L. Mathier and A. G. Roy
Nous présentons ici une méthode simple qui permet de mesurer simultanément l'eau et les sédiments en transit à la surface du sol en milieu agricole. Des hydrogrammes de ruissellement superficiel sont comparés pour évaluer la variabilité saisonnière et spatiale du ruissellement et de l'érosion lors de trois précipitations naturelles (1,16, 1,76, 1,78 mm h-1). Les mesures ponctuelles sur le terrain sont utilisées pour caler une équation de transport des sédiments compatible avec les modèles hydrologiques distribués. Le débit solide (Qs) est exprimé par:
Qs = k Qm lp
où Q est le débit liquide et l l'intensité des précipitations. Les hydrogrammes montrent que les débits varient dans l'espace selon la distance entre deux points d'échantillonnage et, dans le temps selon l'état de la surface. Pendant la saison, la concentration moyenne des sédiments dans l'eau et le taux de transport demeurent constants. Le transport par le ruissellement sur la parcelle est faible et semble limité par la capacité du processus à maintenir les sédiments en mouvement. Ce comportement affecte le paramètre m associé au débit liquide dans l'équation de transport, qui est en dessous des valeurs théoriques proposées dans la littérature. Le transport de sédiments est également fonction de l, qui par le biais de l'impact des gouttes de pluie sur la surface détache et facilite le transport des particules.
Data are needed to validate a storm by storm model of sheetwash erosion, to assess spatial and temporal variations of runoff and erosion and to calibrate a sediment transport equation on agricultural fields. This remains a major problem in the development of distributed hydrological models. This paper presents a simple method to measure simultaneously water and sediment discharges on hillslopes. Hillslope hydrographs and sediment transport rates are used to investigate spatial and seasonal variations in runoff and erosion. Measurements are also used to calibrate a sheetwash sediment transport equation compatible with distributed hydrological models. Sediment discharge (Qs) is expressed by :
Qs = k Qm lp
where Q is water discharge, S is hillslope gradient and l rainfall intensity. Parameters k, m, n and p are constants for a given context.
The experimental site is located in the Eastern Townships (Québec, Canada). It is a corn field (1 000 m2) where sheetwash erosion is active. Simultaneous measurements of water and sediment discharges are collected using hydraulically efficient samplers specially designed to minimize direct rainsplash input and to prevent sediment accumulation within the receptacle. Data were collected during three natural rainfalls with low average intensities monitored in June (l -1,76 mm h-1), September (l = 1,78 mm h-1) and October (l = 1,16 mm h-1) 1987. Because rainfall intensity varies within a precipitation, each rainfall event was subdivided into distinct measurement periods of short duration (5 to 60 min) with intensities ranging from 0,12 to 8,9 mm h-1. For each precipitation two samplers were operated simultaneously over the field. One of the sampler occupied a fixed spatial location, which allows comparison between event according to variations in vegetation cover and soil compaction. The second sampler is located at a different location for each event in order to sample different spatial contexts. Overall 80 samples with a measurable amount of water and sediments were obtained.
Firstly, our results show that peaks in sediment transport rates are in most cases associated with peaks of water discharges, which occur simultaneously or just after the maxima in rainfall intensities. For the first and second rainfall events similar hydrological and erosional responses of the field were observed at the two spatial locations. For there events, the distance between the two samplers on the field M short (respectively 7 and 4 meters). For a given event, the two hydrographs have the same shape, although the downslope hydrograph is target and there is a lag (5 min) between peak discharges. These characteristics suggest a kinematic response of runoff on the field. For the third precipitation, the two samplers are located 13 meters apart. There is an important diminution of runoff and erosion downslope. The inversion of the size (volume of water) of the hydrographs is attributed to the divergence of runoff caused by the microtopography and the presence of obstacles on the surface.
Over the season there is a difference in the hydrological response of the field. In September, the vegetation cover is dense and the mean infiltration and interception rates are high. Mean water and sediment discharges are low in comparison with those measured in June and October where the vegetation is sparse or absent (no interception). In October, the compaction of the soil surface is high and the infiltration capacity is low. Despite the tact that the mean rainfall intensity is slightly lower for this event, the highest amount of water and sediment discharges are observed. Average sediment concentration in water is constant for the three precipitation events. This suggests that the amount of loose sediments on the surface ready to be transported is always sufficient. The comparison of hill slope hydrographs on the field and at different times showed that water and sediment vary : i) in space according to slope length between sampling locations, and ; ii) over the season according to vegetation cover and soil surface properties.
Secondly, parameters of the sediment transport equation are estimated for the three events. The results show that water discharge and rainfall intensity are positively related with sediment discharge, but also that the effect of hillslope gradient is negligible. This is explained by the fact that only four low gradient values are used in this study and that a wide range of discharges are measured on each gradient. The overall measurements yields the empirical equation
Qs = 0,02 Q0,97 l0,6
There is a good agreement between water discharge, rainfall intensity and sediment transport (R2 = 0,91). Over the season, variation in mean discharges are not followed by fluctuations in the rate of sediment discharge which remained constant for the three events. Sediment transport appears to be low and under the limit of transport for rainwash erosion. As a result, the rate of increase in sediment transport (m = 0,97) is under the lower limit of the theoretical range of values (from 1,4 to 2,4) proposed in literature. Sediment discharge is also influenced by the contribution of rainsplash to particle detachment and transport. This process is evaluated by the incorporation of rainfall intensity into the sediment transport equation. The value obtained from the empirical estimation of p (0,6) is not significantly different from the theoretical value (p = 0,5) proposed in the literature. The contribution of rainfall intensity to the prediction of sediment transport is low but significant.
