this program. Six campaigns were carried out, each comprising measurements at 100 outdoor sites and the participation of 50 non-smoking volunteers who wore personal samplers and had passive monitors installed in their homes. Iso-concentration maps were drawn for each campaign and the results showed that outdoor concentrations were significantly lower than indoors. Almost 75% of the volunteers were exposed to mean concentrations higher than the limit value of 5µgm³. It is demonstrated that personal exposure levels cannot be deduced simply by combining indoor and outdoor background concentrations. It is also shown that there is need for better knowledge of the contributions to overall exposure of outdoor microenvironments and the authors hope that future European directives will take this into account.     

Changing effluent chemistry affect survival,growth and physiological function of <Emphasis Type="Italic">Acacia nilotica</Emphasis> seedlings in northwestern region of India

Recycling and conservation efforts for water are the need of the day because of the lack of new water sources and the ever-increasing demand for drinking water. Seedlings of Acacia nilotica L. were irrigated with: canal water (T₁, control); municipal effluent (T₂); textile effluent (T₃); steel effluent (T₄); textile + municipal effluent in 1:1 ratio (T₅); steel + municipal effluent in 1:2 ratio (T₆); steel + textile in 1:2 ratio (T₈) and steel + municipal + textile in 1:2:2 ratio (T₇) with views to observe effluents effect on the seedlings and its adaptability and to recommend safe disposal of these effluents. Seedlings in T₆, T₇ and T₈ showed 50% lesser height and collar diameter than those in control. Seedlings in T₂ attained greatest height, collar diameter, numbers of branches and produced 140 g dry biomass seedling⁻¹. Highest concentration of manganese (Mn), iron (Fe), copper (Cu) and zinc (Zn) and lowest concentration of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) in the seedlings of T₄, T₆, T₇ and T₈ resulted in nutritional imbalance, mineral toxicity and reduction in photosynthetic (Pn) and transpiration (E) rates and caused seedling mortality. Seedlings of T₃ had highest sodium concentration and low concentration of Ca, Mg and micronutrients resulting in nutritional imbalance, augmented chlorosis and reduced gas exchange and biomass by half as compared to control. Increased growth, Pn and E and biomass in seedlings of T₅ over T₃ and survival period in T₆, T₇ and T₈ seedlings suggested a beneficial effect of effluents mixing. Unscientific disposal should be avoided and toxic concentration of metal ions␣may be reduced for long-term application and harmless disposal of effluents in afforestation and urban development.