Name: bioindication testing of stream environment suitability for young freshwater pearl mussels using in situ exposure methods
Uploaded: 2018-09-05T15:30:00
Description: Watch this Scientific Journal Video about Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods at JoVE.com

Michal B&#237;l&#253; and Ond&#345;ej P. Simon were supported by grants from the Czech University of Life Science [Internal Grant Agency of Faculty of Environmental Sciences, CULS Prague (42110 1312 3175 (20164236))]. Support for Karel Douda came from the Czech Science Foundation (13-05872S). Data on the bioindication and present occurrence of pearl mussels were collected during the implementation of the Czech Action Plan for Freshwater Pearl Mussels managed by the Nature Conservation Agency of the Czech Republic, which is funded by the government of the Czech Republic and is available at

1. Mesh Cage
Note: See Figure 1.
<ol>
	<li>Prepare material
	<ol>
		<li>Prepare the material for the in-laboratory part of the experiment: ~1 - 2 L of river water per mesh cage, mesh cages (1 main plastic body, 2 plastic covers, 2 sheets of special technical sieves with 340 &#181;m pores, 4 bolts and 4 nuts per cage), pliers, a spanner, Pasteur pipettes, a strainer, a digital camera, a trinocular dissecting zoom stereo microscope, a calibration ...

<ol>
	<li>Goldberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mussel+watch-a+first+step+in+global+marine+monitoring.">The mussel watch-a first step in global marine monitoring.</a> Marine Pollution Bulletin. 6 (7), 111-114 (1975).</li><li>Vaughn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecosystem+services+provided+by+freshwater+mussels.">Ecosystem services provided by freshwater mussels.</a> Hydrobiologia. , (2017).</li><li>Lopes-Lima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+status+of+freshwater+mussels+in+Europe:+state+of+the+art+and+future+challenges.">Conservation status of freshwater mussels in Europe: state of the art and future challenges.</a> Biological Reviews. 92 (1), 572-607 (2017).</li><li>Strayer, D. L., Malcom, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+recruitment+failure+in+freshwater+mussel+populations+in+southeastern+New+York.">Causes of recruitment failure in freshwater mussel populations in southeastern New York.</a> Ecological Applications. 22 (6), 1780-1790 (2012).</li><li>Douda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nitrate+nitrogen+pollution+on+Central+European+unionid+bivalves+revealed+by+distributional+data+and+acute+toxicity+testing.">Effects of nitrate nitrogen pollution on Central European unionid bivalves revealed by distributional data and acute toxicity testing.</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 20 (2), 189-197 (2010).</li><li>Modesto, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+and+mussels:+importance+of+fish+for+freshwater+mussel+conservation.">Fish and mussels: importance of fish for freshwater mussel conservation.</a> Fish and Fisheries. , (2017).</li><li>Bauer, G., W&#228;chtler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ecology and evolution of the freshwater mussels Unionoida. 145, 1-394 (2001).</li><li>Neves, R. J., Widlak, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habitat+ecology+of+juvenile+fresh-water+mussels+(Bivalvia,+Unionidae)+in+a+headwater+stream+in+Virginia.">Habitat ecology of juvenile fresh-water mussels (Bivalvia, Unionidae) in a headwater stream in Virginia.</a> American Malacological Bulletin. 5, 1-7 (1987).</li><li>&#352;vanyga, J., Simon, O. P., Min&#225;rikov&#225;, T., Spisar, O., B&#237;l&#253;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Z&#225;chrann&#253; program pro perlorodku &#345;&#237;&#269;n&#237; v &#268;R (Action plan for the endangered freshwater pearl mussel in the Czech Republic). , (2013).</li><li>Schmidt, C., Vandr&#233;, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+experience+in+the+rearing+of+young+freshwater+pearl+mussels+(Margaritifera+margaritifera).">Ten years of experience in the rearing of young freshwater pearl mussels (Margaritifera margaritifera).