In conclusion, results of this experiment show that spatially and temporally distributed data can be used to increase our knowledge on runoff and erosion at the scale of an agricultural field. The rote of low rainfall intensities on runoff and erosion is also important despite the presence of vegetation. These events contribute to the transfer of particles downslope and they increase the amount of loose sediments ready to be transported on the surface. Notwithstanding the fact that the net erosion on the field is negligible, sediment transport is active and predictable using a simple sediment transport equation.
D. Bussières, A. Leclerc and F. H. Wand
La rivière Saguenay est un affluent majeur du fleuve Saint-Laurent, Québec, Canada. La rivière Saguenay draine une région très industrialisée et se divise en deux sections : la section supérieure est peu profonde et constituée d'eau douce, tandis que la section en aval renferme un fjord profond caractérisé par une thermohalocline à environ 25 m. Nous avons caractérisé la capacité de complexation (CC) et la constante de stabilité critique (CSC) de ses eaux douces, dans la section supérieure de la rivière. Cinq différentes stations ont été échantillonnées le même jour; ces échantillons ont été fractionnés en fonction de la masse moléculaire nominale (NMM) des ligands dissous à l'aide de quatre colonnes de chromatographie par perméation de gel (GPC) Séphadex G-10, G-15, G-25 et G-50 utilisées en série. Pour les échantillons globaux, la CC diminue d'amont en aval passant de 0,32 à 0,14 µM. Nous n'avons pu identifier la cause de cette diminution qui pourrait être un simple effet de dilution ou une augmentation d'ions métalliques en solution. Une fois fractionnés, nous trouvons que la CC augmente avec NMM; par contre, la CC normalisée par unité de carbone est plus grande pour les ligands de plus faible NMM. Les CSC obtenues sont toutes similaires, environ 5 x 107 L mol-1, sauf pour les ligands ayant une NMM entre 700 et 1 800 g mol-1 avec une CSC de 27 x 107 L mol-1.
The Saguenay River is a major affluent of the St. Lawrence River, Quebec, Canada. The Saguenay River which drains a heavily industrialized region can be subdivided into two sections: the upper section is rather shallow and contains freshwater as the lower one is a deep fjord characterized by a thermohalocline at about 25 m. This work aimed at identifying the possible modifications brought up by anthropogenic sources upon the complexation capacity of the freshwater of this River. Five different stations were sampled for surface water the same day on the upper section of the River. The samples were filtered on 0,4 µm membrane (pre-cleaned with HNO3). A portion was analyzed and other ones were fractionnated as a function of the nominal molecular mass (NMM) of dissolved ligands by using in series four gel permeation chromatographic (GPC) columns filled with Sephadex G-10, G-15, G-25 and G-50 respectively, the elution being dope by purified 18MOhms water. The complexation capacity (CC) and critical stability constant (CSC) of the different fractions have been characterized using a method based on free Cu2+ back-titration by Differential Pulsed Anodic Stripping Voltammetry (DPASV) and a 1:1 complexation scheme. Because copper was giving two unresolved peaks on the tailing of the oxygen peak, all polarograms have been deconvolved by a PASCAL computer program based on a least-sqares nonlinear fit using the Taylor differential correction technique. All results compiled were from the peak centered at - 60 mV against an Ag/AgCl reference. By manipulating the usual equations to determine CC and CSC with the free Cu2+ back-titration, we were able to calculate CC by three different routes and CSC by two different routes ; when enough reliable data were available for each route, all values obtained were concordant. So we observed that, going downstream, the CC decreased from 0,32 to 0,14 µM for whole samples. At this point, we cannot identity the cause of this decrease wether it is due to simple dilution or by addition of new dissolved metallic ions into the stream. Once fractionnated, CC measured was seen increasing with NMM but normalized CC per unit of carbon has been found to be greater for ligands with small NMM (normalized CC decreased with increasing NMM). The CSC obtained were all similar, about 5 x 107 L mol-1, excepted for ligands with NMM between 700 and 1 800 g mol-1, the CSC being 27 x 107 L mol-1 from the inverse linearized method.
Bioaccumulation d'aluminium chez la truite Salmo truffa fario soumise au retombées des pluies acides : étude structurale, ultrastructurale et microanalytique
C. Chassard-Bouchaud, Ch. Galle and E. Lopez-Rabereau
Des études microanalytiques ont été menées sur des truites Salmo trutta fario, âgées de deux ans, récoltées dans une rivière des Vosges soumise aux retombées des pluies acides et sur des truites témoins récoltées dans une rivière d'Auvergne, non soumise aux pluies acides. La rivière des Vosges est caractérisée par un pH de 5,42 et par une concentration en aluminium de 200 µg/L-1. Notre but étant de déterminer les tissus, cellules et organites cibles de bioaccumulation éventuelle de l'aluminium, nous avons analysé rein, foie, branchie et tractus digestif. Deux méthodes microanalytiques ont été utilisées pour localiser l'aluminium à l'échelle cellulaire et subcellulaire et connaître les éléments avec lesquels il peut être associé; ce sont la spectrométrie de masse par émission ionique secondaire (microscope ionique associé à un système informatisé de traitement d'images) et la spectrométrie des rayons X (microsonde électronique de Castaing associée à un microscope électronique à transmission).
La microanalyse des rein, foie, branchie et tractus digestif montre l'existence de deux processus conduisant à la bioaccumulation de l'aluminium. Le premier, classiquement connu pour d'autres métaux, met en évidence une insolubilisation de l'aluminium sous forme de phosphate, dans des organites limités par une membrane : les lysosomes et les granules pigmentaires des mélanocytes. Le second, démontre la formation de volumineux dépots extra-cellulaires, atteignant 100 µm de long et entraînant la destruction du tissu. Aucune bioaccumulation significative d'aluminium n'a été observée chez des truites témoins, récoltées dans le centre de la France, où l'eau à pH 7.9 est dépourvue d'aluminium.
