Volume 6, numéro 3, 1993

FR :

Les activités exoenzymatiques participent de façon importante à la transformation des composés organiques dans les milieux aquatiques, en particulier par hydrolyse de composés à haut poids moléculaire qui sont réduits en monomères ou en petits oligomères assimilables par les bactéries. Cette étape est un processus clé dans le fonctionnement de la boucle microbienne. De nature très diverse, les exoenzymes ont une localisation variable selon le Gram de la bactérie ; leur régulation se situe soit au niveau de leur synthèse, soit au niveau de l'expression de leur activité. Bien que certaines exoenzymes soient constitutives, l'inductibilité semble être le mode de fonctionnement le plus fréquent. L'intégration de ce processus dans des études globales sur le potentiel hydrolytique de milieux aquatiques nécessite quelques précautions dans la réalisation expérimentale et dans l'interprétation des données : variabilité intrinsèque différente selon l'exoenzyme considérée (de 7 % pour la glucosidase à 16 % pour la phosphatase), variabilité géographique importante (pouvant atteindre +/- 50 % à un mètre de distance) dans les milieux sédimentaires, ce qui nécessite la connaissance topographique du milieu étudié et la définition d'une stratégie d'échantillonnage préalable. Les méthodologies de prélèvements, en particulier en sédiments ou en substratums grossiers, présentent une certaine complexité de mise en oeuvre que n'ont pas, au moins à faible échelle, les prélèvements en colonne d'eau (hétérogénéité du milieu à l'échelle centi- voire millimétrique, densité de population très variable, diffusion de substrat nutritifs,...). Une standardisation du protocole expérimental est proposé.

Dans l'optique d'études sur les capacités d'assimilation et de biodégradation de matière organique des systèmes aquatiques, les données d'activités exoenzymatiques nécessitent d'être couplées à d'autres mesures biologiques ou biochimiques : biomasse bactérienne et/ou phytoplanctonique (en particulier pour la phosphatase), analyse fine de la matière organique assimilable (par exemple, par classes : lipides, glucides, protides, dans leurs fractions dissoutes et particulaires).

EN :

Exoenzymes play an important role in the transformation of organic compounds in aquatic environments : these biomolecules convert high molecular weight compounds by hydrolysis into monomeric or oligomeric compounds that are then assimilable by bacteria. This step is a key process in the microbial food web and microorganisms that produce exoenzymes are probably good competitors in aquatic environments.

The different exoenzymes are located in different places on the cell membrane with respect to the bacterial cell wall type : exoenzymes of the gram negative bacteria are rather located on the outside of the cytoplasmic membrane or in the periplasm. Their regulation can be either at the level of exoenzyme synthesis or at the level of enzymatic expression. Their activity is generally described with a Michaelis-Menten equation. Most exoenzymes are inducible (phosphatase), but some are constitutive (aminopeptidase).

The use of exoenzyme methods in the aquatic environment needs some care, both during application and interpretation. The intrinsic variability of enzyme activity differs with the type of exoenzyme : the more the activity is inducible, the higher is its variability : e. g. phosphatase (inducible : 16 %) in comparison with glucosidase (constitutive : 7 %). Experimental enzymatic substrates are in tact « model » molecules that are supposed to have the same behaviour as « natural » substrates : this is not always true.

Spatial variability is also important in sedimentary environments : significant variations in activity exist, especially with depth and stratification of sediment, at a scale of 1 - 10 cm (± 50 %). A topographie knowledge of the studied environment and the definition of a suitable sampling strategy are thus very important. Sampling methodotogies in sediment, silt or coarse substrates are more difficult to set up than are those for the water column (heterogenous environment at the centimetre scale, variation of microbial population density, nutrient diffusion, gradients). The sampling of sediments with a corer, and the subsequent fractionation of the sediment into different layers for incubation in the laboratory, may modify the physicochemistry of the sediment and could influence bacterial activity (experimental artefact). A technique has been developed for incubation inside the corer in order to minimize perturbations but this technique is limited by the sediment granulometry. Conversely, homogenization of sediments allows a better standardization of experiments and yields more reproducible results.

