We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1. Préparation de la verrerie et de logements de filtre <ol style="margin-left: 40px;"><li> Sauf indication contraire, tous les stériliser la verrerie, des boîtiers et des solutions de filtrage à 121 ° C pendant 15 min. Pour assurer la stérilité, couvrir toutes les ouvertures ou les surfaces exposées avec soit une feuille d&#39;aluminium ou de papier de bande sécurisé avant la stérilisation. </li><li> Assembler appareil de filtration en attachant boîtier de filtre (diamètre 47 mm) à une branche latérale L erlenmeyer. Recueillir les filtres nécessaires: diamètre 47 mm filtres à disques électropositifs / électronégatif. </li><li> Préparer 1 L de 1,5% d&#39;extrait de boeuf (floculation; produit des particules de floc lorsque le pH de la solution est abaissé à 3,5, ou non-floculant, ce qui ne est pas globale à pH 3,5), avec 0,05 M de glycine, en dissolvant 15 g d&#39;extrait de boeuf dans 1 L d&#39;eau désionisée et en ajoutant 3,75 g de glycine. REMARQUE: extraits non-floculants boeuf nécessiteront l&#39;ajout de célite avant concentration tertiaire. </li><li> Ajouter élution polyph additif de sodiumosphate d&#39;extrait de boeuf à une concentration de 0,1%. </li><li> Ajouter solution 1 M d&#39;acide chlorhydrique, goutte à goutte, le mélange extrait de boeuf, solution de pH à pH désiré. Extrait de boeuf autoclave pendant 15 à 30 min à 121 ° C. </li><li> Mesurer 0,1 g de fines, moyennes ou grandes particules de célite (pour une utilisation avec non-floculation extrait de boeuf seulement). </li><li> Préparer vide / aspiration par fixation de tubes compatible Erlenmeyer bras latéral ballon et à la sortie de vide. </li></ol> 2. Préparation des solutions et Virus Stock <ol style="margin-left: 40px;"><li> Préparer 1 L de HCl 1. </li><li> Préparer 1 litre de 1 M de NaOH. </li><li> Préparer la solution 1x PBS (NaCl 137 mM, 2,7 mM de KCl, 1,47 mM K 2 PO 4 et 4,3 mM NaH 2 PO 4). <ol><li> Ajuster le pH de 1 x PBS à 9,0 en utilisant du NaOH 1 M. </li></ol></li><li> Préparer et diluer stock de virus à 10 4 -10 5 nombre le plus probable (NPP) ml -1 en mélangeant 1 ml de stock de virus avec 9 ml de PBS (pH7), comme précédemment décrit 8,13. Conserver à -80 ° C en aliquots de 1 ml. <ol><li> Stériliser stock de virus par filtrage de la seringue (0,22 um de taille de pores) pour éliminer les contaminants potentiels. </li></ol></li><li> Déchlorée eau du robinet <ol><li> Mesure 1 L d&#39;eau du robinet stérile en utilisant une solution stérile graduée et verser dans un bêcher de 2 L contenant une barre d&#39;agitation magnétique. </li><li> Ajouter 0,7 g de thiosulfate de sodium et mélanger jusqu&#39;à ce que les granules se dissolvent. Ajuster le pH de l&#39;eau du robinet à 7-7,5 si nécessaire l&#39;aide de M HCl 1 ou une solution M NaOH. </li><li> Pic de virus Thaw (1 ml) et ajouter du volume entier de pic à 1 L d&#39;eau du robinet déchlorée stérile. Mélanger pendant 10 minutes. </li><li> Préparer un blanc de méthode (de l&#39;eau du robinet déchlorée sans adénovirus) avec chaque expérience et le processus de la même manière que l&#39;échantillon enrichi. </li></ol></li></ol> 3. Préparation de Millipore Filtre Appareil <ol style="margin-left: 40px;"><li> Retirer couche stérile sur le boîtier du filtre. Assurerbon ajustement entre le boîtier de filtre bol supérieur et l&#39;écran inférieur du filtre pour éviter les fuites échantillon lors de la filtration. </li><li> Retirez protection stérile de 1 L erlenmeyer. Assurez-vous que le bouchon de l&#39;unité de filtre