The major harm caused by acidic precipitation is shown by a disappearance of fish. Other factors besides acidity such as aluminium levels are significantly harmful and many studies have shown that aluminium ions are toxic to fish. The only sensible course of action is to investigate the basic mechanisms by which each of the metal pollutants enters and attacks living systems. For this, one needs to be a combination of physicist, chemist, biochemist, physiologist and toxicologist. Investigations on metal bioaccumulation require very sensitive analytical instrumentation. Total analytical methods commonly used are inadequate : absorbed and adsorbed elements cannot be distinguished.
Therefore, interesting information can be obtained by using physical methods of chemical microanalysis : two available microanalytical techniques are particularly suitable, X-ray spectrometry and secondary ion mass spectrometry.
X-ray spectrometry, also called Electron Probe X-ray Microanalysis or Electron Microprobe (EMP) provides a means for studying the local chemical composition and structure of biological specimens. EMP can be used in association with a photon microscope or with a transmission electron microscope allowing the detection of elements at subcellular level.
The Secondary Ion Mass Spectrometry (SIMS), also called Ion Microscopy, allows to visualize, analyse and photograph the microscopical distribution of the stable or radioactive isotopes of the elements present in a histological section. The sensitivity of the method is very high, ranging from 0.1 to 1 ppm. In association with SIMS, a processing of secondary ion images is used.
Two years-old samples of Salmo trutta fario, from wild populations living in acidified waters of Eastern France (near Cornimont in the Vosges moutains) were studied for aluminium detection at cellular and subcellular levels. The acidified waters were characterized by a low pH (5,42) and high aluminium level reaching 200 µg/L-1. Control trouts living in non acidified waters (pH : 7.9 and aluminium free) of central France (near Clermont-Ferrand) were used for comparison. In order top determine the tissues, cells and organelles of a possible aluminium concentration, the following organs were investigated : kidney, liver, gill and digestive tractus, using both microanalytical techniques described above.
In the kidney ion images showed aluminium emission from tubule lysosomes with a ring-shaped localization along the apical border of the epithetial cells; aluminium emissions from the tubule lumen and from the pigment granules of the melanocytes were also observed. Using the electron microprobe, X-ray emission spectra of aluminium associated with phosphorus were obtained from lysosomes and pigment granules. In the liver and in the gills, ion images showed a high aluminium emission from the same organelles and X-ray spectra of aluminium and phosphorus were also obtained. Moreover, in the pyloric caeca, large extracellular deposits of aluminium were detected : they measured about 100 µm in length and were located in places where tissues had been destroyed.
The same structural, ultrastructural and microanalytical investigations were performed on the control trouts from which non aluminium detection was obtained.
In conclusion, two processes appear to be involved in the aluminium accumulation in the brown trout. The first one corresponds to a well known insolubilisation of aluminium phosphate inside the lysosomes, due to an acidic phosphatase enzymatic activity; aluminium is also trapped inside pigment granules. Both of these mechanisms of storage inside membrane-limited organelles, prevent cells from any interior damage. The second one corresponds to the formation of large extracellular deposits which are likely to provoke injuries leading to the tissue destruction. Such data demonstrating basic mechanisms of aluminium accumulation in a fish, could not have been obtained using total analytical methods.
Rôle de la fréquence des prélèvements de la biomasse produite sur les capacités épuratrices de Lemna minor L.
M. Radoux and D. Kemp
Les Lemnaceae en général, Lemna minor L. et L. gibba L. en particulier, font l'objet de recherches importantes au niveau international sur le rôle positif ou négatif que ces espèces peuvent jouer dans le domaine de l'épuration des eaux usées urbaines par les diverses techniques extensives.
Selon les travaux scientifiques réalisés en vraie grandeur comme en pilote, les auteurs insistent soit sur l'intérêt de ces espèces (productivité élevée, haute teneur en protéines, capacité de bioaccumulation de métaux lourds, prélèvements périodiques aisés, bonnes capacités épuratrices en N et P. ...) soit sur leurs inconvénients (effets néfastes sur l'épuration dans les lagunages à microphytes, anaérobiose de la nappe aquatique, prélèvements périodiques difficiles - ! -, capacités épuratrices médiocres, voire nulles, ...).
Dans ce contexte de résultats apparemment contradictoires, qui ne se limitent d'ailleurs pas aux seules Lemnaceae - loin s'en faut-, nous avons comparé, sous climat local, les rendements épuratoires de deux systèmes de bassins miniatures en série, rigoureusement identiques, alimentés par le même débit des mémos eaux usées urbaines durant toute la période de végétation : l'un des systèmes était peuplé de Lemna minor, l'autre n'en contenait pas.
Nos résultats font apparaître que l'efficacité épuratrice de Lemna minor dépend, entre autres, de la fréquence des prélèvements périodiques de la biomasse produite.
1. Lorsque les prélèvements sont peu fréquents (7 fois sur la période de végétation), le système à Lemna épure sensiblement mieux que le témoin non « planté » en ce qui concerne les matières en suspension et la charge organique. Il influence défavorablement, par contre, l'épuration tertiaire au niveau de l'azote et surtout du phosphore.
2. Lorsque les prélèvements sont fréquents (24 fois sur la période de végétation), le système à Lemna améliore considérablement la rétention des matières en suspension et celle de la charge organique. II augmente nettement l'épuration tertiaire au niveau de l'azote et reste sans effet pour le phosphore.
The affect of Lemnaceae, and in particular of Lemna miner L. and L. gibba L. on various extensive urban sewage treatment techniques is currently the subject of intense international research.
