Benzo(a)pyrene inhibits the role of the bioturbator Tubifex tubifex in river sediment biogeochemistry.

The interactions between invertebrates and micro-organisms living in streambed sediments often play key roles in the regulation of nutrient and organic matter fluxes in aquatic ecosystems. However, benthic sediments also constitute a privileged compartment for the accumulation of persistent organic pollutants such as PAHs or PCBs that may affect the diversity, abundance and activity of benthic organisms. The objective of this study was to quantify the impact of sediment contamination with the PAH benzo(a)pyrene on the interaction between micro-organisms and the tubificid worm, Tubifex tubifex, which has been recognized as a major bioturbator in freshwater sediments. Sedimentary microcosms (slow filtration columns) contaminated or not with benzo(a)pyrene (3 tested concentrations: 0, 1 and 5 mg kg(-1)) at the sediment surface were incubated under laboratory conditions in the presence (100 individuals) or absence of T. tubifex. Although the surface sediment contaminations with 1 mg kg(-1) and 5 mg kg(-1) of benzo(a)pyrene did not affect tubificid worm survival, these contaminations significantly influenced the role played by T. tubifex in biogeochemical processes. Indeed, tubificid worms stimulated aerobic respiration, denitrification, dehydrogenase and hydrolytic activities of micro-organisms in uncontaminated sediments whereas such effects were inhibited in sediments polluted with benzo(a)pyrene. This inhibition was due to contaminant-induced changes in bioturbation (and especially bio-irrigation) activities of worms and their resulting effects on microbial processes. This study reveals the importance of sublethal concentrations of a contaminant on ecological processes in river sediments through affecting bioturbator-microbe interactions. Since they affect microbial processes involved in water purification processes, such impacts of sublethal concentrations of pollutants should be more often considered in ecosystem health assessment.

Use of slow filtration columns to assess oxygen respiration, consumption of dissolved organic carbon, nitrogen transformations, and microbial parameters in hyporheic sediments.

Biogeochemical processes mediated by microorganisms in river sediments (hyporheic sediments) play a key role in river metabolism. Because biogeochemical reactions in the hyporheic zone are often limited to the top few decimetres of sediments below the water-sediment interface, slow filtration columns were used in the present study to quantify biogeochemical processes (uptakes of O2, DOC, and nitrate) and the associated microbial compartment (biomass, respiratory activity, and hydrolytic activity) at a centimetre scale in heterogeneous (gravel and sand) sediments. The results indicated that slow filtration columns recreated properly the aerobic-anaerobic gradient classically observed in the hyporheic zone. O2 and NO3- consumptions (256 +/- 13 microg of O2 per hour and 14.6 +/- 6.1 microg of N-NO3- per hour) measured in columns were in the range of values measured in different river sediments. Slow filtration columns also reproduced the high heterogeneity of the hyporheic zone with the presence of anaerobic pockets in sediments where denitrification and fermentation processes occurred. The respiratory and hydrolytic activities of bacteria were strongly linked with the O2 consumption in the experimental system, highlighting the dominance of aerobic processes in our river sediments. In comparison with these activities, the bacterial biomass (protein content) integrated both aerobic and anaerobic processes and could be used as a global microbial indicator in our system. Finally, slow filtration columns are an appropriate tool to quantify in situ rates of biogeochemical processes and to determine the relationship between the microbial compartment and the physico-chemical environment in coarse river sediments.

Chemical and bacterial changes during laboratory conditioning of formulated and natural sediments.