</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 20 (7), 735-747 (2010).</li><li>Gum, B., Lange, M., Geist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+reflection+on+the+success+of+rearing+and+culturing+juvenile+freshwater+mussels+with+a+focus+on+the+endangered+freshwater+pearl+mussel+(Margaritifera+margaritifera+L.).">A critical reflection on the success of rearing and culturing juvenile freshwater mussels with a focus on the endangered freshwater pearl mussel (Margaritifera margaritifera L.).</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 21 (7), 743-751 (2011).</li><li>Denic, M., Taeubert, J. E., Lange, M., Thielen, F., Scheder, C., Gumpinger, C., Geist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+stock+origin+and+environmental+conditions+on+the+survival+and+growth+of+juvenile+freshwater+pearl+mussels+(Margaritifera+margaritifera)+in+a+cross-exposure+experiment.">Influence of stock origin and environmental conditions on the survival and growth of juvenile freshwater pearl mussels (Margaritifera margaritifera) in a cross-exposure experiment.</a> Limnologica. 50, 67-74 (2015).</li><li>&#268;ern&#225;, M., Simon, O. P., B&#237;l&#253;, M., Douda, K., Dort, B., Galov&#225;, M., Volfov&#225;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-river+variation+in+growth+and+survival+of+juvenile+freshwater+pearl+mussels+assessed+by+in+situ+exposure+methods.">Within-river variation in growth and survival of juvenile freshwater pearl mussels assessed by in situ exposure methods.</a> Hydrobiologia. , (2017).</li><li>Denic, M., Taeubert, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+relationships+between+the+larvae+of+two+freshwater+mussels+and+their+fish+hosts.">Trophic relationships between the larvae of two freshwater mussels and their fish hosts.</a> Invertebrate Biology. 134 (2), 129-135 (2015).</li><li>Douda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-dependent+vitality+of+juvenile+freshwater+mussels:+implications+for+breeding+programs+and+host+evaluation.">Host-dependent vitality of juvenile freshwater mussels: implications for breeding programs and host evaluation.</a> Aquaculture. 445, 5-10 (2015).</li><li>Buddensiek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+juvenile+freshwater+pearl+mussels+Margaritifera+margaritifera+L.+in+cages:+a+contribution+to+conservation+programmes+and+the+knowledge+of+habitat+requirements.">The culture of juvenile freshwater pearl mussels Margaritifera margaritifera L. in cages: a contribution to conservation programmes and the knowledge of habitat requirements.</a> Biological Conservation. 74 (1), 33-40 (1995).</li><li>Hru&#353;ka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+of+semi-natural+breeding+program+of+freshwater+pearl+mussel+in+the+Czech+Republic.">Experience of semi-natural breeding program of freshwater pearl mussel in the Czech Republic.</a> Die Flussperlmuschel in Europa: Bestandssituation und Schutzma&#223;nahmen. , 69-75 (2001).</li><li>Barnhart, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buckets+of+muckets:+a+compact+system+for+rearing+juvenile+freshwater+mussels.">Buckets of muckets: a compact system for rearing juvenile freshwater mussels.</a> Aquaculture. 254 (1), 227-233 (2006).</li><li>Simon, O. P., Van&#237;&#269;kov&#225;, I., B&#237;l&#253;, M., Douda, K., Patzenhauerov&#225;, H., Hru&#353;ka, J., Pelt&#225;nov&#225;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+status+of+freshwater+pearl+mussel+in+the+Czech+Republic:+several+successfully+rejuvenated+populations+but+the+absence+of+natural+reproduction.">The status of freshwater pearl mussel in the Czech Republic: several successfully rejuvenated populations but the absence of natural reproduction.</a> Limnologica. 50, 11-20 (2015).</li><li>. A language and environment for statistical computing Available from: <a target="_blank" href="https://www.r-project.org/">https://www.r-project.org/</a> (2013)</li><li>Hastie, L. C., Yang, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conservation of the freshwater pearl mussel I: captive breeding techniques. 