Researches on both full-scale and pilot plants stress either these species' advantages (high productivity, high protein content, heavy metal bioaccumulation, easy harvesting, high N and P purification efficiency, ...) or disadvantages (detrimental effect on microphyte ponding, anaerobiosis in the water layer, difficult harvesting, poor or non-existent purification capacity).Against this background of apparently conflicting results (and in this respect, Lemnaceae are far from being the only case), we have compared the performances of two systems under local climate, bath consisting of 4 strictly identical ponds in series (4 x 0.96 m2) supplied with an identical influx of the came urban sewage, one with a population of Lemna miner and the other without.
Each series of ponds was supplied with a hydraulic load of wastewater corresponding to the use of 6 square metres of pond per capita.
The two series of ponds were monitored in 1985 and 1986 during the periods of vegetative growth (from May to October inclusive).
In 1985, the duckweed biomass was harvested 7 times during the experimental period : each lime, 50 % of the plant cover was removed; this frequency amounts to roughly one harvest every fortnight in June, July, August and September.
In 1986, the duckweed biomass was harvested 24 times during the experimental period in the same manner as the previous year; this frequency amounts to 1 or 2 harvests every week in May, June, July, August and September.
The parameters chosen to characterize the pollutant load at the inflow and the outflow of each ponding system were measured twice monthly.
An amount of wastewater proportional to the volume in circulation was sampled automatically at each level. Our protocol required on sample of inflow every 30 minutes and one sample per litre of outflow. Consequently, average concentrations of the various parameters were assessed on the basis of a mixture of 14 x 48 inflow samples and a mixture of outflow samples equal in number to the volume in litres flowing through the system in two weeks.
Taking into account the automatically recorded hydraulic flow rates, theses average concentrations were then converted into absolute loads (flow rates) for each parameter and for every 14-day period. They are expressed in gram per 14 days.
Four parameters are discussed :
- suspended solids (S.S.)
- total chemical oxygen demand (total COD) on unfiltered samples, i. e. the total organic load
- total nitrogen on unfiltered samples (total N)
- total phosphorus on unfiltered samples (total P).
Our results show that the purification efficiency of Lemna minor does depend on the frequency of the biomass production removal.
1. When the removal is less frequent (7 times during the period of vegetative growth), the system with Lemna minor has purification rates that are significantly better than those of the unplanted control system as far as suspended solids and organic load are concerned. However, it has a negative impact on tertiary retention for nitrogen and especially phosphorus.
2. When the biomass production is frequently removed (24 times during the period of vegetative growth, the system with Lemna minor considerably improves the retention of suspended solids and of organic load. It also improves significantly tertiary purification as far as nitrogen is concerned, but bas no effect on phosphorus.
Observation et étude expérimentale de mycobactéries atypiques en aquariums d'eau douce et d'eau de mer
M. Dailloux, C. Henry and D. Terver
L'eau des aquariums est source de Mycobactéries atypiques qui peuvent être pathogènes pour l'homme et les poissons.
Une étude a été réalisée à l'aquarium du Musée de Zoologie de Nancy. La recherche de Mycobactéries a été effectuée dans 40 aquariums équipés de lampes germicides à UV : 11 bassins étaient alimentés en eau douce et 29 en eau de mer. Deux aquariums non équipés de système de désinfection ont également été analysés, les propriétaires de ces derniers ayant présenté un granulome cutané à M. marinum. L'action des UV sur M. marinum en suspension dans l'eau a été testée expérimentalement dans des bassins d'eau douce peuplés de Cichlidés.
Pour chaque aquarium, un échantillon de 250 ml a été prélevé. Les cultures après décontamination au lauryl sulfate de soude ont été réalisées sur milieu de Loewenstein.
Les résultats indiquent que, quel que soit l'aquarium, la présence de mycobactéries est constante. L'isolement des mycobactéries peut être gêné par la présence d'une flore bactérienne ou fongique importante (≥ 103 U.F.C./ml).
Les espèces les plus fréquemment isolées sont M. gordonae et M. fortuitum ; M. kansasii et M. marinum ont rarement été isolées (6140 aquariums). Différents facteurs peuvent intervenir sur la sélection des espèces. La salinité de l'eau limite le développement de certaines espèces, alors qu'elle permet la croissance de M. fortuitum. Dans notre étude, la température de l'eau n'a pas été un facteur sélectif. L'utilisation de lampes UV limite le nombre de Mycobactéries. Dans les bassins expérimentaux, les radiations UV se sont révélées très actives sur M. marinum en présence ou en l'absence de poissons. A l'inverse de M. gordonae, M. fortuitum est rarement isolée en présence d'UV. Un nombre important de poissons par aquarium augmente la flore bactérienne et mycobactérienne.
La prévention des infections à Mycobactéries atypiques chez l'homme comme chez les poissons devrait pouvoir être assurée par des mesures d'hygiène élémentaire.
Water is a natural habitat of mycobacteria. In aquaria 3 species of atypical mycobacteria are frequently present : M. marinera, M. kansasitand M. fortuitum. They are potential pathogen for fishes and men. Tuberculosis has been recognized as the cause of mortality in marine and fresh water fishes. Clinical signs of fish tuberculosis are variable : ascites, skin ulcerations, skeletal deformities. The human infection is cutaneous granuloma occuring after in jury in aquaria.
In the aquaria, of which two patients with cutaneous lesions due to M. marinum were analysed, UV lamps were not used. Many factors have an influence on the number of mycobacterial organisms in aquaria waters : number of fishes per tank, decontamination system, salinity, temperature.
