Cumulative Stressors Trigger Increased Vulnerability of Diatom Communities to Additional Disturbances.

Chronic, non-lethal stressors occurring gradually (in space or time) can result in cumulative impacts that are more dramatic than higher intensities or occasional critical levels of any single one of these stressors. The negative effects of the chronic stressors trigger lasting impacts that may grow in intensity and become problematic over time and/or to higher trophic levels. In rivers, aquatic organisms experience this type of cumulative stress along the up- to downstream gradient in natural and anthropogenic contaminants generally observed in inhabited watersheds. Diatoms are a major component of the periphyton in rivers; their richness and diversity in natural communities are directly related to their varied ecological preferences and sensitivity to disturbance. In this study, we monitored from 2003 to 2008 the changes in the diversity of taxonomic and non-taxonomic features along a small river (Riou-Mort, South West France), at three sites: one site upstream considered as a reference for this watershed, one intermediate site with high nutrient load, and one downstream site exposed to both nutrient and metal pollution. The cumulative impacts of nutrients plus metals led to a gradual decrease in species richness and diversity, and in a potential capacity to cope with additional stresses, e.g., climate change-related ones. This is reflected by a decrease in species richness downstream, more dramatic in the hot summer of 2003 than in cooler summers. With the increasingly protective environmental regulations (e.g., Water Framework Directive in Europe), accumulation of stresses on aquatic resources are recommended to receive increasing attention, in particular considering the expected changes in climate.

Use of diatom assemblages as biomonitor of the impact of treated uranium mining effluent discharge on a stream: case study of the Ritord watershed (Center-West France).

The rehabilitation of French former uranium mining sites has not prevented the contamination of the surrounding aquatic ecosystems with metal elements. This study assesses the impact of the discharge of treated uranium mining effluents on periphytic diatom communities to evaluate their potential of bioindication. A 7-month survey was conducted on the Ritord watercourse to measure the environmental conditions of microalgae, the non-taxonomic attributes of periphyton (photosynthesis and biomass) and to determine the specific composition of diatom assemblages grown on artificial substrates. The environmental conditions were altered by the mine waters, that contaminate the watercourse with uranium and with chemicals used in the pit-water treatment plants (BaCl2 and Al2(SO4)3). The biomass and photosynthetic activity of periphyton seemed not to respond to the stress induced by the treated mining effluents whereas the altered environmental conditions clearly impacted the composition of diatom communities. Downstream the discharges, the communities tended to be characterized by indicator species belonging to the genera Fragilaria, Eunotia and Brachysira and were highly similar to assemblages at acid mine drainage sites. The species Eunotia pectinalis var. undulata, Psammothidium rechtensis, Gomphonema lagenula and Pinnularia major were found to be sensitive to uranium effluents whereas Neidium alpinum and several species of Gomphonema tolerated this contamination. The relevance of diatoms as ecological indicator was illustrated through the changes in structure of communities induced by the discharge of uranium mining effluents and creates prospects for development of a bioindicator tool for this kind of impairment of water quality.

Recovery potential of periphytic biofilms translocated in artificial streams after industrial contamination (Cd and Zn).