se adapte parfaitement dans l&#39;ouverture supérieure du erlenmeyer, créant une bonne étanchéité. </li><li> Retirer la partie supérieure du bol unité de filtre de l&#39;écran de filtre inférieure et placer 47 mm disque filtrant électropositif carrément sur l&#39;écran avec des pinces stériles. Fixez logements cuve du filtre à l&#39;écran en bas et le verrouiller en place en tournant dans le sens antihoraire. </li></ol> 4. Appuyez de filtration d&#39;eau (concentration primaire) <ol style="margin-left: 40px;"><li> Mesurer 100 ml de virus dopés l&#39;eau du robinet en utilisant stérile graduée. </li><li> Verser 100 ml d&#39;eau du robinet virus ensemencés dans le boîtier de filtre contenant soit un / filtre de cellulose 47 mm verre à plis ou un filtre à disque en fibres nano-alumine / verre. </li><li> Permettre à l&#39;eau de passer lentement à travers le filtre. Appliquer une dépression douce pour enlever les résidus restants of eau du robinet à partir de la surface du filtre. </li><li> Si l&#39;on utilise un traitement à la trypsine, ajouter 5 ml de trypsine à 0,25% pour filtrer surface et incuber pendant 5 min. </li><li> Laver la trypsine à partir de la surface du filtre avec 50 ml d&#39;eau désionisée stérile. </li></ol> 5. Virus élution de filtre <ol style="margin-left: 40px;"><li> Déposer le boîtier de filtre de 2 L Erlenmeyer et placez sur 250 ml bras latéral flacon. </li><li> Mesurer 100 ml d&#39;extrait de boeuf en utilisant graduée. Verser lentement 100 ml d&#39;extrait de boeuf dans le boîtier de filtre. </li><li> Laisser extrait de boeuf à verser à travers un filtre, capturant extrait de boeuf dans un petit (250 ml) Erlenmeyer. Appliquer le vide pour passer à travers l&#39;extrait résiduelle de boeuf du filtre. </li></ol> 6. Celite Concentration secondaire <ol style="margin-left: 40px;"><li> flacons de transfert contenant les virus élues à une plaque d&#39;agitation. Ajouter barreau magnétique stérile et lieu sur la plaque d&#39;agitation, le mélange pour créer légère vortex. </li><li> Ajouter 0,1 g de célitepoudre à 100 ml d&#39;extrait de boeuf et laisser célite pour disperser. Placez la sonde de pH stérile dans l&#39;extrait de boeuf. </li><li> Ajouter goutte à goutte de l&#39;HCl 1 à l&#39;extrait de boeuf pour obtenir un pH 4,0. Laisser mélanger lentement pendant 10 min. </li><li> Placer 47 mm pré-filtre sur le boîtier de filtre (comme décrit précédemment) en utilisant un flacon Erlenmeyer de 2 litres. </li><li> Verser 100 ml d&#39;extrait de boeuf / mélange de Celite sur pré-filtre. Laisser extrait de boeuf de verser à travers. Appliquer une légère vide pour tirer à travers extrait résiduelle de boeuf du filtre. </li><li> Passer clip métallique sur l&#39;extrémité de boîtier de filtre bec. Attacher 15 ml collection de polypropylène tube stérile de clip en métal. Placez boîtier de filtre dans erlenmeyer. </li><li> Ajouter 5 ml de tampon 1 x PBS, pH 9,0 sur de la Celite recueillies sur le pré-filtre. </li><li> Laisser se écouler à travers PBS pré-filtre. Appliquer une légère vide pour tirer à travers PBS résiduelle dans 15 ml tube de collecte. </li></ol> 7. floculation organique Concentration secondaire <ol style="margin-left: 40px;"><li>Transférez les béchers contenant le virus élué à une plaque d&#39;agitation. Ajouter barreau magnétique stérile pour 100 ml d&#39;extrait de boeuf et mélanger pour créer légère vortex. </li><li> Placez la sonde de pH stérile dans l&#39;extrait de boeuf. </li><li> Ajouter 1 M HCl goutte à goutte à l&#39;extrait de boeuf et ajuster le pH à 3,5. Mélanger