To determine the consequence of each factor, a study has been conducted at the “Aquarium du Musée de Zoologie de Nancy”. Research of mycobacteria was carried out in the water of 40 tanks : 11 were supplied with fresh water and 29 with salt water. Each tank was equipped with germicide UV (λ : 253,7 nm) : the intensity was 15 watts for aquaria smaller than 1 000 liters and 36 watts for aquaria larger than 5 000 liters. The effectiveness of UV radiation against M. marinum was tested in 3 experimental fresh water tanks of 280 liters. The first part of the experiment was tested without fish. Tank n° 1 was a control, lamp was switched on during the complete study, M. marinum was not added. In tank n° 2 (with UV) and n° 3 (without UV), 2 ml of M. marinum (of suspension 107 CF/ml) was added. Samples of water were analysed every two weeks. After six weeks tanks n° 2 and n° 3, were prepared for the next study : UV lamps were switched on in n° 2 and switched off in n° 3 both of which were contaminated by M. marinum. After 4 weeks 27 fishes, Cichlids, were introduced in the three aquaria. The day after, M. marinum was added to tanks n° 2 and n° 3. Every week water analysis was done, as well as an identification and quantification of all species of mycobacteria.
From each tank 250 ml of water were collected. The water was passed through a 0,2 mµ membrane. The filters were introduced in distilled water and decontaminated by lauryl sulfate. The culture of mycobacteria was grown with Loewenstein medium at 30 and 37 °C. Each colony type was identified by cultural and biochemical characteristics.
This study shows the richness in aquaria of mycobacteria; whatever the tanks, mycobacteria presence was constant. In non-treated home aquaria, the presence of mycobacteria was very important, 4 to 6 species per tank, (but in this case M. marinum was not found). In aquaria with UV lamps, the number of species per tank was lower (1 to 3).
The growth of mycobacleria could be prevented when the samples were contaminated by fungi and bacteria. However, inability to recover mycobacteria from water occurred only when a massive over-growth by non-mycobacterial contaminant was present (103 CFU/ml). This was the case of non-treated tanks, belonging to patients who developed a chronic granuloma on their hands, M. marinum was not isolated in these aquaria. The evaluation of slowly growing mycobacteria could be altered by the important development of fast growing mycobacteria on the same culture tube. Among isolated species, M. fortuitum and M. gordonae saprophytic strains were frequent; M. kansasii and M. marinum involved in human cutaneous granuloma were unusual, as were the non-pigmented strains of groupe III of Runyon ; M. avium was not isolated.
During this study, we observed a relationship between the mycobacteria presence and the cleanness of tank and the fishes population. A great number of fishes per tank was a factor which increased the bacterial and mycobacterial contamination. During this experiment fishes didn't present tubercular-lesions but when a dead fish was examinated, the culture from post-modem samples revealed the presence of M. marinum. The microbiological examination of skip and viscera was negative.
The comparison of results in non-treated home tanks and UV treated tanks of the Museum indicates the role of water treatment by UV lamps on the number of isolated mycobacteria.
The germicide UV camps are frequently used for the decontamination of tanks. The efficiency is good for bacteria, but unknown for mycobacteria. This study shows that UV radiation decreased the mycobacterial contamination. The species of mycobacteria differ in their sensitivity to UV radiation. In experimental tanks, the results showed the great susceptibility of M. marinum to UV lamps such they were used in aquaria. Presence of fish dues not change the results. If the addition of M. marinum and the lighting of lamps were simultaneous, M. marinum was not isolated in water. If the contamination by M. marinum preceded the lighting of UV lamps, must of bacteria was eliminated in one week and the totality in 4 weeks. For the other species, we observed that the mycobacterial sensitivity to UV light decreases in the following order : quickly growing mycobacteria, photochromogen and scotochromogen strains. During our experimental study, M. gordonae was isolated more frequently when UV lamps were switched on. The results obtained in the 40 tanks with UV lamps allowed the evaluation of the influence of salinity and temperature of water on mycobacterial survival and the selection of species. We did not observed a difference in the concentration of mycobacteria in two types of aquaria, fresh water and salt water. Na Cl is known as an inhibitor of the mycobacterial growth. The sensitivity of strains differs. The salinity of water appears to be a selection factor. M. forfuitum was isolated more frequently in salt water.
M. marinum was isolated only in salt water and M. kansasii in fresh water. These results are surprising, as these strains have about the same metabolism. The temperature of water can also be a selection factor for mycobacteria, but in our study the temperature was similar in each aquarium (25°-26°). In this study, we did not observe thermophile strains such as M. avium.
Aquarists must be informed of the aquarium contamination by atypical mycobacteria and their role in the evolution of skin lesions after injury of hands and arms. The use of germicide UV lamps improves the bacteriological quality of water.
Biodégradation anaérobie de l'acide crotonique par une biomasse bactérienne spécialisée dans la dégradation de l'acide butyrique
P. Martel-Naquin, C. Comel, R. Gourdon and J. Veron
La connaissance, actuellement très limitée, du métabolisme des bactéries acétogènes intervenant dans la biodégradation anaérobie de l'acide butyrique et d'un de ses sous-produits, l'acide crotonique, est à l'origine de cette étude.
Après avoir mis au point un réacteur anaérobie à biomasse fixée, cette dernière a, dans un premier temps, été adaptée à la biodégradation exclusive du butyrate. La dégradation du crotonate a ensuite été étudiée, selon différents protocoles expérimentaux (pulses de crotonate en alimentation continue avec du butyrate puis alimentation continue avec du crotonate). Des injections de crotonate ont également été effectuées en circuit fermé, avec une biomasse adaptée dans un premier temps à la dégradation d'un mélange d'AGV, le réacteur étant ensuite alimenté avec du propionate puis du butyrate seuls.