Metal wastes can significantly disturb aquatic communities, particularly photosynthetic organisms, the main primary producers in freshwater running ecosystems. In this study, biofilms and diatoms were used as bioindicators to characterize the kinetics of biofilm recovery. An experimental decontamination study was conducted under laboratory conditions, after biofilm colonisation at a site subject to discharge of industrial metals (Zn and Cd) and in parallel at an upstream site, metal-free, considered as a control. After 24 days of colonisation, biofilms were translocated and maintained in the laboratory for 56 days under clean conditions (control and decontamination) or metal contamination. Various tests were conducted from the community level--measures of metal bioaccumulation, cell densities and taxonomic investigations, to the individual level--measures of teratological forms. After 56 days of decontamination, Zn and Cd concentrations in decontaminated biofilms showed a sharp decline, respectively ranging from 6.7 ± 2 to 4 ± 2.5 mg Zn g⁻¹ DW and from 207.6 ± 24.5 to 45.4 ± 9.9 µg Cd g⁻¹ DW. However, at the end of the experiment bioaccumulations remained significantly higher than concentrations in control biofilms. Despite a diatom evolution in biofilm assemblages, taxonomic inventories did not demonstrate a complete restoration of diatom communities in biofilms under decontamination conditions compared with controls, since metal-resistant species initially present after colonisation at the contaminated site, such as Eolimna minima, persisted in high abundance in decontaminated biofilms. Biofilms kept under metal pressure showed very high bioaccumulation capacities and a sharp decline of species diversity which allowed identification of some resistant species. Regarding these first results on the behaviour of diatom biofilms under experimental decontamination conditions, improvement of the natural hydrosystem's chemical state appears quickly, but an eventual return to good ecological status appears delayed, with the persistence of metal-tolerant species even after 56 days.

Assessing the impact of chloroacetanilide herbicides and their metabolites on periphyton in the Leyre River (SW France) via short term growth inhibition tests on autochthonous diatoms.

The Leyre River is the main tributary to the Bassin d'Arcachon lagoon. Herbicides belonging to the chloroacetanilide class have been found in the river (S-metolachlor and acetochlor) as well as some of their metabolites at higher concentrations. As the environmental toxicity of these molecules is not well known, ecotoxicological tests have been carried out on river periphyton at different levels of biological diversity: from the clone of one diatom species (Nitzschia nana) to the population of the same species (several clones) up to the multi-specific species community dominated by diatoms. Moreover, tests were performed on diatoms coming from an unpolluted upstream site and from a contaminated downstream site, in order to investigate possible tolerance acquisition to pollutants. The method consisted in measuring diatom growth inhibition at different doses of each substance from the increase of chlorophyll-a concentration after 4 days. It resulted that acetochlor was clearly more toxic than S-metolachlor at all levels of biological diversity. EC(50) values estimated from the tests suggest no effect of contaminants on diatom growth or biomass in the river. The toxicity of the metabolites appeared very low compared to that of their parent compounds. No difference in tolerance to the herbicides was demonstrated between summer diatom communities from the two sites in spite of different specific compositions. However, concerning the populations of N. nana isolated in winter following the highest herbicide concentrations in the river (about 0.5 µg L(-1)), the downstream population showed a higher tolerance to acetochlor but there was no co-tolerance to S-metolachlor. Thus, it appeared that acetochlor represents the highest toxic pressure on periphyton among the other contaminants in the Leyre River.

Ecological assessment of French Atlantic lakes based on phytoplankton, phytobenthos and macrophytes.

Biological elements, including phytoplankton, phytobenthos, macrophytes, benthic invertebrates and fish, are employed by the EU Water Framework Directive (WFD) 2000/60/EC as ecological indicators for the assessment of surface waters. The use of primary producers (phytoplankton, phytobenthos and macrophytes) for water quality assessment has a long history, and several methods have been developed worldwide. In this study, we used these three communities to assess the ecological status of five natural lakes located in the Aquitaine region (southwest France). Several biological indices used in lakes from other European countries or in French rivers were employed and compared among the three communities. Each primary producer provided complementary information about the ecological status of the lakes, including the invasiveness of exotic taxa. Regardless of the producer community used, the response to the environment, as reflected by the indices (adequate for each community), was similar: Lakes Cazaux, Lacanau and Hourtin showed the best ecological status and Parentis and Soustons the worst. Phytoplankton diagnosis reflected and integrated unambiguously the water quality of the lakes, as demonstrated by the strong relationships between the phytoplankton index and the trophic status criteria. This community appeared as the best indicator, especially when macrophytes were absent. The methods applied here represent a potential tool for the assessment of the ecological status in the context of WFD, but they need to be refined. We propose modifications for phytobenthos index initially tailored for running waters for adequate use in lentic ecosystems. Indices for the three primary producers should be modified to incorporate exotic species which may provide information on potential biodiversity losses.