pendant 30 minutes. </li><li> Verser l&#39;éluat dans 250 ml tubes coniques centrifugeuse et centrifuger à 2500 g pendant 15 min à 4 ºC. </li><li> Verser le surnageant en prenant soin de ne pas de ne pas perturber le culot. Remettre en suspension le culot dans 5 ml de 1 x PBS, pH 9. </li><li> Centrifuger la suspension à 4000 xg pendant 10 min à 4 ºC. Verser le surnageant et jeter le culot. </li><li> Ajuster le pH à 7-7,5, stériliser par filtration à travers un filtre à seringue de 0,22 pm et de congélation à -80 ° C. </li></ol> 8. Viral Nucleic Acid Extraction <ol style="margin-left: 40px;"><li> Extraire les acides nucléiques viraux en utilisant un kit commercial approprié conformément aux instructions du fabricant. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 9. AdV 40/41 qPCR <ol style="margin-left: 40px;"><li> Utilisation des amorces et des sondes, ainsi que son mélange réactionnel et les profils de cycles thermiques tel que publié 8,13. NOTE: limite estimée de détection du test est d&#39;environ 5 unités qPCR par réaction. </li><li> Ajouter des composants de PCR mélanges d&#39;amplification à des concentrations de la manière suivante: Tris 10 mM (pH 8,3), 50 mM de KCl, 4,5 mM MgCl 2 mM dNTP 10, 10 uM des amorces et une sonde uM et 0,5 ul de polymerase de sorte que les volumes finaux de 45 pi peut être ajouté au nombre approprié de puits dans une plaque de réaction de PCR de 96 puits. </li><li> Charge 45 pi de mélange d&#39;amplification par PCR dans chaque puits approprié d&#39;un puits 96, une plaque de réaction de PCR rapide. </li><li> Ajouter 5 ul d&#39;ADN extraites échantillon dans les puits appropriés de la plaque PCR. </li><li> Couvrir la plaque avec joint de couvercle résistant à la chaleur, tourner la plaque de déplacer les gouttelettes en bas du puits de la plaque PCR. </li><li> Définissez cycles d&#39;amplification par PCR que Follux: 95 ° C pendant 10 min, suivie de 40 cycles de 95 ° C pendant 15 sec et 60 ° C pendant 1 min. </li></ol> 10. Les analyses de données <ol style="margin-left: 40px;"><li> Analyser les mesures triple des dilutions en série de 5 fois des échantillons couvrant 1: 5-1: gamme 625 de dilution afin de déterminer le nombre le plus probable (NPP) des particules virales, comme décrit précédemment 8,13. </li><li> Analyser des dilutions en série de 5 fois de pointes expérimentales adénovirus couvrant 1: 5-1: gamme de dilution 15 625 afin de déterminer le nombre le plus probable (NPP) des particules virales. </li><li> Utilisation de la calculatrice EPA MPN (http://www.epa.gov/nerlcwww/online.html), régler la calculatrice pour tenir compte de type de dilution (1: 5 ou 01h10), le nombre de dilutions effectuées, et le nombre de répliquer des échantillons en série de dilution. </li><li> Effectuer log 10 transformation des données, suivi par la normalisation à une unité de volume (par exemple, 1 ml, 100 ml, etc. </em&gt;). Calculer recouvrements expérience donnée en nombre de particules virales présentes dans l&#39;échantillon en divisant les concentrés avec le nombre de particules virales présentes dans les pics expérimentaux. </li><li> Évaluer l&#39;effet des différentes variables expérimentales (par exemple, différents types CELITE gammes de pH, différents types de filtres et différentes techniques de concentration secondaire) sur les recouvrements AdV en effectuant des tests statistiques paramétriques ou non paramétriques (par exemple, test t apparié, un ou deux -way ANOVA) en fonction de la distribution des données et la conception expérimentale. </li></ol>