Contrairement à ce que laissait penser la bibliographie, il a été constaté que les bactéries adaptées à la dégradation exclusive du butyrate sons très rapidement à même de dégrader le crotonate.
Les résultats obtenus permettent d'approcher les spécificités bactériennes, la voie catabolique suivie par le crotonate, son mode de régulation enzymatique et les équilibres qui la gouvernent. C'est ainsi qu'il est possible de proposer un modèle explicatif relativement simple du mécanisme de biodégradation du crotonate.
Volatile Fatty Acids (VFAs) are intermediate metabolites formed in the anaerobic biodegradation of organic matter. They are commonly found in sewage, municipal sanitary landfill leachate and effluents from agricultural and food-processing industries. A good knowledge of the microorganisms involved in VFA biodegradation is necessary to operate satisfactory biotreatment of those effluents.
The objective of the present study is to better understand the metabolism of the anaerobic bacteria responsible for the degradation of butyric acid and one of its metabolites (crotonic acid), which is still poorly known.
Syntrophomonaswolfei is one of the few butyrate-degrading acetogenic bacteria that bas been documented. First studios have shown that this microorganism is not capable of degrading crotonic acid (MCINERNEY et al., 1979, 1981). This is surprising since crotonyl-Coenzyme A, in its activated form, is an intermediate metabolite of n-butyrate ß-oxidation, which is the most common mechanism of butyrate biodegradation. In addition, ß-oxidatlon of crotonate is thermodynamically possible, even under standard conditions.
These observations are al the origin of the present study, which investigates the anaerobic biodegradation of crotonate. Other Investigators have followed a similar approach and isolated S. wolfei in pure culture on crotonate.
The degradation of crotonate was studied in a bench-scale up-flow anaerobic filter of twenty liters, operated in the dark, at 35 °C.
A first set of experiments was carried out with a biomass exclusively adapted to the biodegradation of butyrate. Heat-expansed vermiculite was used as a packing medium. Various experimental protocols were successive followed. First, pulses of crotonate were injected into the reactor under conditions of continuous feeding with butyrate, and then, the reactor was continuously fed with crotonate. The objective was to determine whether a bacterial population exclusively adapted to butyrate biodegradation would be capable of degrading crotonate.
It was found that crotonate was actually biodegraded in the reactor. Woth the first protocol, when pulses of crotonate were injected into the reactor, crotonate was totally removed in 55 hours (fig. 3). Butyrate and acetate concentrations increased as crotonate was degraded, but no significant increase in biogas production was observed. On the other hand, under the same conditions, it was found that iso-butyrate was not degraded, which is consistent with other published data (MCINERNEY et al., 1979, 1981 ; STIEB and SCHINK, 1985,1989).
With the second protocol (continuous feeding with crotonate at 5.2 gg/l), crotonate was totally biodegraded in 48 hours after a 24 hours lag period. This biodegradation resulted in the accumulation of acetate and, in a lower extend, butyrate (fig.4).
Following this stage, the reactor was fed with a higher crotonate concentration (12 g/l), and it was observed that crotonate was totally degraded in 20 hours, without any lag period (fig. 5).
These results showed that butyrate-degrading bacteria were capable of degrading crotonate effectively after a short period of adaptation.
Further experiments were conducted with a biomass previously adapted to the degradation of a mixture of VFAs (acetate, propionate, iso-butyrate, butyrate and caproate). Berl saddles were used as a support for bacterial growth. The reactor was operated in a recirculated batch mode and spiked with crotonate. Finally, the reactor was successively fed for four weeks with propionate and for two weeks with butyrate, before being spiked with crotonate.
In all these experiments, crotonate biodegradation was observed, but, in contrast to the results obtained with the “vermiculite reactor”, no butyrate accumulation occured (fig.6).
These results show that a bacterial population adapted to the degradation of a mixture of VFAs or to the degradation of individual VFAs such as propionate and n-butyrate, is capable of degrading crotonate.
Based on the present study and on literature data, the following mechanism can be proposed for the biodegradation of crotonate (fig.7). The first stage is the activation of crotonate into crotonyl-Coenzyme A by an acetyl-CoA/crotonyl-CoA transferase, as recently isolated from S. wolfei (BEATY and MCINERNEY, 1987). When present at low concentrations, crotonate is probably directly degraded into acetate, as shown by the results obtained with the “selles de Berl reactor”, in which no intermediate metabolite has been detected. At higher concentrations, enzymatic sites may be saturated and an equilibrium be established with butyrate, which is then released into the medium. This has been shown by the accumulation of butyrate under conditions of continuous feeding with crotonate. In addition, another intermediate metabolite has been formed, which has not been identified in the present study. This product is most probably poly-ß-hydroxy-butyrate, which has been found in S.wolfei (MCINERNEY et al, 1979) although if is not very common in chemiotrophic bacteria.
Détermination par bioessai de la biodisponibilité des ressources azote et phosphore, dans les eaux du Golfe du Morbihan
B. Le Rouzici and G. Bertru
Des dosages physico-chimiques et des numérations du phytoplancton sont réalisés sur des prélèvements d'eau du Golfe de Morbihan (France), conjointement à des bioessais en milieu non renouvelé :
1. Avec le peuplement autochtone, par enrichissement en azote et en phosphore de l'eau filtrée sur 200 µm.
2. Avec deux algues tests épuisées en azote et en phosphore, sur l'eau filtrée stérilement. Les quotas cellulaires des algues tests en fin de croissance sont comparés aux quotas cellulaires minimaux et maximaux préalablement établis.