For studies on the assimilation capacity of aquatic ecosystems and biodegradation of organic matter, exoenzymatic activities data need to be associated with other biological or biochemical parameters : bacterial and/or phytoplanctonic biomass; precise analysis of assimilable organic matter in the particulate or dissolved phases; physicochemical data... Data concerning the modelling of exoenzyme activities in relation to parameters such as temperature or oxygen level are lacking. Integration of these data will afford a better overall understanding of the role of exoenzymes in the metabolism of aquatic environments, and will help establish the limits of the validity of this technique for global studies of the assimilation capacities and organic matter biodegradation in aquatic ecosystems.

FR :

La technique de comptage par microscopie en épitluorescence est la méthode la plus performante pour dénombrer la totalité des bactéries présentes dans les milieux aquatiques. Cependant cette technique est longue, fastidieuse et subjective. Afin d'automatiser et de rendre objectif le dénombrement, le microscope à épifluorescence est couplé à un analyseur d'images. Si les systèmes d'analyse d'images sont utilisés pour les mesures de taille des bactéries aquatiques, très peu d'études font état de comparaison entre les dénombrements par analyse d'image et ceux réalisés de façon traditionnelle. Cet article présente les résultats des dénombrements de souches bactériennes de référence et de bactéries des milieux aquatiques, par la technique de microscopie en épifluorescence des cellules bactériennes marquées au DAPI, réalisés simultanément par observation microscopique visuelle (visuel) et par analyse d'images automatisée (automatique).

Le système d'analyse d'images est composé d'une caméra vidéo (Lhesa LH40036) de sensibilité de 510^-4 lux, d'une carte de numérisation (512 x 512 pixels, 8 bits, cyclope v 2.32, Digital vision) d'un micro-ordinateur 80-386 et d'un logiciel de dénombrement (Mudicam®. EAU). Le système est couplé à un microscope en épilluorescence Olympus BH2.Les dénombrements ont été réalisés d'une part sur des suspensions de souches bactériennes de référence (n = 30) à différents états physiologiques et sur des échantillons d'eaux (n = 50) d'origines diverses (fleuve, eaux saumâtre, marine et résiduaire). La comparaison des deux méthodes est réalisée par un modèle de régression linéaire et une analyse de variance. Les tests statistiques associés permettent de conclure à une bonne concordance entre les deux méthodes. A partir de l'ensemble des dénombrements réalisés, 18 d'entre eux pris au hasard ont été dénombrés de façon manuelle par deux opérateurs et par le système d'analyse d'image. Il apparaît que les différences de comptage les plus élevées correspondent aux dénombrements effectués par chacun des deux opérateurs. Ceci met en évidence que non seulement le système d'analyse d'image permet une quantification rapide des abondances bactériennes, mais en outre il supprime la subjectivité de l'opérateur tout en réalisant des dénombrements aussi précis.

EN :

Direct counting by epifluorescence microscopy is the best method available to determine total counts of aquatic bacteria. However, microscopic observation is tedious and time-consuming. A more rapid and certainly less subjective way of counting bacteria is to combine epifluorescence microscopy with an image analysis system. Surprisingly, although image analysis is now a relatively common method to measure the size of aquatic bacteria, very few studies have been devoted to the validation of total counts by image-analysis systems. In this paper, we present data on simultaneous determination of total counts of 4'6-diamidino-2-phenylindole (DAPI) stained bacteria by visual means and by image-analysed (Mudicam® system) epifluorescence microscopy methods.

The Olympus microscope BH2 is equipped for epifluorescence with a 100 W Hg lamp and a 100x oil immersion objective (Apo UVFL 160/1.3). The image analysis system consists et a high performance (5 x 10^-4 lux) video camera (Lhesa LH40036) and an image processor which digitalizes the video image in a grey scale extending from 0 (black) to 255 (white) into a binary image with 512 x 512 pixels (8 bit, cyclope v 2.32, Digital Vision), and image analysis software (MUDICAM®. EAU). The samples were stained with DAPI (final concentration 2.5 µg/ml) and filtered through polycarbonate inters (0.22µm, Nuclepore Corporation). The surface area of the video image is 76 x 111 µm².

