Microalgal whole-cell biomarkers as sensitive tools for fast toxicity and pollution monitoring of urban wet weather discharges.

Urban wet weather discharge (UWWD) management is an important issue. UWWD often represents a significant source of pollution in all aquatic bodies. The occurrence of this pollution is difficult to predict due to the variability of storm events and the unknown contents of urban watershed leached out by rain. Previous studies have tried to demonstrate the ecotoxic impact of UWWD. However, most of them merely highlight the limitations of classic monospecific bioassays, given the high dilution of micropollutants or the presence of nutrients masking toxic effects. Overcoming this problem is therefore of great interest. In this study, we demonstrated the utility of a battery of biomarkers (e.g. membrane permeability, chlorophyll fluorescence, esterase and alkaline phosphatase activities) on the microalgae Chlorella vulgaris to detect the toxic effects of 7 UWWD samples after short exposures (2 and 24 h). These biomarkers are linked to microalgal life traits. Complementarily, monospecific bioassays were carried on Pseudokirchneriella subcapitata, Chlorella vulgaris, Daphnia magna and Heterocypris incongruens to compare their sensitivity to the UWWD samples. No toxic effect was detected in any of the bioassays. Yet, algal biomarkers indicated a disturbance in microalgae physiology, and particularly a perturbation of chlorophyll fluorescence, which was observed in all of the samples tested. While algal membrane permeability was affected by only one UWWD, these two enzymatic activities were stimulated or inhibited depending on the sample. Finally, this study demonstrates the sensitivity of algal biomarkers and the need to develop new, fruitful approaches to characterizing UWWD toxicity.

Xurography-based microfluidic algal biosensor and dedicated portable measurement station for online monitoring of urban polluted samples.

A critical need exists to develop rapid, in situ, and real-time tools to monitor the impact of pollution discharge toxicity on aquatic ecosystems. The present paper deals with the development of a novel, simple-to-use, low-cost, portable, and user-friendly algal biosensor. In this study, a complete and autonomous portable fluorimeter was developed to assess the A-chlorophyll fluorescence of microalgae, inserted by capillarity into low-cost and disposable xurography-based microfluidic chips. Three microalgae populations were used to develop the biosensor: Chlorella vulgaris, Pseudokirchneriella subcapitata, and Chlamydomonas reinhardtii. Biosensor feasibility and sensitivity parameters, such as algal concentration and light intensity, were optimized beforehand to calibrate the biosensor sensitivity with Diuron, a pesticide known to be very toxic for microalgae. Finally, the biosensor was employed in 10 aqueous urban polluted samples (7 urban wet-weather discharges and 3 wastewater) in order to prove its reliability, reproducibility, and performance in the detection of toxic discharges in the field.

Assessment of long term ecotoxicity of urban stormwaters using a multigenerational bioassay on Ceriodaphnia dubia: A preliminary study.

Standardized ecotoxicity bioassays show some limits to assess properly long-term residual toxicity of complex mixture of pollutants often present at low concentration, such as stormwaters. Among invertebrate organisms used for ecotoxicity testing, the microcrustacean Ceriodaphnia dubia (C. dubia) is considered as one of the most sensitive, especially regarding reproduction impairment as a toxicity endpoint. Consequently, this work explores the interest to perform a multigenerational assay based on the study of the reproduction of C. dubia to assess long-term ecotoxicity of complex mixture, using stormwater samples. With this in mind, a battery of standardized bioassays (Daphnia magna mobility, Pseudokirchneriella subcapitata population growth, Heterocypris incongruens growth and one generation C. dubia reproduction inhibition assays) was performed in parallel to a three generation C. dubia reproduction inhibition assay on 2 stormwater samples. Results highlighted that while all standardized bioassays failed to reveal residual toxicity in the stormwater samples, the C. dubia multigenerational assay exhibited an higher sensitivity than the previous ones. No adverse effect was observed for the first exposed generation, but an increase in mortality and a reproduction disturbance was obtained in the second and third exposed generation depending of the sample. Further experiments are now needed to optimize the exposure protocol of this multigenerational assay.