Within the framework of toxicity testing using formulated sediment, a conditioning treatment prior to toxic contamination has been examined. This preliminary step enables the bacterial colonisation of the sediment, the initiation of organic matter degradation, and the establishment of stable biological and physico-chemical conditions. The treatment involved in keeping the formulated sediment under water in conditions similar to that chosen for toxicity tests. The behaviour of a formulated sediment was compared with a natural sediment. The monitoring of physico-chemical and biological parameters of sediment and water column was carried out over a 30-day incubation in two laboratories. The parameters of pH and redox, dissolved organic carbon (DOC), NH4 and NO2, total organic carbon (TOC) were measured. The bacterial community was characterised by the determination of bacterial density, in total bacteria number or colony forming units (CFU), several exoenzymatic activities (P-glucosidase, xylosidase, leucine-amino-peptidase phosphatase and sulfatase activities), and three gas productions (CO2, N2O and CH4). The same experiment was carried out with a natural sediment. A 10- to 15-day conditioning allowed a physico-chemical stabilisation and corresponded to kinetic changes in hydrolysis activities. As compared to data of the natural sediment, the biological activity of the formulated sediment showed a different dynamic with lower activity levels. For both sediments, an important decrease of activities levels was observed after 15 days because of a substrate limitation. The work showed that a preliminary conditioning treatment of a formulated sediment provides the stabilisation of parameters that can affect toxicant bioavailability. Additional research is needed to determine the real influence of conditioning on the bioavailability of contaminants. The possible advisability of organic matter input, to maintain the sediment bacterial activity, has to be studied.

Interactions between a polycyclic aromatic hydrocarbon mixture and the microbial communities in a natural freshwater sediment.

The toxicity of a polycyclic aromatic hydrocarbon (PAH) mixture was assessed on the indigenous microbial communities of a natural freshwater sediment. The fate and effects of the PAH mixture (phenanthrene, fluoranthene and benzo(k)fluoranthene) were studied over 28 days. Bacterial communities were described by bacterial counts (total bacteria and viable bacteria), and by some hydrolytic enzyme activities (beta-glucosidase and leucine-aminopeptidase), PAH concentrations were measured in the overlying waters and in the sediments. No effect of PAH was detected at 30 mg/kg for all bacterial parameters. At 300 mg/kg, the quantity of total bacteria and the proportion of viable bacteria markedly decreased, compared to the control (0 mg PAH/kg). At 300 mg/kg, an increase of the beta-glucosidase activity and a decrease of the leucine-aminopeptidase activity were observed. For all treatments, the benzo(k)fluoranthene concentration in the sediment was stable over 28 days whereas, in the same time, only 3-6% of the initial concentrations of phenanthrene and fluoranthene remained. This study shows that (1) PAH induce perturbations of sediment microbial communities in terms of density and metabolism (but not always as an inhibition), (2) indigenous bacteria of sediments might be used for toxicity assessment of specific organic pollutants, (3) native microorganisms of sediment seem to have a high capacity for PAH degradation, depending on the physico-chemical properties and the bioavailability of the substance encountered.

Pyrene degradation by two fungi in a freshwater sediment and evaluation of fungal biomass by ergosterol content.

Mucor racemosus var. sphaerosporus and Phialophora alba were investigated for their abilities to degrade pyrene in a freshwater sediment, with or without glucose supply as nutrient or carbon source, during 90 days. The ergosterol contents in sediment were quantified to estimate fungal biomass and to assess the correlation between fungal activity and biodegradation of pyrene. Results showed that, in an heterogeneous environment, these fungi presented different abilities to degrade pyrene. P. alba increased the degree of pyrene degradation by 9%, compared to the native micro-organisms, but a supply of glucose acted as an inhibitor to pyrene disappearance. M. racemosus var. sphaerosporus was not efficient at sediment bioremediation (with or without glucose added), because it reduced the rate of pyrene degradation by the native microflora. In any case, there was no increase of ergosterol in boxes during bioremediation experiments. In our experimental conditions, ergosterol content could not be correlated to pyrene degradation.

Liquid chromatography study of pyrene degradation by two micromycetes in a freshwater sediment.

Pyrene biodegradation in a freshwater sediment without fungi supply, or inoculated with two sediment micromycetes, Mucor racemosus var. sphaerosporus and Phialophora alba was studied after 0, 5, 13, 28, 60 and 90 days. The influence of glucose addition was estimated, and a liquid chromatographic method for simultaneous quantitative determination of residual anthracene, fluoranthene and pyrene in the sediment was developed. Samples with PAHs were extracted in Soxhlet with ethyl acetate, and LC analysis was performed on a 5 microm Supelcosil column (150 x 4.6 mm I.D.) with gradient elution (2 ml min(-1)) of acetonitrile-water and UV detection at 254 nm. Recoveries of anthracene, fluoranthene and pyrene were 90.3%+/-1.1%, 93.2%+/-0.9% and 90.42%+/-1.9%, respectively, without interference. The native sediment microorganisms (with or without glucose added) have shown 35% pyrene degradation and sediment with glucose inoculated by the strains revealed 40%.