2, (2003).</li><li>Hru&#353;ka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nahrungsanspr&#252;che+der+Flu&#223;perlmuschel+und+deren+halbnat&#252;rliche+Aufzucht+in+der+Tschechischen+Republik.">Nahrungsanspr&#252;che der Flu&#223;perlmuschel und deren halbnat&#252;rliche Aufzucht in der Tschechischen Republik.</a> Heldia. 4 (6), 69-79 (1999).</li><li>Scheder, C., Lerchegger, B., Jung, M., Csar, D., Gumpinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+experience+in+the+rearing+of+freshwater+pearl+mussels+(Margaritifera+margaritifera):+advantages+of+a+work-saving+infection+approach,+survival,+and+growth+of+early+life+stages.">Practical experience in the rearing of freshwater pearl mussels (Margaritifera margaritifera): advantages of a work-saving infection approach, survival, and growth of early life stages.</a> Hydrobiologia. 735 (1), 203-212 (2014).</li><li>Braun, A., Auerswald, K., Geist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drivers+and+spatio-temporal+extent+of+hyporheic+patch+variation:+implications+for+sampling.">Drivers and spatio-temporal extent of hyporheic patch variation: implications for sampling.</a> PLoS ONE. 7 (7), e42046 (2012).</li><li>Franken, R. J. M., Storey, R. G., Williams, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological,+chemical+and+physical+characteristics+of+downwelling+and+upwelling+zones+in+the+hyporheic+zone+of+a+north-temperate+stream.">Biological, chemical and physical characteristics of downwelling and upwelling zones in the hyporheic zone of a north-temperate stream.</a> Hydrobiologia. , 183-195 (2001).</li><li>Roley, S. S., Tank, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+water+physicochemical+constraints+on+the+endangered+clubshell+mussel+(Pleurobema+clava).">Pore water physicochemical constraints on the endangered clubshell mussel (Pleurobema clava).</a> Canadian Journal of Fisheries and Aquatic Sciences. 73 (12), 1712-1722 (2016).</li><li>Larson, J. H., Eckert, N. L., Bartsch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+variability+in+shell+and+soft+tissue+growth+of+the+freshwater+mussel+Lampsilis+siliquoidea.">Intrinsic variability in shell and soft tissue growth of the freshwater mussel Lampsilis siliquoidea.</a> PLoS ONE. 9 (11), e112252 (2014).</li><li>Lavictoire, L., Moorkens, E., Ramsey, A. D., Sinclair, W., Sweeting, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+substrate+size+and+cleaning+regime+on+growth+and+survival+of+captive-bred+juvenile+freshwater+pearl+mussels,+Margaritifera+(Linnaeus,+1758).">Effects of substrate size and cleaning regime on growth and survival of captive-bred juvenile freshwater pearl mussels, Margaritifera (Linnaeus, 1758).</a> Hydrobiologia. 766 (1), 89-102 (2015).</li><li>Hru&#353;ka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+of+semi-natural+breeding+programme+of+freshwater+pearl+mussel+in+the+Czech+Republic.">Experience of semi-natural breeding programme of freshwater pearl mussel in the Czech Republic.</a> Die Flussperlmuschel in Europa: Bestandssituation und Schutzmassnahmen. , 69-75 (2000).</li><li>Bayne, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+components+of+growth+differences+between+individual+oysters+(Crassostrea+gigas)+and+a+comparison+with+Saccostrea+commercialis.">Physiological components of growth differences between individual oysters (Crassostrea gigas) and a comparison with Saccostrea commercialis.</a> Physiological and Biochemical Zoology. 72 (6), 705-713 (1999).</li><li>Tamayo, D., Azpeitia, K., Markaide, P., Navarro, E., Ibarrola, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+regime+modulates+physiological+processes+underlying+size+differentiation+in+juvenile+intertidal+mussels+Mytilus+galloprovincialis.">Food regime modulates physiological processes underlying size differentiation in juvenile intertidal mussels Mytilus galloprovincialis.</a> Marine Biology. 163 (6), (2016).</li></ol>

