Comparability of river quality assessment using macrophytes: a multi-step procedure to overcome biogeographical differences.

This paper exposes a new methodological approach to solve the problem of intercalibrating river quality national methods when a common metric is lacking and most of the countries share the same Water Framework Directive (WFD) assessment method. We provide recommendations for similar works in future concerning the assessment of ecological accuracy and highlight the importance of a good common ground to make feasible the scientific work beyond the intercalibration. The approach herein presented was applied to highly seasonal rivers of the Mediterranean Geographical Intercalibration Group for the Biological Quality Element Macrophytes. The Mediterranean Group of river macrophytes involved seven countries and two assessment methods with similar acquisition data and assessment concept: the Macrophyte Biological Index for Rivers (IBMR) for Cyprus, France, Greece, Italy, Portugal and Spain, and the River Macrophyte Index (RMI) for Slovenia. Database included 318 sites of which 78 were considered as benchmarks. The boundary harmonization was performed for common WFD-assessment methods (all countries except Slovenia) using the median of the Good/Moderate and High/Good boundaries of all countries. Then, whenever possible, the Slovenian method, RMI was computed for the entire database. The IBMR was also computed for the Slovenian sites and was regressed against RMI in order to check the relatedness of methods (R(2)=0.45; p<0.00001) and to convert RMI boundaries into the IBMR scale. The boundary bias of RMI was computed using direct comparison of classification and the median boundary values following boundary harmonization. The average absolute class differences after harmonization is 26% and the percentage of classifications differing by half of a quality class is also small (16.4%). This multi-step approach to the intercalibration was endorsed by the WFD Regulatory Committee.

Least Disturbed Condition for European Mediterranean rivers.

The present report describes a three-step approach that was used to characterize and define thresholds for the Least Disturbed Condition in Mediterranean streams of four different types, regarding organic pollution and nutrients, hydrological and morphological alterations, and land use. For this purpose, a common database composed of national reference sites (929 records) from seven countries, sampled for invertebrates, diatoms and macrophytes was used. The analyses of reference sites showed that small (catchment <100 km(2)) siliceous and non-siliceous streams were mainly affected by channelization, bank alteration and hydropeaking. Medium-sized siliceous rivers were the most affected by stressors: 25-43% of the samples showed at least slight alterations regarding channelization, connectivity, upstream dam influence, hydropeaking and degradation of riparian vegetation. Temporary streams were the least affected by hydromorphological changes, but they were nevertheless affected by alterations in riparian vegetation. There were no major differences between all permanent stream types regarding water quality, but temporary streams showed lower values for oxygenation (DO) and wider ranges for other variables, such as nitrates. A lower threshold value for DO (60%) was determined for this stream type and can be attributed to the streams' natural characteristics. For all other river types, common limits were found for the remaining variables (ammonium, nitrate, phosphate, total P, % of artificial areas, % of intensive and extensive agriculture, % of semi-natural areas in the catchment). These values were then used to select the list of reference sites. The biological communities were characterized, revealing the existence of nine groups of Mediterranean invertebrate communities, six for diatoms and five for macrophytes: each group was characterized by specific indicator taxa that highlighted the differences between groups.

The role of UV-B radiation in aquatic and terrestrial ecosystems--an experimental and functional analysis of the evolution of UV-absorbing compounds.

