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Turner Designs News, March 2005

Note from the Director
Instruments In Action: Center for Embedded Network Sensing (CENS) Using Cyclops-7 Sensors for Algae Bloom Monitoring
Instruments In Action:
Bacterial Source Tracking in Puerto Rico with the 10AU
Instruments In Action: Cornell Researchers use AquaFluor to Measure Ammonia Extraction by Tropical Fish
Jim's Corner: Dye Selection for Groundwater Studies & The Use of Multiple Dyes
Technically Speaking: Quantitative Results with in vivo Chlorophyll Measurements

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Note From the Director

Thank you for taking the time to read the latest edition of TD News. In this issue you will see articles on a wide variety of fluorescence applications you may not be familiar with. Please take some time to learn about the multiple uses of fluorometers in order to make the most of your instrument and to discover unrealized possibilities of fluorescence sensors. In recent years we have developed sensors for new applications such as; sensors for the detection of optical brighteners to detect leaking septic tanks in coastal waters, detection of cyanobacterial (blue-green algae) pigments, and a sensitive ammonium technique. In most cases customers can choose from our entire product line for any one of these applications. In addition to the articles in this newsletter, real-world examples of Turner Designs instruments being used for a wide range of applications can be found at our Data Bank site. We feel strongly that presenting real-world examples and data is the most effective marketing strategy for our customer base.

I hope you enjoy this edition of the TD Newsletter. We are always interested in hearing from you; please do not hesitate to contact us with feedback on the newsletter or our products and services


Yours truly,
Rob Ellison
Director of Sales and Marketing

fluorometer

Center for Embedded Networked Sensing (CENS) Using Cyclops-7 Fluorometer in Monitoring of Algal Blooms

About CENS:
CENS, a NSF Science & Technology Center, is developing Embedded Networked Sensing Systems and applying this revolutionary technology to critical scientific and social applications. Like the Internet, these large-scale, distributed, systems, composed of smart sensors and actuators embedded in the physical world, will eventually infuse the entire world, but at a physical level instead of virtual.

Project Overview:
A specific area in which CENS technology is being applied is the monitoring of harmful algal blooms (HABs). A variety of naturally-occurring and introduced microorganisms adversely impact marine ecosystems and uses of marine resources. They can affect human health, fisheries and even tourism. However, conditions under which HABs develop are not well understood, and methods for detecting microorganisms are too slow and complex for timely intervention. With the development of technology, sensor networks provide a method to monitor the microorganisms in real time and solve the problem. The goal of this project is to deploy large numbers of sensors and robots operating in a semi-autonomous but coordinated fashion in the marine environment (Figure 1). The system should be able to follow, identify and investigate the behavior of microorganisms in situ and in real time.

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Figure 1: (center) Schematic of autonomous, coordinated network of mobile sensors. (upper left) Testing of wireless communication between nodes in tank testbed. (upper right) Autonomous, mobile node equipped with computer-controlled mobility, communications, sampling system, and Turner Designs Cyclops-7 fluorometer. (center bottom) Submersible node to be used as a data mule or for obtaining vertical profiles.

Approach:
We are studying the dynamics of blooms of the alga Aureococcus anophagefferens in the waters off Long Island, NY. Existing monitoring efforts are time-consuming and involve manual sampling and analysis. We are constructing a network of sensors and samplers, consisting of both stationary and mobile “nodes,” to allow for spatially- and temporally-rigorous monitoring and adaptive sampling. For example, changes in temperature or chlorophyll fluorescence can serve as triggers for sampling (Figure 2).

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Figure 2: Concentration of Aureococcus anophagefferens (BT) and temperature with depth in a column testbed. Note the decrease in BT concentration (black profile) around the point of the thermocline (gray profile).

The stationary nodes consist of temperature and salinity sensors, Turner Designs Cyclops-7 chlorophyll a fluorometers, and associated processors and communications equipment (Figure 3).


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Figure 3: Schematic of stationary node components.