Exposure time:
Even one-month exposed mesh cages show a visible growth reflecting differences between localities (Figure 3), so they are very usable for the quick and easy detection of a locality characterization. Nevertheless, the relevance of the results depends on the short-term state of the conditions, which can oscillate. In particular, short rainfall events can play a role. In contrast, unpredictable episodic pollution may not always be recorded. In locality V ...

The exposure of experimental organisms in situ with the subsequent evaluation of their condition is one possible way to get information about the environmental quality and (especially) the site suitability for a species. Within animals, such a bioindication is applicable primarily for small invertebrates which are able to live in a limited bounded space. Young stages of bivalves (Bivalvia) are one such suitable organism group1.
Bivalves of the family Unionidae are a very important component of aquatic ecosystems2. However, these species are often critically endangered, especially in streams and rivers. Some of them are characterized as &#39;umbrella species&#39; whose conservation is closely related to the conservation of the whole stream biotope and which require a comprehensive approach3. These animals have a life cycle associated with many environment components, from water chemistry4,5 to changes in the populations of fish which serve as mussel larvae hosts6. Because mussel juveniles often represent a critical phase of the mussel life cycle, the site suitability for their development at this stage is crucial for a successful species population development in a locality.
The freshwater pearl mussel (FWPM, Margaritifera margaritifera; Unionida, Bivalvia) is a critically endangered bivalve occurring in oligotrophic European streams. Their numbers have fallen drastically during the 20th century across the occurrence area. It seems that the current decline in species reproduction in the majority of the central European populations is primarily caused by very low to zero survival of juveniles during the first few years of their life. It is assumed that juvenile FWPMs live for many years in the shallow hyporheic zone7, of which the conditions and their variability are still not well described. Moreover, until their second year of life, the juveniles only have a dimension of up to about 1 mm, so they are very difficult to find in large volumes of sediment under natural conditions8. Therefore, experiments with captive juveniles are necessary for the study of their ecology.
Within the Czech Action Plan for Freshwater Pearl Mussel9, there are thousands of juveniles rising every year from a semi-natural breeding program. Nevertheless, there is a question of which localities and habitats are suitable for successful population support by these juveniles or for eventual species reintroduction. In situ bioindications present a way of finding the answer.
Despite the fact that inconsistent survival rates of juvenile mussels in exposure cages were observed in some earlier works that questioned the suitability of juvenile mussels as bioindicators10, several recent studies have confirmed the applicability of juvenile exposure methods for water quality testing11,12,13. Additionally, it has been demonstrated that several factors need to be considered when interpreting the results of these particular studies, such as the stock origin14 and the persisting effects of larval conditions15.
The question arises of how to install experimental juveniles in tested localities and how to most effectively evaluate their condition. The first rigorous application of in situ exposure methods with juvenile FWPMs was published by Buddensiek16. Juvenile FWPM individuals were kept in sheet cages, exposed in the free-flowing water of streams, and their survival and growth were quantified after several weeks of exposure. The approach was originally developed as a semi-artificial breeding method, but the author also highlighted its applicability for the assessment of habitat requirements and water quality. Although the FWPM juvenile survival is naturally very low on a scale of months/years and only a very small number of animals will survive, the survival rate can be a good marker of the environmental effect on a scale of several weeks16. Over years of research, exposure methods were developed further to hold experimental juvenile mussel in-stream habitats and to evaluate their growth and survival rates; these include sandy boxes17, mussel silos based on an upwelling principle18, and various other exposure cages (summarized by Gum and colleagues)11. Because juveniles occur naturally in shallow hyporheic zone7, the application of experimental devices within the stream bottom is very desirable.
In our article, we describe the use of two exposure devices for FWPMs: i) modified Buddensiek sheet cages (&#34;mesh cages&#34;) also enabling bioindication testing in hyporheal conditions; and ii) Hru&#353;ka sandy boxes (&#34;sandy cages&#34;). The protocol describes the application of both methods in open water and hyporheic conditions (i.e., four variants of exposure are described). The methods were gradually modified and expanded over more than 15 years of application within the Czech Action Plan for Freshwater Pearl Mussel9 and verified by a set of experiments.