Les bioessais avec enrichissement révèlent une limitation de la croissance des algues par l'azote. Les bioessais avec Fragilaria elliptica présentent une relation linéaire croissante simple entre la concentration en azote assimilée par l'algue test et la densité cellulaire du peuplement phytoplanctonique naturel. L'azote total en solution représente la fraction utilisée par ces algues : azote biodisponible. Par contre, la fraction « phosphore soluble » ne représente qu'une partie du phosphore biodisponible, et se trouve consommée de manière très diverses par les algues testées. Cependant, l'analyse des quotas cellulaires en fin de croissance des algues tests montre que le phosphore est l'élément impliqué dans la limitation de leur croissance.
Les bioessais exposent des résultats contradictoires de limitation de la croissance. Les algues autochtones utilisent des ressources en phosphore supérieures à celles qui sont mises à disposition des algues tests, par stockage interne ou par consommation du phosphore minéral adsorbé sur les particules en suspension.
In estuarine water, the knowledge of the resources bioavailability is indispensable for understanding phytoplankton dynamics. For assessing nitrogen and phosphorus bioavailability, algal bioassays are the most indicated. Nutrient limitations of an alga exhibit a threshold rather than an additive or a multiplicative response (ELRIFI et al., 1985). In contrast, numerous nutrient limitations are described for an algal population (TILMAN,1982).
Samples are taken monthly at 1 meter’s depth from two French river estuaries, Noyalo River and Vannes River, in the Morbihan Gulf from April to July. Main physico-chemical measures (temperature, salinity, nitrate, nitrite, ammonia, total nitrogen, reactive phosphorus and total phosphorus) were taken. Total soluble nitrogen and total soluble phosphorus concentrations were determined after filtration en 0,2 µm, for comparison with bioassays. In addition two kinds of batch bioassays with 250 ml erlenmeyers into controlled environment (17 °C and 180~200 µEinstein), were employed :
- Estuarine waters filtered on 200 µm, to remove most of the zooplankton, were enriched with sodium nitrate and/or dipotassium hydrogen phosphate. Algal Growth response, was recorded by fluorescence « in vivo », against a blank.
- With the total growth on sterilely filtered (0,2 µm) estuarine waters of two test algae, starved simultaneously in phosphorus and nitrogen in artificial sea water, it was possible to determine the bioavailability of these two elements in the soluble traction. The cellular density of the two test algae : Fragilaria elliptica Schumann and Phaeodactylum tricornutum Bohlin, was related to optical density measures at 750 nm. Nitrogen and phosphorus intracellular concentrations, in the whole culture at the end of the growth, gave their bio-availability for the test algae provided. Nitrogen and phosphorus cell quotas, intracellular concentration per cell, were then compared with their higher and lower cell quotas. Higher and lower cell quotas were measured at the end of the test algae growth in an artificial medium without phosphorus or nitrogen resources. They were similar to those described by DAUTA (1982).
In 1989, an exceptional drought reduced the river flow. Consequently, nutrient flux decreased and phytoplankton density, mainly diatoms, was reduced to between 100 and 800 cells/ml. Sea water dominated in the Morbihan Gulf with salinities higher Chan 30 %o.
First, waters filtered on 200 µm and added to which was nitrate and/or phosphate revealed a nitrogen limitation of natural phytoplankton.
Secondly, phosphorus limitation were found in bioassays on sterilely filtered waters, in all the test algae starved for phosphorus and nitrogen. At the end of the growth in bioassays, phosphorus cell quotas of the test algae were generaly similar to the lower ones. The growth of the tests algae was limited by that resource.
These contrary results are related to the intracellular storage by the natural phytoplankton and/or the major role of particulate fractions in phosphorus bioavailability.
Phosphorus and nitrogen concentrations assimilated by the test alga : Fragilaria elliptica, were inversely related. From May to July 1989 the results showed a simple linear relation between soluble nitrogen source (filtered on 0.2 µm) which was bioavailable for the test alga and natural phytoplankton density (R2 = 0.98). The two measurements of April did not agree with the latter relation, rather because of environmental factors such as temperature (11 °C against 20~21 °C for the next ones), than because of the resources. Moreover, the station B in July exhibited a natural phytoplankton density significantly lower than the station A and independent of the nitrogen bioavailability for the two test algae. The bioavailable phosphorus for Phaeodactylum tricornutum, was also significantly lower at station B. That resource can explain the difference in density monitored in July.
The total soluble nitrogen source, inorganic and organic, and just a fraction of the total soluble phosphorus source, were assimilated. The soluble phosphorus assimilated by test algae, after sterile filtration, is not the total bioavailable phosphorus in the water of the Morbihan Gulf and seems to be inversely related to the natural phytoplancton density. A part from mineral phosphorus adsorbed on suspended matter is also bioavailable (BERLAND et al., 1973). However, for the phosphorus resource, bioassays have to be performed on particulate fractions.
Efficacité des méthodes d'adsorption-élution utilisant la poudre ou la laine de verre pour la concentration des virus dans les effluents de stations d'épuration
B. Hugues, M. André and H. Champsaur
Pour rechercher les virus dans les eaux usées traitées, une nouvelle méthode d'adsorption-élution sur laine de verre a été appliquée comparativement à la méthode d'adsorption-élution sur poudre de verre. Lorsque la technique de concentration sur laine de verre est utilisée, c'est dans les 25 premiers ml de l'éluat que la majorité des virus poliomyélitique est retrouvé (89 à 94 %). La comparaison des méthodes de concentration des virus indigènes à partir d'échantillons d'effluents provenant de deux stations d'épuration biologique de la Côte d'Azur (Cagnes-sur-Mer et Nice), a mis en évidence la supériorité de cette nouvelle méthode : les taux de positivité ont été respectivement de 85 % vs 38 % pour l'effluent de Cagnes-sur-Mer et 100 % vs 44 % pour l'effluent de Nice. De même, les titres en virus indigènes après concentration ont varié de 0 à 250 NPPUC/l pour la méthode sur laine de verre contre 0 à 25,5 NPPUC/l pour la méthode sur poudre de verre. La différence constatée entre les méthodes est statistiquement significative après analyse de variance (p = 0,0119 pour l'effluent de Cagnes-sur-mer et p < 0,0001 pour l'effluent de Nice). De plus, la technique sur laine de verre ne nécessite ni l'abaissement du pH, ni le changement de la composition ionique de l'échantillon d'eau à analyser.