Identification and assessment of ecotoxicological hazards attributable to pollutants in urban wet weather discharges.

Urbanization has led to considerable pressure on urban/suburban aquatic ecosystems. Urban Wet Weather Discharges (UWWD) during rainfall events are a major source of pollutants leached onto and into urban surfaces and sewers, which in turn affect aquatic ecosystems. We assessed the ecotoxicity of the different compounds identified in UWWD and identified the hazard represented by each of them. To this end, hazard quotient (HQ) values were calculated for each compound detected in UWWD based on their predicted no effect concentration (PNEC) values and their maximum measured effluent concentrations (MECmax) found in the dissolved part of UWWD. For the 207 compounds identified in UWWD, sufficient data existed for 165 of them to calculate their PNEC. The ecotoxicity of these compounds varied greatly. Pesticides represented a high proportion of the wide variety of hazardous compounds whose HQ values were calculated (94 HQ values), and they were among the most hazardous pollutants (HQ > 1000) transported by stormwater. The hazard of combined sewer overflows (CSO) was linked mainly to heavy metals and pharmaceutical compounds. Consequently, the monitoring of these pollutants should be a priority in the future. The hazard level of certain pollutants could have been underestimated due to their adsorption onto particles, leading to their low concentration in the dissolved phase of UWWD. Hence, an in-depth study of these pollutants will be required to clarify their effects on aquatic organisms.

Methods for evaluating the pollution impact of urban wet weather discharges on biocenosis: A review.

Rainwater becomes loaded with a large number of pollutants when in contact with the atmosphere and urban surfaces. These pollutants (such as metals, pesticides, PAHs, PCBs) reduce the quality of water bodies. As it is now acknowledged that physico-chemical analyses alone are insufficient for identifying an ecological impact, these analyses are frequently completed or replaced by impact studies communities living in freshwater ecosystems (requiring biological indices), ecotoxicological studies, etc. Thus, different monitoring strategies have been developed over recent decades aimed at evaluating the impact of the pollution brought by urban wet weather discharges on the biocenosis of receiving aquatic ecosystems. The purpose of this review is to establish a synthetic and critical view of these different methods used, to define their advantages and disadvantages, and to provide recommendations for futures researches. Although studies on aquatic communities are used efficiently, notably on benthic macroinvertebrates, they are difficult to interpret. In addition, despite the fact that certain bioassays lack representativeness, the literature at present appears meagre regarding ecotoxicological studies conducted in situ. However, new tools for studying urban wet weather discharges have emerged, namely biosensors. The advantages of biosensors are that they allow monitoring the impact of discharges in situ and continuously. However, only one study on this subject has been identified so far, making it necessary to perform further research in this direction.

Feasibility of using a translucid inorganic hydrogel to build a biosensor using immobilized algal cells.

Anthropic activities generate contaminants, as pesticides and other pollutants, in the aquatic environment which present a real threat to ecosystems and human health. Thus, monitoring tools become essential for water managers to detect these chemicals before the occurrence of adverse effects. In this aim, algal cell biosensors, based on photosystem II activity measurement, have been designed for several years in previous studies. In this work, we study a new immobilization technique of algal cells in the aim of improving the performance of these biosensors. Immobilization was here achieved by encapsulation in a hybrid alginate/silica translucid hydrogel. The feasibility of this process was here assessed, and the biosensor designed was tested on the detection of chemicals in urban rainwaters.

Tamoxifen ecotoxicity and resulting risks for aquatic ecosystems.