The analysed samples come from culture collections of different bacterial strains (n = 30) submitted to different conditions and incubation times to obtain various physiological states (Table 1). The nature water samples were collected from several aquatic ecosystems : Rhône river, Mediterranean sea, Thau lagoon and Montpellier sewage waters (n = 50). The bacterial abundances ranged from 105 to 108 cells/ml and the size range of the cells varied from 0.63 to 17 µm². Comparisons between the image analysis and visual counts were made on the basic of thirty fields per filter. The image analysis counts are based on a two step procedure. The video image of each microscopie field is first numerised and stored on a hard disk (153 Mo). When all the fields have been stored, the digitized images are submitted to an automatic thresholding which allows background substraction. Automatic counting of bacterial cells is then performed on the basis of object specifications defined by the operator. These specifications concern the minima and maxima values of the area (expressed in pixel numbers) and the fluorescence (expressed in gray levels) of the objects. The MUDICAM®EAU software also provides the mean number of cells per millilitre and the associated variance.

Average concentrations and confidence limits are shown in Table 2 for bacterial collection strain cultures and in Table 3 for water samples. When we compared visual and image analysis counts by- linear regression, the ability of the image analysis system to enumerate bacterial cells was clearly demonstrated. With bacterial culture (Fig. 2) and with water samples (Fig. 3), the coefficients of correlation were respectively r = 0.997 and r = 0.996 (p = 0.0001). The slopes of the models are not significantly different from unity and the Y-intercepts are not different from zero. Moreover we have compared the total visual counts of two experimenters and the image-analysed counts on eighteen random samples (Table 4). The variance analysis shows that there is no difference between the three methods, with mean value of 6.09, 6.08 and 6.11 for the image-analysed method, experimenter n° 1 and experimenter n° 2, respectively. While non significant, the greatest difference in counts was obtained between the two experimenters.

If may be concluded that the image analyser tested for total counts by epifluorescence microscopy is a precise and rapid procedure for the determination of total bacterial counts. This method may be standardized and its automation allows the analysis of many samples, an important advantage in ecological studies. Storage of the samples also allows one to treat a posteriori some complementary aspects of the total count, such as the double staining of bacteria. The image analyser tested is appropriate for bacterial ecology studies which require epifluorescence microscopy.

FR :

Cet article est une revue des essais in vitro et in vivo utilisés pour évaluer le caractère génotoxique des micropolluants des milieux environnementaux relatifs aux eaux continentales et marines, rejets liquides d'origine domestique, industrielle ou agricole, sédiments de rivières et boues de stations de traitement d'épuration.

Les essais in vitro réalisés sur cellules eucaryotes ou procaryotes sont fondés sur la détection des mutations géniques et chromosomiques, ou la mesure des adduits à l'ADN. Ils constituent des systèmes d'épreuve miniaturisés qui requièrent des volumes d'échantillons faibles; ils se prêtent ainsi au dépistage à grande échelle de la génotoxicité et à l'étude des concentrats et des extraits préparés à partir des milieux contaminés. Ils sont cependant moins bien adaptés à la prédiction de l'impact des micropolluants sur l'environnement.

La recherche de conditions d'essai plus proches de la réalité environnementale a conduit au développement des essais in vivo réalisés sur organismes supérieurs, mollusques, poissons ou amphibiens, qui évaluent un potentiel génotoxique à partir d'études cytogénétiques ou d'études du caryotype des organismes exposés.

Les critères de génotoxicité étudiés in vitro peuvent être utilisés dans le cadre d'études écoépidémiologiques, sur le terrain, afin d'évaluer l'impact réel des micropolluants présents dans les milieux environnementaux sujets à des contaminations d'origines diverses.

EN :

This review deals with in vitro and in vivo genotoxicity bioassays carried out to evaluate the genotoxic potential of polluted environmental samples : continental and marine waters, domestic and industrial wastewaters, aquatic sediments and sludges of urban or industrial wastewater treatment plants.