<table>
	<tbody>
		<tr>
			<td>biological material maintenance and care</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freshwater pearl mussel juveniles</td>
			<td>any</td>
			<td>NA</td>
			<td>from a FWPM breeding programme</td>
		</tr>
		<tr>
			<td>plastic boxes</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>thermobox</td>
			<td>MERCI</td>
			<td>212,070,600,030</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>field thermobox (ca25 l)</td>
			<td>any</td>
			<td>NA</td>
			<td>cold box (insulated box) commonly used for food transport</td>
		</tr>
		<tr>
			<td>river water</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic Pasteur pipettes with balloon bulb (droppers)</td>
			<td>any</td>
			<td>NA</td>
			<td>hole diameter 1 mm</td>
		</tr>
		<tr>
			<td>hydrogen peroxide</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic container (ca 50 l) for river water</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic tea strainer</td>
			<td>any</td>
			<td>NA</td>
			<td>commonly used in kitchen</td>
		</tr>
		<tr>
			<td>mesh cages construction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>main plastic bodies</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plactic covers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>special technical sieves 340 &#181;m</td>
			<td>Silk &#38;Progress</td>
			<td>UHELON 20 T</td>
			<td></td>
		</tr>
		<tr>
			<td>special technical sieves 100 &#181;m</td>
			<td>Silk &#38;Progress</td>
			<td>UHELON 67 M</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber hose (diameter&#160; 5.5 mm)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>steel bolts</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>steel nuts</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>spanner</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>steel spikes</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>pliers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>beakers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic dishes (ca. 25x15x3-5cm)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>squirt bottle</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>field protocols</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>stationery</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic container</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>string</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>hammer</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>sandy cages construction and use</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sieve 1 mm</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>sieve 2 mm</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>special technical sieves 340 &#181;m</td>
			<td>Silk &#38;Progress</td>
			<td>UHELON 20 T</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic boxes with tight-fitting lid</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>hot melt adhesive</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic box (ca 250 x 150 x 100 cm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>big plastic box (ca 25 l)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>flat stone</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>net</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>river sand</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>round containers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>magnifying glasses</td>
			<td>Carson</td>
			<td>Carson CP 60</td>
			<td>There ar many possibilities. This is one example only</td>
		</tr>
		<tr>
			<td>cages installation and maintenance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>field temperature dataloggers</td>
			<td>ONSET</td>
			<td>UA-001-64</td>
			<td>http://www.onsetcomp.com/products/data-loggers/ua-001-64</td>
		</tr>
		<tr>
			<td>spade</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>toothbrush</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>experiment evaluation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>trinocular dissecting zoom stereo microscope</td>
			<td>Bresser optic</td>
			<td>ICD 10x-160x</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>digital camera/ electronic eyepiece</td>
			<td>Bresser optic</td>
			<td>MikroCamLab 5M</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>Calibration&#160; gird</td>
			<td>Am Scope</td>
			<td>SKU: MR100</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>external power source with two movable light guides</td>
			<td>Arsenal</td>
			<td>K1309010150021</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>Image software</td>
			<td>ImageJ software</td>
			<td></td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>table processor</td>
			<td>MS excel</td>
			<td></td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
	</tbody>
</table>

bioindication testing of stream environment suitability for young freshwater pearl mussels using in situ exposure methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

The four bioindication methods (open water sandy cages, within-bed sandy cages, open water mesh cages, and within-bed mesh cages) were applied to investigate the environment condition suitability for FWPMs in the upper Vltava River Basin (Bohemian Forest, Czech Republic). This river represents one FWPM residual locality within central Europe19. Here, we present a specially selected set of results illustrating the most important aspects of the four methods. Further ...

Watch this Scientific Journal Video about Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods at JoVE.com