Comparison of periphytic biofilm and filter-feeding bivalve metal bioaccumulation (Cd and Zn) to monitor hydrosystem restoration after industrial remediation: a year of biomonitoring.

Despite a significant decrease in the metallic waste emissions from an industrial site and a remediation process initiated in 2007, the Riou-Mort watershed (southwest France) still exhibits high Cd and Zn concentrations. Metal wastes have long been proven to significantly disturb aquatic communities. In this study, bioaccumulation capacities and responses to the chemical improvement of the hydrosystem were assessed for a year along the contamination gradient through the comparison of two biological models: Corbicula fluminea and periphytic biofilms, both considered as good bioindicators. Bioaccumulation results confirmed the persistence of water contamination in Corbicula fluminea and biofilms with, respectively, maximum Cd concentrations reaching 80.6 and 861.2 µg gDW(-1), and Zn concentrations 2.0 and 21.3 mg gDW(-1). Biofilms exhibited bioaccumulation in close correlation with water contamination, while Corbicula fluminea presented Cd bioaccumulation clearly regulated by water temperature and metal concentrations, affecting the ventilatory activity, as revealed by condition indices measurements. Also, a linear regression using Cd bioaccumulation and temperature () showed that below approximately 6 °C Corbicula fluminea did not appear to accumulate metals. Bioconcentration factors (BCFs) were higher in biofilms in comparison with Corbicula fluminea and showed the great accumulation capacity of suspended particulate matter in biofilms. However, bioaccumulation capacities are known to be influenced by many factors other than metal concentrations, such as temperature, water oxygenation or plankton and nutrient concentrations. Thus, this study demonstrates the power of a combined assessment using both Corbicula fluminea and biofilms as bioindicators to give a more integrated view of water quality assessment. Finally, when comparing our results with previous studies, the start of hydrosystem restoration could be shown by decreasing bioaccumulation in organisms.

Variations of periphytic diatom sensitivity to the herbicide diuron and relation to species distribution in a contamination gradient: implications for biomonitoring.

Diatoms are commonly used as bioindicators of trophic and saprobic pollution in rivers. However, more knowledge is needed concerning their sensitivity to toxicants such as agricultural herbicides. In this study, seven species of periphytic diatoms were isolated from the Morcille River (Beaujolais area, France) which presents a streamward contamination gradient by pesticides and particularly diuron. The sensitivity of these species to diuron was assessed through ecotoxicological tests based on short-term growth inhibition of monospecific cultures. After application of an appropriate toxicological model, EC50 were determined and the species were ranked according to their tolerance. EC50 values ranged from 4.5 to 19 µg L(-1). Finally, the results were related to field periphyton samples from an upstream and a downstream site in order to check if variations in specific relative abundance between sites are consistent with differences in tolerance to diuron. Species distribution between sites was only partially in accordance with toxicological results suggesting that other factors (toxic or trophic) have an important influence on diatom communities in the river. Nevertheless, diatoms showed their potential to indicate water contamination by pesticides and toxic indices could be developed in complement to existing trophic indices.

Effects of pesticides on freshwater diatoms.