We analysed and compared the functioning of UV-B screening pigments in plants from marine, fresh water and terrestrial ecosystems, along the evolutionary line of cyanobacteria, unicellular algae, primitive multicellular algae, charophycean algae, lichens, mosses and higher plants, including amphibious macrophytes. Lichens were also included in the study. We were interested in the following key aspects: (a) does the water column function effectively as an 'external UV-B filter'?; (b) do aquatic plants need less 'internal UV-B screening' than terrestrial plants?; (c) what role does UV screening play in protecting the various plant groups from UV-B damage, such as the formation of thymine dimers?; and (d) since early land 'plants' (such as the predecessors of present-day cyanobacteria, lichens and mosses) experienced higher UV-B fluxes than higher plants, which evolved later, are primitive aquatic and land organisms (cyanobacteria, algae, lichens, mosses) better adapted to present-day levels of UV-B than higher plants? Furthermore, polychromatic action spectra for the induction of UV screening pigments of aquatic organisms have been determined. This is relevant for translating 'physical' radiation measurements of solar UV-B into 'biological' and 'ecological' effects. From the action spectra, radiation amplification factors (RAFs) have been calculated. These action spectra allow us to determine any mitigating or antagonistic effects in the ecosystems and therefore qualify the damage prediction for the ecosystems under study. We summarize and discuss the main results based on three years of research of four European research groups. The central theme of the work was the investigation of the effectiveness of the various screening compounds from the different species studied in order to gain some perspective of the evolutionary adaptations from lower to higher plant forms. The induction of mycosporine-like amino acids (MAAs) was studied in the marine dinoflagellate Gyrodinium dorsum, the green algal species Prasiola stipitata and in the cyanobacterium Anabaena sp. While visible (400-700 nm) and long wavelength UV-A (315-400 nm) showed only a slight effect, MAAs were effectively induced by UV-B (280-315 nm). The growth of the lower land organisms studied, i.e. the lichens Cladina portentosa, Cladina foliacaea and Cladonia arbuscula, and the club moss Lycopodiumannotinum, was not significantly reduced when grown under elevated UV-B radiation (simulating 15% ozone depletion). The growth in length of the moss Tortula ruralis was reduced under elevated UV-B. Of the aquatic plants investigated the charophytes Chara aspera showed decreased longitudinal growth under elevated UV-B. In the 'aquatic higher plants' studied, Ceratophyllum demersum, Batrachium trichophyllum and Potamogeton alpinus, there was no such depressed growth with enhanced UV-B. In Chara aspera, neither MAAs nor flavonoids could be detected. Of the terrestrial higher plants studied, Fagopyrum esculentum, Deschampsia antarctica, Vicia faba, Calamagrostis epigejos and Carex arenaria, the growth of the first species was depressed with enhanced UV-B, in the second species length growth was decreased, but the shoot number was increased, and in the latter two species of a dune grassland there was no reduced growth with enhanced UV-B. In the dune grassland species studied outdoors, at least five different flavonoids appeared in shoot tissue. Some of the flavonoids in the monocot species, which were identified and quantified with HPLC, included orientin, luteolin, tricin and apigenin. A greenhouse study with Vicia faba showed that two flavonoids (aglycones) respond particularly to enhanced UV-B. Of these, quercetin is UV-B inducible and mainly located in epidermal cells, while kaempferol occurs constitutively. In addition to its UV-screening function, quercetin may also act as an antioxidant. Polychromatic action spectra were determined for induction of the UV-absorbing pigments in three photosynthetic organisms, representing very different taxonomic groups and different habitats. In ultraviolet photobiology, action spectra mainly serve two purposes: (1) identification of the molecular species involved in light absorption; and (2) calculation of radiation amplification factors for assessing the effect of ozone depletion. Radiation amplification factors (RAFs) were calculated from the action spectra. In a somewhat simplified way, RAF can be defined as the percent increase of radiation damage for a 1% depletion of the ozone layer. Central European summer conditions were used in the calculations, but it has been shown that RAF values are not critically dependent on latitude or season. If only the ultraviolet spectral region is considered, the RAF values obtained are 0.7 for the green alga Prasiola stipitata, 0.4 for the dinoflagellate Gyrodinium dorsum, and 1.0 for the cyanobacterium Anabaena sp. In the case of P. stipitata, however, the effect of visible light (PAR, photosynthetically active radiation, 400-700 nm) is sufficient to lower the RAF to about 0.4, while the PAR effect for G. dorsum is negligible. RAFs for some damage processes, such as for DNA damage (RAF=2.1 if protective effects or photorepair are not considered [1]), are higher than those above. Our interpretation of this is that if the ozone layer is depleted, increased damaging radiation could overrule increased synthesis of protective pigments. In addition to investigating the functional effectiveness of the different screening compounds, direct UV effects on a number of key processes were also studied in order to gain further insight into the ability of the organisms to withstand enhanced UV-B radiation. To this end, the temperature-dependent repair of cyclobutane dimers (CPD) and (6-4) photoproducts induced by enhanced UV-B was studied in Nicotiana tabacum, and the UV-B induction of CPD was studied in the lichen Cladonia arbuscula. Also, photosynthesis and motility were monitored and the response related to the potential function of the screening compounds of the specific organism.

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus.

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.

Effects of 5,7-DHT Injection into the Optic Lobe on the Circadian Locomotor Rhythm in the Cricket, Gryllus bimaculatus.

The effect of direct 5,7-dihydroxytryptamine (5,7-DHT) injection into the medulla region of the optic lobe on the locomotor activity was investigated in the adult male cricket, Gryllus bimaculatus. After a 6 hr phase advance of a light-dark cycle, the 5,7-DHT injected animals needed significantly longer time for resynchronization to the new cycle (6.55 +/- 0.62 days) than the control, Ringer's solution injected animals (3.17 +/- 0.15 days; P < 0.001, t-test). Light induced a bout of activity (i.e., masking effect) when light-dark cycle was phase advanced by 6 hr and the duration of the masking effect was significantly longer in 5,7-DHT injected animals. An initial bout of the nocturnal activity was significantly greater in the 5,7-DHT injected animal. Under constant darkness, the freerunning periods of both groups were not significantly different. Under constant light, a significantly higher percentage of 5,7-DHT injected animals showed arrhythmicity compared with the control group. An analysis carried by high-pressure liquid chromatography with electro-chemical detection (HPLC-ECD) revealed that the serotonin content in the optic lobe was significantly reduced to less than 50% in the 5,7-DHT injected animals, even one month after the injection. These results suggest that serotonin plays important roles in the regulation of circadian locomotor rhythms of the cricket mainly by regulating the sensitivity of the photoreceptive system.