The end-points of the in vitro and in vivo assays are : genetic alterations, i.e. reverse and forward mutations, DNA adducts or chramosomic damages, i.e. chromosomic aberrations (AC), micronuclei (MN) and sister chromatid exchanges (SCE).

The in vitro assays generally detect adverse effects on DNA only alter concentration or extraction of micropollutants. They constitute miniaturized tools, rapid and easy to use, thus well-suited for large screening studies. In vitro genotoxicity bioassays requiring only small volumes of samples are therefore systems of choice for testing concentrates or extracts from environmental contaminated samples. Among the in vitro assays reviewed, the Salmonella typhimurium gene mutation test is the most often used to assess the genotoxic potential of contaminated samples. However, genotoxicity tests performed on eukaryotic cell cultures are more relevant than those using bacteria for evaluating environmental pollution. The use of fish cell fines appears superior to the use of mammalian cells for assessing an aquatic impact.

In vitro bioassays, whether performed on prokaryotic or eukaryotic cells, are limited for predicting the possible impact of genotoxic pollutants on the environment. It is clear that it is difficult to extrapolate in vitro bioassay results to higher organisms in which the response obtained integrates effects of complex metabolizing systems, hormonal regulation and immunological defenses.

Therefore, genotoxicity studies performed with aquatic organisms such as molluscs (Mytilus sp.), fish (Umbra pygmaea, Notobranchius rachowi) or amphibians (Pleurodeles waltl) appear more representative of environmental conditions. The genotoxicity end-points of in vivo assays are mainly cytogenetic damage such as the SCE, AC or MN but also take into account DNA adducts. Direct testing of environmental samples without preconcentration is possible with in vivo assays. This means that factors such as bioavailability and metabolism will be integrated direcrly in the response of these assays. Hence, these in vivo assays are more sensitive titan in vitro genotoxicity tests. However, in vivo tests require important volumes of sample and it will be difficult or almost impossible to apply them for testing concentrates or sample extracts, generally only available in small quantities. An interesting area of application of in vivo assays is field studies and ecoepidemioiogy. In this respect, they would constitue an a posteriori control system of pollution effects, assuming that suitable control areas are available to eliminate the influence of confounding factors.

As a general conclusion, if is important to emphasize the interest of using both in vitro and in vivo bioassays for evaluating the genotoxicity of contaminated environmental samples. This rationale is based on the fact that in vitro bioassays are well adapted for genotoxicity screening or concentrates and extracts testing, white in vivo tests are interesting because of their better representativity in terms of environmental conditions of exposure to pollutants.

FR :

Le recensement de la charge polluante rejetée dans la rivière Vienne (France) par les usines et les stations d'épuration de Limoges à Confolens a été effectué. Des campagnes de prélèvement et d'observations visuelles ont permis de localiser les lieux d'apparition de mousses en aval d'usines de fabrication de pâte à papier et de cartons. L'étude du pouvoir moussant des mélanges des deux principaux rejets polluants (papeterie et cartonnerie) a permis de mettre en évidence des phénomènes de synergie entre certains mélanges se traduisant à la fois par une augmentation du pouvoir moussant et de la stabilité de la mousse dans le temps. L'étude par « HPLC » montre l'apparition de pics supplémentaires confirmant l'interaction entre les constituants des rejets; le principal effluent a pu être suivi à l'aide de ses caractéristiques chimiques dans la rivière et dans les mousses jusqu'à Confolens.

EN :

The study reported here considers of the formation and stability of foam on the Vienne river. Foaming is frequently encountered in relation to the discharge of industrial effluents, especially from the paper industry (CRAIG and al., 1990). Earlier papers have investigated the consequences of such discharges (NEILSON and al., 1990; KALLQVIST and al., 1989; SRIVASTAVA and al. 1988).

The extent of foam formation is determined by a number of factors, including effluent composition, turbulence of the stream, etc. Foams stability requires the presence of long chain fatty acids, amine acids, tannins etc. Many industries discharge their effluents into the Vienne river (paper and cardboard industries, leather dressing plants and tanneries).