The study of pesticide effects on algae, and diatoms in particular, was focused on photosynthesis and biomass growth disturbances. Few studies have been performed to investigate the effects of these toxic agents on intracellular structures of diatom cells. Nuclear alterations and cell wall abnormalities were reported for diatoms exposed to toxic compounds. Nevertheless, the cellular mechanisms implicated in the development of such alterations and abnormalities remain unclear. Sensitivity to pesticides is known to be quite different among different diatom species. Eutrophic and small species are recognized for their tolerance to pesticides exposure. More pronounced cell defenses against oxidative stress may explain this absence of sensitivity in species of smaller physical size. Notwithstanding, on the whole, explaining the rationale behind tolerance variations among species has been quite difficult, thus far. In this context, the understanding of intracellular toxicity in diatoms and the relation between these intracellular effects and the disturbance of species composition in communities represent a key target for further research. The original community species structure determines the response of a diatom community to toxic agent exposure. Diatom communities that have species capable of switching from autotrophic to heterotrophic modes, when photosynthesis is inhibited (e.g., after pesticide exposure), can continue to grow, even in the presence of high pesticide pollution. How diatoms respond to toxic stress, and the degree to which they respond, also depends on cell and community health, on ecological interactions with other organisms, and on general environmental conditions. The general structural parameters of diatom communities (biomass, global cell density) are less sensitive to pesticide effects than are the specific structural parameters of the unicellular organisms themselves (cell density by species, species composition). For benthic species, biofilm development and grazing on this matrix as a source of food for invertebrates and fishes may also modify the response of diatom communities. Environmental parameters (light exposure, nutrient concentrations, and hydraulic conditions) affect, and often interfere with, the response of diatoms to pesticides. Therefore, the complexity of aquatic ecosystems and the complexity of pesticide to easily detect the effects of such pollutants on diatoms. Clearly more research will be required to address this problem.

Field transfer of periphytic diatom communities to assess short-term structural effects of metals (Cd, Zn) in rivers.

Short-term effects of metal pollution were assessed by transferring periphytic diatom communities developed on artificial substrates from a reference site to a site polluted by heavy metals (around 15 microg Cd L(-1) and 800 microg Zn L(-1)), located in neighbouring streams. Metal sensitivity of mature assemblages (aged 1 month) was evaluated by studying changes in diatom density and taxonomic composition 2 and 4 weeks after the transfer. Resident communities of both sites were simultaneously collected on similar artificial substrates, in order to compare short-term effects of metals within transferred communities with long-term effects observed at the polluted site. Field transfer induced a marked shift of community structure towards that of the resident community of the polluted site: diatom density rapidly decreased after the transfer, from 35,000+/-4000 to 15,000+/-300 cells cm(-2) after 2 weeks, and taxonomic composition changed. Relative abundances of species characteristic of the reference site rapidly decreased, e.g. Nitzschia dissipata and Gomphoneis minuta, whereas species characteristic of the polluted site progressively increased within transferred communities, e.g. Gomphonema parvulum, Pinnularia sp. or Fragilaria crotonensis. The structure of periphytic diatom communities could therefore be an indicator of metal pollution, with marked taxonomic changes being identified within reference assemblages after only 2 weeks under relatively low metal exposure.

Cadmium decontamination and reversal potential of teratological forms of the diatom Planothidium frequentissimum (Bacillariophyceae) after experimental contamination.

While the induction of teratology by cadmium (Cd) on diatoms is already known, reversal kinetics are not well documented. This study aims to understand the viability of diatoms exhibiting teratological frustules and their reproduction capacities within a Cd-impacted population to predict their return to normal diatom forms. We worked on a frequently encountered species in French hydrosystems: Planothidium frequentissimum (Lange-Bertalot) Round & L. Bukhtiyarova. First, a 21-d contamination phase highlighted increasing inductionof different teratological types in response to two levels of Cd contamination: 20 and 100 µg · L(-1) . The deformity counting indicated that Cd firstly generated striae and mixed teratologies, then affected the central area and the valves. Second, a 28-d decontamination phase demonstrated the Cd depuration capacity of Planothidium frequentissimum. Cd half-lives appeared relatively low, ~6 d for the 100 µg · L(-1) condition. Moreover, the decontamination phase showed a decrease in teratology abundances, but a still incomplete recovery after 28 d. Deformations of the striae appeared to be the most sustainable phenotype since they were still significantly higher than in reference cultures at the end of the decontamination phase for both Cd cultures.

KOBAYASIELLA BEBOURENSIS, A NEW FRESHWATER DIATOM SPECIES FROM REUNION ISLAND AND FURTHER OBSERVATIONS ON K. MICROPUNCTATA AND K. JAAGII(1).