Tamoxifen, a drug used to treat cancer, is regularly found in hydrosystems at concentrations of several hundred ng L(-1). To characterize its ecotoxicity, we implemented a battery of bioassays on organisms belonging to 3 different trophic levels: Pseudokirchneriella subcapitata, Chlorella vulgaris and Chlamydomonas reinhardtii, for primary producers, Daphnia magna (immobilization, grazing and reproduction) for primary consumers, and Danio rerio for secondary consumers (embryotoxicity test). In view of the results obtained and the ecotoxicity values of tamoxifen available in the literature, we established a PNEC (Predictive No Effect Concentration) equal to 81 ng L(-1) for continental water. This PNEC allowed us to calculate Risk Quotients (RQ) for 4 continental hydrosystems in 4 different countries in which measures of tamoxifen had already been performed on surface waters. In two of the situations studied, RQs were higher than 1, reaching a maximum of 2.6. These results show the need to deepen the characterization of ecotoxicological risks linked to the discharge of tamoxifen in surface waters. In addition, we propose applying this approach to other drug residues detected in the environment.

Rhodamine B doped silica encapsulation matrices for the protection of photosynthetic organisms.

An advanced encapsulation matrix that efficiently protects microalgae from harmful UV light without causing toxicity to the entrapped culture is developed based on the electrostatic adsorption of the dye Rhodamine B on silica preformed particles during sol-gel synthesis. The three microalgae (Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii) were previously immobilized in alginate following the Two-step procedure. Once entrapped in the silica gel, Rhodamine B act as an inner cut-off filter, protecting the encapsulated organisms from UV radiation. This matrix allows the sterilization of encapsulation devices without affecting the viability of the entrapped microalgae cells. The condensation of Si(IV) in the presence of silica particles with adsorbed dye generates silica matrices with good mechanical stability. Furthermore; no appreciable differences in microstructure, as assessed by SAXS (Small Angle X-ray Scattering), are caused by the addition of the dye.

Sol-gel silica platforms for microalgae-based optical biosensors.

An advanced hybrid biosensing platform with improved optical quality is developed based on the acidic encapsulation of microalgi in silica matrices synthesized by TAFR (tetraethoxysilane derived alcohol free route). The three microalgi (Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii) were previously immobilized in alginate following the two-step procedure. Tuning the alginate protecting function with the aid of Tris-HCl buffer, the sol-gel synthesis was conducted at pH 4.0 well below the tolerance limit imposed by the encapsulated microalgae. The acidic condensation of Si(IV) generates silica matrices with outstanding optical properties that suit the requirements of biosensors based on optical detection methods.

Co-encapsulation of Daphnia magna and microalgae in silica matrices, a stepping stone toward a portable microcosm.

We report on the first silica encapsulation of a metazoan (Daphnia magna), with a high initial viability (96% of the population remained active 48 h after encapsulation). Moreover, the co-encapsulation of this crustacean and microalgae (Pseudokirchneriella subcapitata) was achieved, creating inside a silica monolith, the smallest microcosm developed to present. This artificial ecosystem in a greatly diminished scale isolated inside a silica nanoporous matrix could have applications in environmental monitoring, allowing ecotoxicity studies to be carried out in portable devices for on-line and in situ pollution level assessment.

Development of a biosensor for environmental monitoring based on microalgae immobilized in silica hydrogels.

A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU) and various concentrations of the common herbicide atrazine. Microalgae are very sensitive to herbicides and detection of fluorescence enhancement with very good efficiency was realized. The best detection limit was 0.1 µM, obtained with the strain C. reinhardtii after 40 minutes of exposure.

Ecotoxicological risk assessment linked to infilling quarries with treated dredged seaport sediments.