These environmental sensors measure at a high frequency and signal bloom events (e.g. elevated chlorophyll levels). We are then be able to deploy the more complex mobile node (Figure 4) to make more sophisticated measurements and retrieve relevant samples.

fluorometer

Figure 4: Mobile node equipped with wireless communication, computer-controlled mobility and navigation, sampling system, and Turner Designs Cyclops-7 fluorometer.

The Turner Designs Cyclops-7 chlorophyll fluorometer is an integral part of the network of sensors. As a proxy for photosynthetic biomass, chlorophyll measurement is one of the most biologically-significant parameters to measure in marine systems. Previous blooms of A. anophagefferens have shown chlorophyll a levels between 5 and 50 ìg/L, although higher values are possible. We chose to use the Turner Designs Cyclops-7 fluorometer because of its flexible sensitivity ranges, low power requirements and small size.

Further information:
For additional information on this project, please contact:

Beth Stauffer
Research Lab Technician
Dept of Biological Sciences
University of Southern California
Phone: (213) 821-2123
E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it
URL: www.usc.edu

Or:

Carl Oberg
Engineering Technician
University of Southern California
Computer Science
SAL 206
941 West 37th Place
Los Angeles, California 90089-0781
Phone: (213) 740-0541
E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

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Bacterial Source Tracking in Puerto Rico with Turner Fluorometers

This January we brought two Turner 10-AU field fluorometers to southwestern Puerto Rico to see how well they worked for bacterial source tracking. Bacterial source tracking tries to identify sources of fecal contamination using a variety of phenotypic, genotypic, and chemical methods. We wanted to use the fluorometers to detect optical brighteners- the colorless, fluorescent dyes in laundry detergents that make clothing "whiter than white." Because laundry detergent residues are often associated with human sewage, the combination of high fecal bacterial counts and the presence of optical brighteners in surface waters suggests that human fecal contamination is present (Table 1)

Bacterial Count
Optical Brightener
Likely Result
High
 
High
 
Malfunctioning septic system or leaking sewer pipe
High
 
 
Low
Non-human warm-blooded animals
 
Low
High
 
Gray water in storm water system
 
Low
 
Low
No evidence of fecal contamination at the time

Table 1. Four possible outcomes when fluorometry for optical brighteners is combined with counts of fecal indicator bacteria.

To detect human fecal contamination, we combined field fluorometry with targeted sampling for Escherichia coli, a bacterium widely used as an indicator of fecal contamination. Targeted sampling is much like the children's game of "hot and cold," and requires sampling and resampling for fecal bacteria until a persistent source of the bacteria is identified (Kuntz et al., 2003). We used the IDEXX Colilert system to identify high numbers of E. coli.

fluorometer

This was our first study to combine fluorometry and targeted sampling beyond our preliminary work on the Georgia coast (Gates et al., 2004). Overall, the system seemed to work well. For example, we observed high counts of E. coli and high fluorometric signals in the Yaguez River, which flows through the city of Mayagüez. We are confirming these data with PCR analysis for the presence of human virulence factor. The clear waters in Puerto Rico were an ideal testing ground for fluorometry because organic matter didn't interfere with the fluorometric signal. We are continuing to develop the combination of fluorometry and targeted sampling for Georgia waters, where high amounts of organic matter in the water do interfere with the fluorometric signal. However, for the moment, the data are encouraging for places like Puerto Rico.

fluorometer

References:
Kuntz, R. L., P. G. Hartel, D. G. Godfrey, J. L. McDonald, K. W. Gates, and W. I. Segars. 2003. Targeted sampling protocol with Enterococcus faecalis for bacterial source tracking. J. Environ. Qual. 32:2311-2318.

Gates, P., P. Hartel, K. Payne, J. McDonald, K. Austin, K. Rodgers, J. Fisher, S.N.J. Hemmings, and L. Gentit. 2004. Combining targeted sampling and bacterial source tracking to determine sources of fecal contamination to the south beach of Sea Island during calm and stormy conditions. Sea Island Company. 12 p.

fluorometer

Cornell Researchers Use Aquafluor Fluorometer To Measure Ammonia Excretion By Tropical Fish

Pete McIntyre, a researcher at Cornell University, selected a Turner Designs Aquafluor fluorometer to measure ammonia in water as part of a research project investigating the impacts tropical freshwater fish have on nutrient cycling in their ecosystem.