An inventory of the main urban and industrial discharges has been established (Map 1). The effluents from the pulp, paper and cardboard industries provide the main pollution foad in terms of volume, COD, suspended solids (SS) and anionic surfactants.

A visual survey allowed us to locus our investigations on the places where persistant foams appear, especially downstream of Saillat below the discharges from Aussedat Rey and SGPL (Picture 1), and below small waterfalls (Pont de Pilas, Chabanais, Ansac...). At Confolens, the foams are most stable and form stable drifting foam residues.

Synergistic foaming effects have been reported due to the combination of polyamides and tannins (BIKERMAN, 1953). We have chosen to analyze the main effluents (Table 1) and their mixtures in relation to foaming (foaming capacity, foaming stability and surfactant analysis). The method used for foaming capacity determination was based on the hand shaking of 250 ml bottles. The stability of the foam was defined as the time for which the height of foam persists. Anionic surfactants were present at significant concentrations, varying from 1 mg/l (as sodium dodecyl sulphate) in the Aussedat Rey effluent to 4 mg/l in the SGPL effluent and 7 mg/l in the St Junien wastewater treatment plant effluent. The maximum foaming capacity was obtained for a 70/30 Aussedat Rey/SGPL effluent mixture (Fig. 1). The foaming capacity persists river time, remaining practically unchanged for three days. After 6 days, the maximum foaming capacity appears to be reduced. Foam stability is also maximum for the same 70/30 mixture (Fig. 2). After 6 days, the 50/50 and 70/30 mixtures can still produce 3 cm of foam that persist for 2 hours (Fig. 3).

For HPLC analysis (20 µl samples), the effluents from AR and the effluents from SGPL (or the mixture of the two) were diluted in 10 times their volume of distilled water prior to analysis. Concerning the mixture 95 % AR - 5 % SGPL (95/5) the peak that characterizes the SGPL effluent starts appearing and growing at 6.76 min. With the proportions : 90/10 and 70/30, its retention time respectively diminishes from 6.34 min. to 5.58 min. Moreover an extra peak appears with the 70/30 mixture at 5.02 min. This extra peak is at its highest at 4.96 min. for a 50/50 mixture. At the same time the initial AR peak is decreasing. It is thus confirmed that one or more constituent: are formed on mixing the two effluents, as indicated by the synergistic effect described earlier for the foam capacity and stability analysis.

Anionic surfactants were analyzed in the Vienne river (Fig. 5). Their concentration dramatically increase at point (4) (Pont de Pilas), just below the discharges from AR and SGPL. When the river flow increases, dilution masks the phenomenon. A drastic decrease in pollution appears in August when the industrial activity is reduced because of holidays (Fig. 7).

The HPLC Vienne river analysis (Fig. 5) shows an important peak of pollution at point (4) (Pont de Pilas) characteristic of the AR effluent. At Chabanais point (5), the ARISGPL ratio is 95/5 and the peak of SGPL appears, perhaps, et 6.12 min (in the diluted effluents in the same ratio 95/5, it appears at 6.34 min). At Confolens (10), the intensity has diminished (after two days) and the Confolens foams are the same as those produced by a river sample without concentrated effect. No appreciable degradation has occurred, since the height of the peaks point 10 is similar as those of the chromatogram of point (5) in accord with the literature (LESZKIEWICZ and KINNER, 1988 ; COTE and OTIS, 1989).

FR :

Élasticité des consommations d'eau vis-à-vis du prix.

Cet article analyse la façon dont les consommations des usagers domestiques des services d'eau sont influencées par le prix du service.

Les calculs d'élasticité s'appuient sur des données de prix du service et de consommation domestique par habitant issues d'enquêtes qui ont été menées entre 1975 et 1990 auprès d'un grand nombre de services d'eau français (entre 500 et 1 362 services desservant de 8 à 17 millions d'usagers).