The dredged sediments of polluted seaports now raise complex management problems since it is no longer possible to discharge them into the sea. This results in the need to manage them on land, raising other types of technical, economic and environmental problems. Regarding the technical and economic dimensions, traditional waste treatment methods have proved to be poorly adapted, due to very high costs and low absorbable volumes. In this context, filling quarries in coastal areas with treated sediments could represent an interesting alternative for these materials. Nevertheless, for the environmental dimension, it is necessary to demonstrate that this possibility is harmless to inland ecosystems. Consequently, a specific ecotoxicological risk assessment methodology has been formulated and tested on three sediments taken from seaboards of France, in view to providing an operational and usable tool for the prior validation of any operation to fill quarries with treated seaport sediments. This method incorporates the formulation of a global conceptual model of the scenario studied and the definition of protocols for each of its steps: the characterisation of exposures (based on a simulation of sediment deposit), the characterisation of effects (via the study of sediments ecotoxicity), and the final ecotoxicological risk assessment performed as a calculation of a risk quotient. It includes the implementation in parallel of two types of complementary approach: the "substances" approach derived from the European methodology for assessing new substances placed on the market, and the "matrix" approach which is similar to methods developed in France to assess ecological risks in other domains (waste management, polluted site management, …). The application of this dual approach to the three sediments tested led to conclude with reliability that the project to deposit sediments "1" and "2" presented a low risk for the peripheral aquatic ecosystems while sediment "3" presented a high risk.

Unicellular algae used as biosensors for chemical detection in Mediterranean lagoon and coastal waters.

Lagoons and coastal waters are contaminated by a large number of chemicals discharged directly or carried by rivers and runoff water that drain catchment areas in which agricultural activities take place. The inflow of these exogenous compounds constitutes a genuine risk for the health of ecosystems. It is therefore important to detect their presence in the natural environment before they cause irreversible damage. Here we present a study aimed at developing a tool for rapid detection of pesticides and other chemicals in environments liable to be contaminated, in order to propose an early warning system for decision-makers. The study carried out focuses on two herbicides commonly encountered in the environment, i.e. diuron and glyphosate, as well as several of their photodegradation products (DCPU, DCPMU, AMPA). The results presented contribute toward developing a biosensor based on measuring the metabolic activities of immobilized unicellular marine algae. The sensor's operation is based on measuring the esterase localized on the external membrane of the algae cells and chlorophyll fluorescence. The tests carried out show that the signal emitted by the sensor is disturbed by the presence of the two herbicides studied. The system proposed appears useful as a tool for controlling environments requiring monitoring.

Synchronous-scan fluorescence of algal cells for toxicity assessment of heavy metals and herbicides.

Synchronous-scan spectrofluorometry was applied to Chlorella vulgaris cells to assess the toxicity of heavy metals and herbicides in water. Simultaneous scan of both the excitation and emission spectra was done at a constant wavelength difference Deltalambda (20-140 nm) between the emission and excitation wavelengths in the range of 420-700 nm emission, where a peak of fluorescence was observed. Its position depends on Deltalambda. Fluorescence measurements were conducted with algal cells in suspension in water and immobilized in a translucent silica matrix. The influence of toxic chemicals was tested with cadmium as a heavy metal and with atrazine, diuron, DNOC and paraquat as herbicides. The toxic effect of those chemicals mainly results in a quenching of algal cells fluorescence by reducing their photosynthetic activity.

Characterizing the risks to aquatic ecosystems: a tentative approach in the context of freshwater dredged material disposal.

The development of relevant frameworks for assessing ecological risks posed by dredged material management does not only involve an appropriate selection of assessment and measurement endpoints but also requires a sound approach to both risk characterization and the associated uncertainty. A formal methodology addressing both aspects has been developed in France for freshwater sediment deposits in water. Both exposure and effects measurements are 1st transformed into scores or classes. As far as possible, class boundaries are based on existing knowledge or expertise. Benthic organism exposure is based on a ratio of the deposit area to the burrow pit area, whereas pelagic species exposure is based on the ratio of porewater volume to water column volume. The combination of exposure and effect scores yields risk scores, or classes, which are linked to management decisions. Uncertainty is assessed with respect to a set of 4 predetermined criteria for exposure (the strength of association with the assessment endpoint, spatial and temporal representativeness, and the use of standard methods) and 4 criteria for effects (strength of association, the distinction between effect and no effect, sensitivity, and the use of standard methods). This approach was applied to 8 sediments from French canals contaminated to varying degrees.