Assessment of Three Methods for Detection and Quantification of Nitrite-Oxidizing Bacteria and Nitrobacter in Freshwater Sediments (MPN-PCR, MPN-Griess, Immunofluorescence).

> Abstract Nitrification in freshwater, a key process in the nitrogen cycle, is now well known to take place predominantly on suspended particles and in sediment. Nitrobacter is the most commonly isolated nitrite oxidizing bacteria from water environments. Three methods for counting nitrite oxidizing communities (especially Nitrobacter) in sediment were investigated: MPN-Griess, fluorescent antibodies (immunofluorescence), and a more recent molecular method coupling specific DNA amplification by PCR and statistical MPN quantification. After preliminary adjustments of the MPN-PCR technique, the detection level and the yield of each method were determined by inoculating a sediment with a pure Nitrobacter culture. The best recovery yield was obtained with the immunofluorescence technique (21.3%) and the lowest detection level was reached with the MPN-Griess method (10(3) Nitrobacter/g dry weight sediment). The MPN-PCR method resulted in the lowest recovery yields and needs further adaptation to become a reliable and precise tool for investigations of nitrifying bacteria in sediment.

Microbial methods for assessing contaminant effects in sediments.

Contaminated sediments influence drastically the long-term toxicological and ecological properties of aquatic ecosystems. During the past three decades, scientific knowledge about sediment-water exchange processes and the deposition and distribution of pollutants in water and sediment phases has been supplemented by extensive research on the effects of sediment-associated pollutants on aquatic organisms. Basic research in microbiology, ecology, and toxicology has uncovered the crucial role of sediment microorganisms for the biodegradation of organic matter and for the cycling of nutrients, as well as the susceptibility of these processes to toxic pollution events. Microorganisms have been extensively applied in aquatic toxicology, and various microbial toxicity tests are today available that successfully couple microbial toxicity endpoints to the specificity of the sediment matrix. Sediment-associated toxicants can be brought in contact with test bacteria using sediment pore waters, elutriates, extracts, or whole-sediment material. Toxicity indication principles for microorganisms are versatile and comprise growth and biomass determinations, respiration or oxygen uptake, bacterial luminescence, the activity of a variety of enzymes, and a compendium of genotoxicity assays. The border between toxicological and ecological contaminant effect evaluations in sediments is flexible, and long-term ecological predictions should also include an assessment of pollutant degradation capacities and of key reactions in element cycling. Evaluating microbial community structure and function in environmental systems makes use of modern molecular techniques and bioindicators that could trigger a new quality in the assessment of contaminated sediments in terms of indication of subtoxic effects and early-warning requirements.

Impact of wastewater treatment plant discharge on enzyme activity in freshwater sediments.

The biological self-purification processes are a central point for the ecological states of rivers. The degradation efficiency of pollutants is mainly due to the microflora and can be detected with enzyme-activity tests. Extracellular-enzyme activity of freshwater sediments was measured, in a microcosm, versus different pollution levels caused by organic wastewater treatment plant (WTP) discharges. Biochemical evaluation of Vmax values for each exoenzyme appears to be a function of the organic matter content of the WTP effluent (Fig. 2). Glucosidase and peptidase reveal a significant negative correlation (respectively, r = 0.99, with P < 0.02; and r = 0.853, with P < 0.08) of Vmax versus DOC concentration (i.e., an inhibition effect). The same relationships were observed with K(m) values, beta-Glucosidase and aminopeptidase activity are well described by the Michaëlis-Menten equation, but linearization with the Lineweaver-Burk equation does not fit with a simple type of inhibition. Two sediments (sand and silt) have been tested, and the differences in the exoenzyme activity of the two sediments after WTP effluent input could be explained by their physicochemical differences. The effects of WTP effluents on a freshwater sediment indicate that, in the current experiments, the microbial potential exoenzyme activity does not increase: this finding implies that, in rivers, the global hydrolytic potential could remain steady down-stream of a discharge point.