The genus Kobayasiella includes species difficult or even impossible to identify in LM. Only EM allows observation of the ultrastructural features helping in diagnostic species circumscription. Recent surveys of the epilithic diatoms in the running waters of Reunion Island led to the discovery of a new species, K. bebourensis. This species differs from all previously described ones by the uniseriate striae composed of areolae occluded by a cribrum. Besides, two other species, the so rarely recorded K. jaagii (F. Meister) Lange-Bert. and K. micropunctata (H. Germ.) Lange-Bert., were collected in two alpine lakes of the French Pyrenees. K. jaagii lacks an umbilicus in the raphe, which is considered one of the most important diagnostic criterions of the genus. However, this species shows a combination of characteristics including it within the genus Kobayasiella. K. micropunctata is a typical representative of the genus Kobayasiella matching all the ultrastructural features of the genus. K. bebourensis and K. micropunctata have a cingulum composed of at least three connecting bands.

Remediation of a watershed contaminated by heavy metals: a 2-year field biomonitoring of periphytic biofilms.

This study focuses on an industrial contamination site subjected to remediation processes since 2007 in the Riou-Mort watershed (southwest France). The purpose was to assess the first impacts of remediation on periphytic biofilms, and was performed during two years of biomonitoring. Periphytic biofilms were collected on glass slides immersed 24 days at different sites along the contamination gradient for 12 colonisation cycles. Metal contaminations (Cd and Zn) were analysed in biofilms and the evolution of diatom communities was assessed, integrating teratology quantifications. Despite remediation work initiated at the industrial site, this study demonstrated the persistence of metal contamination in water, as well as in biofilms. In addition, our data, showed that the remediation process was initially marked by an increase in metal contamination in the river, with increasing diatom community shifts. Metal-contaminated biofilms presented decreasing species diversities and were dominated by metal-resistant species such as Eolimna minima, whom abundances increased in 2010 reaching 57.2±10%. No significant decrease in metal accumulation was observed and total Cd content in biofilms collected downstream the industrial site ranged from 772.7±88 in July 2009 to 636.9±20 µg/gDW in July 2010. Results obtained on artificial substrates were compared with those of natural substrates and showed similar diatom communities and abundances of deformed diatoms but lower diversities. This ensured that glass slide subtrates gave a good representation of periphytic biofilm health. Finally, results were compared to studies performed before the remediation process and this did not reveal a decrease of metal accumulation in biofilms nor shifts in taxonomic composition of the communities, rather the remaining dominance of metal resistant species such as E. minima was confirmed.

Experimental toxicity and bioaccumulation of cadmium in freshwater periphytic diatoms in relation with biofilm maturity.

A study was undertaken to examine cadmium accumulation in freshwater biofilm, its effects on biofilm development and on diatom community structure in laboratory experimental conditions. A suspension of a biofilm originated from the Riou-Mort River (South West France) was inoculated into three experimental units containing clean glass substrates under laboratory conditions. Settling and already developed biofilms were exposed to a Cd concentration of 100 microg L(-1). Metal accumulation (total and intracellular metal content) in biofilms, dry weight and ash-free dry mass, diatom cell density and diatom community composition were analyzed. Both total and intracellular Cd accumulated by the biofilm throughout the experiment increased with duration of metal exposure. Biofilms in the course of maturation were showed higher Cd content and less effective development than settled biofilms. However diatom communities in younger biofilms exposed to Cd increased their tolerance to Cd by a highly significant development of Nitzschia palea. In contrast, Cd exposure had different effect in installed biofilm and taxonomic composition. These results indicate that mature biofilm may limit Cd accumulation into its architecture and protect diatom communities from the effects of metals.

SCANNING ELECTRON MICROSCOPY OBSERVATIONS OF DEFORMITIES IN SMALL PENNATE DIATOMS EXPOSED TO HIGH CADMIUM CONCENTRATIONS(1).