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No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Filtres électropositifs sont utiles à concentrer virus de l&#39;eau; Cependant, ces filtres peuvent différer dans leur structure et la composition qui pourrait à son tour modifier leur efficacité. Se ajoute à ce problème, les structures et les charges varient capside entre les souches de virus nécessitant des techniques de concentration être adaptés pour assurer une récupération optimale 15. Grâce à des modifications simples des techniques de concentration existants (par exemple, filtre...

Les virus entériques humains sont importants agents responsables de maladies d&#39;origine hydrique 1-3, mais sont généralement présents en faible nombre dans les eaux environnementales contaminées, ce qui rend leur détection difficile sans concentration. Les procédures utilisées pour concentrer les virus comprennent généralement une étape de filtration, suivie d&#39;une élution filtre et secondaire concentration de l&#39;éluat de filtration. Une procédure de filtration commune se fonde sur l&#39;utilisation de membranes chargées telles que des filtres électropositifs (récemment examinés dans 4,5). Ces filtres reposent sur la capture des virus en suspension dans l&#39;eau en utilisant des interactions électrostatiques entre la surface de filtre (chargé positivement) et les particules virales ciblées (chargés négativement). Deux filtres électropositifs qui sont disponibles dans le commerce se appuient sur cette technologie, les filtres en verre / cellulose nano-alumine et / fibres de verre. Les coûts de filtrage verre / cellulose sont jusqu&#39;à 10 fois celle de la / fibres de verre nano-alumine, ce qui limite l&#39;utilisation du verre / cellulose filtres pour la surveillance du virus de routine. Des études récentes ont conclu différences sont nominales entre ces deux filtres dans la récupération des entérovirus de 6,7 de l&#39;eau ambiante, ce qui justifie l&#39;utilisation d&#39;une alternative de filtre moins cher. Autres options de filtres tels que les filtres électronégatifs et de laine de verre ont été étudiés, cependant, ils nécessitent soit le prétraitement de l&#39;eau de source (filtres électronégatifs) ou ne sont pas disponibles dans le commerce (filtres de laine de verre). L&#39;élaboration de procédures de concentration de virus a surtout mis l&#39;accent sur l&#39;optimisation des techniques de concentration primaires (filtres) afin d&#39;améliorer la récupération de virus de l&#39;eau. Toutefois, les procédures de concentration secondaires, qui réduisent le volume d&#39;éluant typiquement de 1 L à millilitre volumes, peuvent également avoir un impact significatif sur les recouvrements de virus 8. Concentration secondaire des virus entériques repose généralement sur un agent de floculation tels que certains types de l&#39;extrait de boeuf (floccu organiquement) ou le lait écrémé floculation 12/09 pour éliminer les particules virales à partir des surfaces de filtrage. Récemment, une autre procédure de concentration secondaire en utilisant l&#39;extrait de boeuf couplé avec l&#39;ajout de célite (particules fines) a montré des résultats prometteurs pour la récupération de l&#39;adénovirus, entérovirus, norovirus et 8,13,14. Celite travaux de concentration en vertu des principes similaires à ceux du procédé de floculation organique en ce que des particules de virus se attachent et sont libérés à partir de particules (floculation ou la célite) en modifiant le pH de la solution de suspension. Les comparaisons entre ces deux techniques de concentration secondaires ont été évalués dans la récupération des adénovirus pointes (adv) types 40 et 41 8. Cette étude a conclu que les deux techniques de concentration secondaires étaient statistiquement similaires dans la récupération des adénovirus. Cependant, le procédé de floculation organique nécessite une 30 min. incubation à un pH de 3,5, tandis que la technique de célite nécessite une incubation plus court (10 min) à un pH de 4,0. Le flocculat organiqueion nécessite également l&#39;utilisation d&#39;équipement de laboratoire coûteux (des centrifugeuses) pour recueillir des particules de floc pendant concentration tertiaire, la technique de la celite en revanche ne utilise que des équipements de laboratoire de base (filtration sous vide) pour séparer les particules de la suspension de célite. Certaines combinaisons de filtres et de techniques d&#39;élution secondaires peuvent également affecter récupérations de virus. Une étude a conclu que certaines combinaisons de primaires (filtres électropositifs) et des techniques de concentration secondaires (célite ou floculation organique) ont eu un impact significatif de la reprise de l&#39;adénovirus 13. Ces résultats suggèrent que l&#39;optimisation est nécessaire pour récupérer le virus de la cible de manière optimale à partir d&#39;une matrice d&#39;eau donnée pour l&#39;utilisation de ces techniques. L&#39;optimisation est beaucoup de temps, processus ardu de nombreux chercheurs évitent activement depuis de nombreuses variables seront évaluées (type de filtre / marque, solution d&#39;élution de pH, célite / floculation organique). Pour ce sTudy, une procédure a été développée pour identifier les conditions optimales pour la concentration du virus dans l&#39;eau en utilisant l&#39;adénovirus humain pointes souches 40 et 41. On peut supposer que, puisque chaque type de virus affiche une morphologie de capside unique et frais de capside spécifique, peuvent avoir besoin d&#39;être optimisé pour tous les virus protocoles de concentration cibler pour parvenir à une reprise virale optimale. Cette étude fournit une approche pour 40 AdV et 41 concentration par: 1) l&#39;évaluation de récupérations de virus dans l&#39;eau du robinet en utilisant des disques de filtres électropositifs suivis par 2) l&#39;évaluation d&#39;une méthode de floculation organique établie par rapport à la technique de célite comme une concentration secondaire, et 3) l&#39;évaluation des tampons d&#39;élution pour la concentration tertiaire.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:295px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 41 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Thiosulfate</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">72051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Celites #577</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">22142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
			<td style="width:143px;">Argonide</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
			<td style="width:143px;">3M</td>
			<td style="width:119px;">6408502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AP-20 Prefilter 47mm</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td style="width:119px;">AP2004700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glycine&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">50046-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Polyphosphate</td>
			<td style="width:143px;">Acros Organics</td>
			<td style="width:119px;">390930010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypsin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">PBS</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hydrochloric Acid</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">A481-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">BBL Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">212303</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Difco Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">211520</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">ABI 7900 Real-time PCR system</td>
			<td style="width:143px;">ABI</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stainless Steel Filter Housing</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td>XX2004720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Blood DNA Extraction Kit</td>
			<td style="width:143px;">Qiagen&#160;</td>
			<td style="width:119px;">51104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EPA MPN Calculator</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.epa.gov/nerlcwww/online.html</td>
		</tr>
	</tbody>
</table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