Biological treatment of sewage in waste water plants does not allow elimination of the whole of the microbial load. Discharge of the treated sewage results in viral pollution of river, lakes and seas, a potential hazard for the health that has to be monitored. The amont of virus in waste water beeing low, concentration from the samples brought to the laboratory is rendered necessary. The aim of this study was to evaluate the efficacy of a new adsorption-elution method on glass wool to recover indigenous viruses from effluents of the cities of Cagnes and Nice (Alpes-Maritimes, France). In order to evaluate its efficiency we compared it to the regular adsorption-elution method on glass powder. As a preliminary we determined upon artificially contaminated 5 liter waste waters samples what detection of virus could be performed only in the first 25 ml of the 100 ml eluate, as in the glass powder concentration method. Results show chat virus titers found in that first fraction of eluate were close to those in the total sample. Thus from 3 samples containing 1.60 108 MPNCU/5 l, 1.96 107 MPNCU/5 l and 4.32 104 MPNCU/5 l we found in that first fraction respectively 1.50 104 MPNCU/5 l (94 %), 1.80 107 MPNCU/5 l (92 %) and 3.85 104 MPNCU/5 l (89 %); these recovery rates are not significantly different by comparison of confidence limits. The glass powder method, necessitates preliminary treatment of the sample : acidification to pH 3.5 and adjunction of AICI3 at a final concentration of 5.10-4 M. After flowing the acidified sample through 100 g of borosilicated glass powder at a rate of 10 l/10 min inside a decantation ampulla. Then adsorbed virus may be eluted from the sedimentated glass powder with 100 ml of borate buffer containing 3 % beet extract, pH9 : the first 25 ml were collected into a flask containing 2.5 ml of a mixture of antibacterial and antifungal antibiotics. For the glass wool adsorption method, a 19 cm3 cartridge was packed with 5 g sodocalcic glass wool at a 0.4 g/cm3 density and rinced sequentially with : 10 ml 1N HCl, 10 ml deionised water, 10 ml 1N NaOH and lastly 40 ml deionised water. It was balanced with 200 ml deionised water. The sample, was pumped at a flow rate ca 10 l/h. Enumeration of viruses was performed by inoculating 40 microplate wells containing KB cells, and performing 3 passages 5 days each, after which the number of wells presenting with CPE was determined. This characteristic number allowed calculation of the most probable number of cytopathic units (MPNCU) with the 95 % confidence limit. The Box and Cox analysis of transformation was applied to the data. Since the calculated value of λ approximated zero (λ = - 0.29 for the Cagnes effluent and λ = - 0,062 for the Nice effluent), transformation of the gross data into logarithm was justified. To allow this transformation, the zero had to be substituted for by a value equal to half the limit sensitivity of the method (I well out of 40), i.e. 0.5. Distribution of the data being roughly log-normal, it was then possible to compare the results of the two methods by two-way analysis of variance, cross classification, without replica. The test for factor method was calculated according to the interaction since this factor is fixed. Overall it appeared that all 31 10-liter samples analysed contained viruses when results from bath methods were combined. Still no single method allowed virus recovery in a 100 % of cases, however the glass wool adsorption method found viruses in 29/31 vs 13/31 with the glass powder method. The new method detected virus in 11/13 (85 %) samples from Cagnes waste waters as well as in 18/18 (100 %) from Nice. Quantitative analysis of the viral titers indicates that, titers were higher following the glass wool adsorption method than following the glass powder adsorption method in 11/13 samples from Cagnes treatment plant and in 17/18 from Nice. Thus virus concentrations varied between 0 and 250 MPNCU/l (MGT= 4.6 MPNCU/l) for the Cagnes effluent and between 2 and 60 MPNCU/l (MGT= 7.5 MPNCU/I) for the Nice effluent. For the same samples virus concentrations obtained following glass powder adsorption method varied between 0 and 8.5 MPNCU/l (MGT 0.9 MPNCU/l) for the Cagnes effluent and between 0 and 25.5 MPNCU/l (MOT= 1.3 MPNCU/l1) for the Nice effluent. This difference is statistically significant (p = 0.0119 for the Cagnes effluent and p < 0.0001 for the Nice effluent). Furthermore, when taking into account the origin of the waters analysed, comparison between observed F0.95 (7.94 for the Cagnes waters and 45.78 for the Nice waters) and theoretical F0.95 (4.75 for the Cagnes waters and 4.45 for the Nice waters) leads to the rejection of the hypothesis of identity of the two methods. The discordances observed are an illustration of the fact that concluding to the absence of viruses in a given sample is a matter of method and should be interpreted with prudence. A few drawbacks inherent to the glass powder adsorption method may explain its poorer efficiency : the necessary acidification of the sample to pH 3.5 may be fatal to a proportion of virions; also the flow rate necessary to maintain the fluid layer of glass powder in suspension during the adsorption step is 6 fold higher than that required in the glass wool method (60 l/h vs 10 l/h). Finally the nature of the adsorbing material, sodocalcic vs borosilicated, may be determinent. We can conclude from the present comparative study, to the statistically significant superiority of the glass wool method for virus concentration from treated waste waters.
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