Application Introduction
Tropical fishes are renowned for their species diversity, interesting behaviors, and beautiful coloration. Most people associate tropical fish primarily with coral reefs, but species inhabiting tropical freshwaters account for 20-25% of the world’s total fish species diversity! Despite their impressive diversity, scientists are only beginning to understand the ways in which these fishes affect the functioning of tropical freshwater ecosystems.

There is a growing list of threats to tropical freshwater fish diversity, including overfishing, habitat degradation, introduced species, and river impoundments. At the same time, the number of humans dependent upon the rich fisheries of tropical rivers and lakes grows every year. These shifts raise critical question about whether every species plays a unique role in its ecosystem, or instead most species are equivalent in their functional roles.

Application Objectives
Pete McIntyre’s research addresses the functional contributions of tropical fishes to their ecosystems through nutrient recycling. Biologists have long recognized that animals are not very efficient at retaining the nutrients in their food, and some of these nutrients are returned to the environment in forms that are readily available to fuel new productivity of plants. In the case of fishes, most species excrete a substantial proportion of their dietary nitrogen as ammonia (NH3) that is released continuously across the gills. In ecosystems where nitrogen availability limits the productivity of algae, the recycling of dietary nitrogen by fishes could be a critical part of the nutrient cycle.

Application Results
In collaboration with Alex Flecker (Cornell University) and Mike Vanni (Miami University, Ohio), he studied the excretion of NH3 and dissolved phosphorus by fishes in a piedmont river in Venezuela. This site is home to around 80 species of fishes, including a variety of catfishes and tetras. They are investigating the determinants of nitrogen and phosphorus recycling rates, including species identity, body size, body composition, and diet. Their work has revealed great variation among species, much of which is explained by body size and composition (see Vanni et al. 2002. Ecology Letters 5: 285-293).

Their field site is not linked into a power grid, so they have always had to rely on portable field equipment that requires little power. For the last two years, they have been using a Turner Designs Aquafluor handheld fluorometer to obtain high-resolution NH3-N data in the field using only battery power. Using the OPA detection method developed by Holmes et al (1999), they have been very pleased with the instrument’s precision and linearity up to ~90 µg N/L when checked against a Turner 10-AU or autoanalyzer. Others in their research team have been equally pleased with the accuracy of low-level measurements (1-8 µg NH3-N/L) taken with the Aquafluor. The Aquafluor’s portability for air-travel and battery power for fieldwork made it a critical part of his research in South America and Africa.

They have now expanded their Venezuelan project to measure NH4 recycling by almost 50 species of fishes, and the results indicate that fishes play a critical role in quickly regenerating nutrients in this N-limited ecosystem. This information is now being combined with surveys of the fish community to determine the importance of individual species in the ecosystem.

fluorometer

We collect fish from riffles by stirring the rocks under which they hide in Rio Las Marias, Venezuela. By exhaustively collecting from many such quadrats, we can estimate the typical density of benthic fishes in the river.

fluorometer

Dozens of species of fish live in small riffles like this one. Some species feed on algae growing on the rocks, others eat the riparian vegetation, and many hunt for insects and crustaceans.

fluorometer

We catch fish from the river and incubate them in bags of water to measure their nutrient excretion.

For further information, please visit:
http://www.eeb.cornell.edu/flecker/flecker.html
http://www.eeb.cornell.edu/

Dye Selection for Groundwater Studies & The Use of Multiple Dyes

Question:
What is the best type of fluorescent tracer dye to use for groundwater studies, and can more than one dye be used simultaneously?