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

This method can help answer key questions in the river ecosystem preservation field such as indications of stages favorable for young mussels that augment as well, as prediction of this which I propagate. The main advantage of this technique is that we can test juvenile growth and survival in hyporheic conditions which represent the natural juvenile environment. Demonstrating the procedures will be my colleagues Simona Nemcikova, Jan Svanyga, and Vojtech Barak.When assembling the cages, always insert one plastic cover first, then one sheet of the plastic sieve and finally the main body on top. Put the cage into the plastic dish containing river water at the FWPM storage temperature. Ensure that the chambers are half full.Next, load the FWPMs into a petri dish. Using a squirt bottle and strainer, sift through the juveniles to clear the detritus. Then, pipet one individual from the dish and transfer it to a dish under the microscope to check it's fitness.Select only FWPM of good fitness. Take two photographs of each laid length wise at about 80 times magnification with the goal of measuring maximum shell length. Proceed to load all the appropriate chambers of the cage with the selected and documented FWPM.Then, put the plastic sieve on the cage followed by the plastic cover and secure all the parts with the nuts. To set up a mesh cage for a hyporheic zone installation, pass one of the hose ends through one of the chambers and fix it in this position. Then, take the anti-clogging mesh and bind it on the bottom end of the hose.Temporarily store the loaded cages in river water in the thermal box. For a sandy cage preparation, collect river sand and sift it through a two millimeter sieve. Then sift it through a one millimeter sieve and keep the particles between one and two millimeters.Place an empty cage in the plastic box and then load the bottom third of the cage with sand. Next, add river water until the water is 10 millimeters above the sand level. Load the sandy cage into a 25 liter water tank and let it equilibrate to the water temperature for 12 hours before loading them with FWPM.Transport the FWPM juveniles to the site in a field thermal box. At the field site, first immerse the thermal box in the river and remove the cage from the field thermal box. Now, start planning the cage positions.Choose conditions typical for FWPMs such as at the edge of the main water flow. Do not place the cage in direct water flow, nor in standing water. To install mesh cages in open water use a pair of the steel spikes.Fix the cage to the river bottom on it's side and level with the river bottom. Angle 45 degrees to the river flow downstream facing the center of the river. The lower horizontal edge should be 10 to 15 centimeters above the river bottom.To install a sandy cage in open water fasten it to a flat stone using a net and place the cage on the river bottom. Angle the larger end against the flow at a 45 degree angle. To install either cage type in the hyporheic zone dig them into the river bottom perpendicularly to the flow of water so that the upper horizontal edge of the cage is parallel to the river bottom surface and the chambers the testing depth in the river bottom.For mesh cages in the hyporheic zone pull out the rubber hose from under the bottom surface for water sampling during the experiment. After the planned exposure period, pull the cages out of the water and clear them of fine sediment, as well as of drifted material. Then, load them into a field thermal box filled with river water.Once back at the lab, evaluate the FWPM by photo documenting them as before. The described methods were used to investigate the environmental suitability of FWPMs in the upper Vltava River Basin in the Czech Republic from 2014-2015. A longitudinal river profile was represented by mainstream localities at sites A through E.Tributaries of different pollution stages were tested at sites R and V.These localities were tested using sandy cages and mesh cages in free flowing water.In addition, a gravel hyporheic zone was tested by within bed sandy cages in localities B, C, and D.The localities could be clearly distinguished based on the growth rate in the open water mesh cages despite the high within cage variability even in different growth favorable periods. In the more growth favorable exposure in 2015, the growth rate increased downstream using the mesh cages. Importantly, the survival rate was equivalently high in both seasons.By contrast, the growth rate of open water sandy cages in 2014 varied by location. The location with the greatest growth, C, was also the location of greatest growth in 2015. While growth overall was about the same in both years, mortality was higher in 2014.The within bed mesh cages showed a significant effect of the substrate composition on mortality. Oxygen saturated stony bottoms had a survival rate close to 100%and poorly oxygenated sand had high mortality. After watching this video, you should have a good understanding of how to install experimental mussel juveniles in the riverside to test local conditions for day

bioindication-testing-stream-environment-suitability-for-young

Research

JoVE Journal

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster (Nephrops norvegicus)

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to individually track cohoused animals. We tested the tracking routine with four cohoused Norway lobsters (Nephrops norvegicus) under light-darkness conditions for 5 days. The lobsters had been individually tagged. The experimental setup and the tracking techniques used are entirely based on the open source software. The comparison of the tracking output with a manual detection indicates that the lobsters were correctly detected 69% of the times. Among the correctly detected lobsters, their individual tags were correctly identified 89.5% of the times. Considering the frame rate used in the protocol and the movement rate of lobsters, the performance of the video tracking has a good quality, and the representative results support the validity of the protocol in producing valuable data for research needs (individual space occupancy or locomotor activity patterns). The protocol presented here can be easily customized and is, hence, transferable to other species where the individual tracking of specimens in a group can be valuable for answering research questions.

Long-term Video Tracking of Cohoused Aquatic Animals: A Case Study of the Daily Locomotor Activity of the Norway Lobster Nephrops norvegicus

Here we present a protocol to individually track animals over a long period of time. It uses computer vision methods to identify a set of manually constructed tags by using a group of lobsters as case study, simultaneously providing information on how to house, manipulate, and mark the lobsters.

We present a protocol related to a video-tracking technique based on the background subtraction and image thresholding that makes it possible to ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...