Les calculs d'élasticité vis-à-vis du prix sont menés suivant les deux méthodes classiquement utilisées : par observation des évolutions des consommations domestiques qui font suite à une forte augmentation du prix du service et par observation des écarts de consommation qui existent à un moment donné entre des services dont les usagers sont soumis à des prix différents. Plusieurs calculs sont ainsi menés suivant des méthodes différentes et portant sur des jeux de données différents.

La taille importante des échantillons permet de mettre en évidence la relation entre consommation et prix sans avoir à connaître les éléments autres que le prix qui influencent les consommations et dont l'évaluation est toujours difficile et critiquable.

Les résultats obtenus sont remarquablement convergents et montrent que les usagers domestiques réalisent un ajustement faible mais réel au prix du service. Ils indiquent également que la diminution des consommations constatée suite à une augmentation du prix du service peut s'étaler sur plusieurs années et qu'elle a un effet durable.

EN :

After a three-year period of drought in France, many debates have been held on water supplies. The price of public water utilities is considered as means to improve solutions of quantitative and even qualitative problems of water supplies. Surprisingly, only one study has been published in France on the real influence of public water utility price on domestic water consumption.

Different evaluations of domestic water consumption price elasticity are presented. They use the (price, consumption) data from a national panel of 500 public water utilities for the years 1975, 1980, 1985 and 1990, and front an even larger sample of 1362 public water utilities for the years 1975 and 1980.

Domestic water consumption for each utility is computed as the volume of water sold to domestic users (industrial and agriculture consumption are ignored) divided by the number of permanent equivalent inhabitants. The number of permanent equivalent inhabitants is the total of bath permanent inhabitants and seasonal inhabitants weighted with the annual duration of their stay.

The measure of the price for the public water utilities used is the price that a domestic user pays for an annual consumption of 100 cubic meters, free of taxes. This price does net include the price paid for public sewerage.

The evaluations of elasticity are carried out in two different ways : time series and cross-sectional. In the time series approach, the evolution of the domestic water consumption for the utilities which have raised their price sharply between one year of observation and the next is compared with that of utilities whose price has remained constant (in constant value) during the same period. For each of the three periods considered (1975-1980, 1980-1985 and 1985-1990), the evolution of the consumption for the utilities that have raised their price appears to be lower. Values of short-term elasticity between - 0,11 and - 0,17 can be derived from this statement. The evolution up to 1990 of the consumption of the utilities that had raised their price between 1975 and 1980 shows that the total effect on consumption of a price increase can take 5 to 10 years to be completed. A long-term price elasticity of -0,33 can then be calculated. The fact that the price for public sewerage is not taken into account is assumed to be a source of small under-estimation for the results of elasticity that are found.

In the cross-sectional approach, thanks to the important quantity of data (price, consumption) that are available for 1975 and 1980 (1336 data points for each year), it is possible to show a relation between domestic water consumption and price for different utilities for bath 1975 and 1980 without any additional information factors other than price that can influence water consumption. The method used consists of dividing the 1336 utilities into 8 groups of services whose prices are similar. For each group, the average price and average consumption is computed. The important number of utilities and users that each group represents minimizes the influence of factors other than price that can influence domestic water consumption. The influence of the price paid for public sewerage is globally taken into account for each group of services through the proportion of users who benefit from public sewerage. Public sewerage price is considered to represent 45 % of the group average price for water utilities. The values of elasticity that can be deduced, which are between short-and long-term elasticity, vary from - 0,23 to - 0,29.

The hypothesis of rational consumer behaviour implies that consumers should react to the marginal price at their own level of consumption, rather than to the average price. However in France, marginal price and average price for one utility are very close, due to the tariff structure and, in tact, consumers probably react as much to average price as to marginal price.

Taxes that can overcharge consumers bill were unknown - it was impossible to take them into account - but they are judged to be low enough not to change the values of elasticity that were found.

The different methods for the calculation of price elasticity, which are presented here for several series of data, give results which are close to each other. Comparison with results from other studios that are available, especially those from the United States, is difficult because the dispersion of the values found is large. Nevertheless our results can be judged rather close to most of the other comparable results found abroad.