<html> Sélection Celite Trois types de célite différents ont été testés avant la sélection de la meilleure variante performante. Celites avec amende à particules moyennes produites les recouvrements d&#39;adénovirus plus élevés. Utilisation de plus grandes celites a entraîné la diminution des recouvrements à la fois pour AdV40 et 41 (entre 32% -100%) (figure 1). Taux moyens de récupération de l&#39;adénovirus 40 étaient 144% ± 52% (fine), 115% ± 28% (moyen...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Une procédure de petit volume pour la concentration virale de l&#39;eau

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Research

JoVE Journal

Environment

10.0K vues.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

Vidéo: A Small Volume Procedure for Viral Concentration from Water

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Déterminé en laboratoire Phosphore Flux de sédiments lacustres en tant que mesure de phosphore interne Chargement en cours

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

Recherche

Environnement

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1&#8211;7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9&#8211;11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

Un essai biologique de petit volume pour évaluer bactérienne / phytoplancton co-culture avec de l&#39;eau-Pulse-modulé en amplitude (EAU-PAM) Fluorimétrie

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Un CO<sub&gt; 2</sub&gt; Facilité Concentration de Gradient pour les essais CO<sub&gt; 2</sub&gt; Enrichissement et du sol Effets sur l&#39;écosystème de prairie Fonction

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Une méthode simple pour l&#39;extraction des échantillons d&#39;eau automatisée en phase solide pour immunologique analyse des petits polluants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB1, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm2) and the volume (in mm3). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

Extraction Metrics pour les systèmes de racines en trois dimensions: Volume et surface Analyse de In-sol X-ray tomodensitométrie données

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Sol Lysimètre Excavation pour Coupled hydrologiques, géochimiques et microbiologiques Enquêtes

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Extraction de pesticides organochlorés à partir de granulés en plastique et analyse de type plastique

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

Capturant pondérée en fonction du débit de l’eau et les particules en suspension de canaux agricole pendant les événements de Drainage

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Une Simple Technique de plantation pour rétablir des arbres où les inondations fréquentes se produit

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

Procédure optimisée pour la détermination de l’Adsorption de Phosphonates sur hydroxyde ferrique granulaire, en utilisant une méthode de dosage de phosphore miniaturisé

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...