Answer:
Rhodamine and Uranine (Fluorescein) are popular fluorescent dyes that have been used for groundwater tracing for over 20 years. The paper at the link below, discusses the properties of the dye and the composition of the ground media to help select the best dye to use to avoid any sorption effects that can retard the dye movement. Dye Properties & Ground Media Paper

There can be advantages in using Rhodamine and Uranine dye at the same time. For example, each dye can be deposited at a different location (well site) and then a fluorometer is used to monitor at a sampling well to determine whether either or both dyes is present. The fluorometer has to be set up with both the Rhodamine and Fluorescein optics, to detect the respective dye. You should expect about 5% overlap on the detection. As an example, if you measured a sample that only contains a 100 ppb concentration of Rhodamine dye on the fluorometer that is configured for Fluorescein, then you should expect to get a reading of approxomately 5 ppb. The same is true for a Uranine sample read on the fluorometer that is configured for Rhodamine.

As long as one dye is not over 10 times the concentration of the other, the reading of a sample containing both dyes can be mathematically corrected to derive at the actual concentration, if quantitative accuracy is required. The majority of groundwater tracer tests, are only interested in determining the time it took for a given dye to be detected in the sampling well and are not concerned with the quantitative accuracy.

 

fluorometer

fluorometerTechnically Speaking, It All Adds Up…….

is a series of articles for people who want to obtain the best possible results from their fluorometer. This month's article will provide information on getting to quantitative results, (absolute values) when making in vivo chlorophyll measurements.

When you consider in vivo chlorophyll measurements, you need to remember that this is a relative measure of algal biomass. If you require quantitative data of in vivo Chlor a, there are several factors that can create a challenge. These factors include variations in the species, physiological effects, ambient light impacts on fluorescence, and the presence of interfering compounds such as dissolved organic matter. (see Guide to In Vivo Chlorophyll Measurement to learn more):

These factors mean that ideally, each sensor would be calibrated before being deployed, (pre-calibration) using the actual water to be measured., Normally, it is not practical for the user to perform an initial primary calibration with natural water samples containing Chlor a concentrations that have been precisely determined. Routinely, a "post calibration" method is performed, where the user performs extractions of the chlorophyll a from water samples collected during the field work. The extracted samples are performed in the Lab as described in the EPA methods, see link below. The extracted chlorophyll a results are then correlated with the in vivo value for a given water sample.

Therefore, before heading into the field to perform in vivo Chlor a sampling with a fluorometer, the user only needs to perform a calibration with a secondary standard, such as Turner Designs solid standards or a fluorescent dye solution. The same secondary standard can later be used to check the instrument performance, and can be used to check multiple instruments at varying times. Using the secondary standard with multiple sensors would ensure consistent and repeatable readings across the instruments. If a digital instrument is being used, a relative value can be assigned to the solid standard during the calibration, such as 100, and this can be checked at different times to check for biofouling, or instrument drift. When using analog sensors, the user would simply note the signal level with the secondary standard installed.

The blank level should also be noted before deployment. The best blank solution is filtered sample water. This will remove all algal cells, but dissolved material that can cause some interference to the fluorescent reading will stay in solution and thus be corrected for by noting the blank level.

With the performance check and blanking complete, the sensor can be deployed. At the time of deployment and at regular intervals during deployment, water samples should be collected. At the time of collection, the in vivo fluorescence of the same water sample needs to be recorded.

NOTE: If you are using a submersible fluorometer, and plan to capture and read samples in a small container, then care needs to be taken to ensure the container itself is not interfering with the reading by a) placing the sensor too close to the bottom or sides or b) the container itself is not fluorescing or reflecting light. Refer to the instrument's User Manual for details.

Once the in vivo reading of the sample has been recorded, some of the water needs to be processed for extracted chlorophyll analysis. This is most commonly done through fluorometry, spectrophotometry, or HPLC analysis. Refer to EPA methods 445,446 and 447 respectively. After the quantitative chlorophyll a concentration has been determined through extracted analysis, the in vivo and extracted values of a given water sample are used to develop a correlation, see graph below.

This is done for all water samples collected in a given environment (different correlations should be developed for different bodies of water), and the average correlation for a given environment is used to correct all of the in vivo data for that region. This is how in vivo fluorometer data is best handled for good quantitative estimates of the Chlor a.


EPA link = http://www.epa.gov/nerlcwww/ordmeth.htm

fluorometer
Figure 1: Once the graph has been generated, it can be used to obtain quantitative results for all the in vivo fluorometer data.