Different types of malformations are likely to affect the morphology of diatoms when exposed to particularly unstable environmental conditions, the most easily identifiable being distortion of the whole frustule. In the present study, we investigated, by means of SEM, valve abnormalities induced by high cadmium contamination (100 µg · L(-1) ) in small pennate diatoms. Changes in the shape of Amphora pediculus (Kütz.) Grunow and anomalous sculpturing of the cell wall of many species, such as Encyonema minutum (Hilse) D. G. Mann, Mayamaea agrestris (Hust.) Lange-Bert., Gomphonema parvulum (Kütz.) Kütz., or Eolimna minima (Grunow) Lange-Bert., were observed, which were not, or almost not, noticeable in the LM. With consideration to current knowledge of diatom morphogenesis, metal uptake by the cell would induce, directly or indirectly, damage to many cytoplasmic components (e.g., microtubules, cytoskeleton, Golgi-derived vesicles) involved in the precisely organized silica deposition. This study confirms that many species, whatever their size, are likely to exhibit morphological abnormalities under cadmium stress, and that this indicator may be valuable for the biomonitoring of metal contamination, even if SEM observations are not necessary for routine studies.

Seasonal effects of cadmium accumulation in periphytic diatom communities of freshwater biofilms.

The relationships between diatom species and cadmium (Cd) accumulated in biofilms of the Riou-Mort River (SW, France) were studied in July 2004 and March 2005. Biofilms were sampled from artificial substrates immersed along a metallic pollution gradient during 20 days. Dynamics of diatom communities and cadmium accumulation were followed by collecting samples after 4, 7, 14 and 20 days of biofilm colonization. Cd accumulation in biofilms during experiment was significantly higher in Cd polluted station (Joanis) than in reference station (Firmi) for both seasons. Periphytic diatom composition varied between sites and seasons. At Firmi station, seasonal dynamics of diatom communities were stable with the dominance of Cyclotella meneghiniana and Melosira varians in July and Surirellabrebissonnii and Navicula gregaria in March. At Joanis station, diatom communities mainly responded to high levels of metal by a high proportion of small, adnate species. Positive correlations between Eolimna minima, Nitzschia palea, Encyonema minutum, Surirella angusta, and Gomphonema parvulum and cadmium accumulation were observed, indicating that these species are tolerant to high levels of cadmium. On the other hand, negative correlations of C. meneghiniana, N. gregaria, Navicula lanceolata, M. varians and Nitzschia dissipata with cadmium qualify them as sensitive diatom species. Periphytic diatom composition through the presence of specific species highlight metal tolerant indicator diatom groups which will be meaningful for biomonitoring pollution in natural aquatic systems.

Cadmium toxicity and bioaccumulation in freshwater biofilms.

A microcosm study was undertaken to examine the effects of dissolved cadmium at various concentrations (0, 10, and 100 microg . L(-1)) on biofilm accumulation and diatom assemblages. A natural biofilm sampled from the Riou-Mort River (Southwest France) was inoculated into three experimental systems, where biofilm settled on glass slides. Samples collected after 1, 2, 4, and 6 weeks of colonization were analyzed for metal accumulation (total metal content and intracellular metal content in the biofilm), biomass (as measured through dry weight and ash-free dry matter), and quantitative as well as qualitative analysis of diatom assemblages. There was a positive correlation between cadmium accumulation and dissolved cadmium concentrations and duration of exposure: a linear relationship was found between concentration factors (CFs) of growing biofilms and time (CFs/day = 0.25 and 0.38 under contaminations of 10 and 100 microgCd . L(-1), respectively). Biofilm settlement, more than photosynthetic activity, was affected by high cadmium concentrations: we observed for all stages of settlement a drastic and significant (p < 0.05) reduction in biofilm biomass and in diatom densities in the highest cadmium contamination, compared to control and low cadmium concentration units.