Assessment of ecotoxicological risks related to depositing dredged materials from canals in northern France on soil.

The implementation of an ecological risk assessment framework is presented for dredged material deposits on soil close to a canal and groundwater, and tested with sediment samples from canals in northern France. This framework includes two steps: a simplified risk assessment based on contaminant concentrations and a detailed risk assessment based on toxicity bioassays and column leaching tests. The tested framework includes three related assumptions: (a) effects on plants (Lolium perenne L.), (b) effects on aquatic organisms (Escherichia coli, Pseudokirchneriella subcapitata, Ceriodaphnia dubia, and Xenopus laevis) and (c) effects on groundwater contamination. Several exposure conditions were tested using standardised bioassays. According to the specific dredged material tested, the three assumptions were more or less discriminatory, soil and groundwater pollution being the most sensitive. Several aspects of the assessment procedure must now be improved, in particular assessment endpoint design for risks to ecosystems (e.g., integration of pollutant bioaccumulation), bioassay protocols and column leaching test design.

A bi-enzymatic whole cell conductometric biosensor for heavy metal ions and pesticides detection in water samples.

A conductometric biosensor using immobilised Chlorella vulgaris microalgae as bioreceptors was used as a bi-enzymatic biosensor. Algae were immobilised inside bovine serum albumin membranes reticulated with glutaraldehyde vapours deposited on interdigitated conductometric electrodes. Local conductivity variations caused by algae alkaline phosphatase and acetylcholinesterase activities could be detected. These two enzymes are known to be inhibited by distinct families of toxic compounds: heavy metals for alkaline phosphatase, carbamates and organophosphorous (OP) pesticides for acetylcholinesterase. The bi-enzymatic biosensors were tested to study the influence of heavy metal ions and pesticides on the corresponding enzyme. It has finally appeared that these biosensors are quite sensitive to Cd2+ and Zn2+ (limits of detection (LOD) = 10 ppb for a 30 min long exposure) while Pb2+ gives no significant inhibition as this ion seems to adsorb on albumin preferably. For pesticides, first experiments showed that paraoxon-methyl inhibits C. vulgaris AChE contrary to parathion-methyl and carbofuran. Biosensors were then exposed to different mixtures (Cd2+/Zn2+, Cd2+/paraoxon-methyl) but no synergetic or antagonist effect could be observed. A good repeatability could be achieve with biosensors since the relative standard deviation did not exceed 8% while response time was 5-7 min. A comparison between inhibition levels obtained with biosensors (after a 30 min long exposure) and bioassays (after a 240 min long exposure) has finally shown a similar LOD for both Cd and Zn (LOD = 10 ppb).

Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae.

A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.

Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides.

An optical biosensor was designed for determination of herbicides as aquatic contaminants. Detection was obtained with immobilised Chlorella vulgaris microalgae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. The algal chlorophyll fluorescence modified by the presence of herbicides was collected at the tip of an optical fibre bundle and sent to a fluorimeter. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimisation. Effects of flow rate, algal density, temperature, and pH on the biosensor response to atrazine were studied. Reversibility and detection limits were determined for DNOC and atrazine, simazine, isoproturon, diuron. Detection of photosystem II (PSII) herbicides was achieved at sub-ppb concentration level.

Optical algal biosensor using alkaline phosphatase for determination of heavy metals.

A biosensor is constructed to detect heavy metals from inhibition of alkaline phosphatase (AP) present on the external membrane of Chlorella vulgaris microalgae. The microalgal cells are immobilized on removable membranes placed in front of the tip of an optical fiber bundle inside a homemade microcell. C. vulgaris was cultivated in the laboratory and its alkaline phosphatase activity is strongly inhibited in the presence of heavy metals. This property has been used for the determination of those toxic compounds.