Fluorescent dyes, particularly those which are non-toxic and not visible, are often added to water systems to provide water discharge and velocity data. Dye tracing studies can also provide useful information for modeling surface and groundwater systems in addition to tracing contaminants. Fluorometers allow researchers to measure ultra-low concentrations of dye. With extreme sensitivity and instrument flexibility researchers can glean powerful data that could potentially be missed when using less sensitive methods.
Fluorescent tracer dyes provide an accurate, cost effective method for measuring water flow levels, mixing zones, time of travel, groundwater transport, leak detection, retention times, etc. Fluorometers are available for common fluorescent dyes such as Rhodamine, Fluorescein, and PTSA, as well as for exotic and custom dye blends that may have unique properties that are beneficial to a specific environment or study. There are several articles that we have that discuss the dye tracing application in depth.
General Overview
Our applicational notes here:
Fluorescent Tracer Studies (Overview of applications)
A Practical Guide to Flow Measurement
Fluorometry in the Water Pollution Control Plant
Flow Measurements in Sanitary Sewers by Dye Dilution
Rhodamine WT
Preparation of Standards for Dye Studies Using Rhodamine WT
Rhodamine WT Dye Worksheet: Amount of Dye and Injection Rate
Using the Turner Designs Model 10AU Fluorometer to Perform Flow Measurements in Sanitary Sewers by Dye Dilution
Fluorescent Tracer Dyes
Fluorescein Dye
PTSA Dye
From A Practical Guide to Flow Measurement, paragraph snippets
MEASUREMENT TECHNIQUES
1. Sample Characteristics
Certain factors may affect the measurement of the sample. The readout of the fluorometer is proportional to a linear concentration from the smallest detectable concentration to about 0.1 parts per million. (See section J on Dyes for further details.) As the concentration is further increased, the readout rises at a decreasing rate, and eventually reverses at a very high concentration. There is no possibility of confusion. At the point where readings become nonlinear, the sample will have a faint but definite pink color when viewed through a one-inch test tube. If you can’t see the pink, it is definitely linear.
At concentrations below 0.1 parts per million, a single-point calibration (or any known lower concentration) may be used to calibrate the instrument because if one sensitivity range is calibrated, all are calibrated. For concentrations between 0.1-0.5 parts per million, a multi-point calibration curve is used or the sample is diluted. Above 0.3 parts per million, dilution will be more accurate. Above 0.5 parts per million, dilute the sample before calibration.
Any other material in the sample that absorbs light will give a reduced reading: dissolved colored material or suspended solids, for example. However, suspended solids that are light in color reflect rather than absorb the green light used for excitation, and may not affect readings at all, even at quite high levels of turbidity. If an effect is suspected, it is easily checked and a correction applied (see section G3 on Turbidity).
The one slight drawback to the fluorometry method is the problem of temperature. All the dyes are inversely affected by temperature: increasing the sample temperature reduces fluorescence, and decreasing temperature increases fluorescence. One can either control the sample temperature (which is easy in a laboratory) or one can correct for it. Because the correction is independent of concentration, it isn’t difficult. Even controlling the temperature in the field is feasible. Simply hang the standard (carefully sealed) over the side of the boat in the water until sample and standard are the same temperature.
Temperature effects on dyes are already widely recognized. Field measurement of discharge rate, as routinely practiced, can also be affected by temperature, and accuracy can be improved substantially. Some of the variations assumed to be due to uncontrollable or unknown effects are due to temperature. The optical filters used in all filter fluorometers show an inverse change in characteristics as a function of temperature calibration is strictly valid only if the ambient temperature is the same and the instrument has been allowed to come to full operating temperature. Depending on the type of photomultiplier tube and the choice of optical filters, the observed change is - 0.15% to -0.33 % in o F. What is important is filter temperature rather than ambient temperature. The Turner Designs Model 10 Series Fluorometer has a uniquely low filter temperature rise (above ambient), but no definite figure can be given because factors such as wind, flowing sample, and direct sunlight will affect this rise. Conveniently, however, the front panel temperature is nearly the same as the filter temperature. This refinement may also explain the perplexing, occasionally reported recoveries of dye in excess of 100%.
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Rhodamine WT is related to rhodamine B, a tracer in common use in the sixties. It was developed to overcome a disadvantage of rhodamine B: absorption on suspended sediment. The same modification was expected to reduce toxicity, and limited testing has confirmed this...
While the EPA has sole responsibility for identifying those substances that may be used as tracers (36), the Food and Drug Administration (FDA) does issue policy statements. The FDA issued such a policy statement on April 22, 1966 concerning rhodamine B (37). A temporary tolerance limit for ingestion of rhodamine B was set at 0.75 mg per day. Based on normally expected water consumption, the tolerance would not be exceeded unless the concentration approaches 370 parts per billion. Noting that 30 ppb may be detected visually in a glass of water, and 10 ppb is visible in a larger volume, such as a clear reservoir, the FDA pointed out that if the dye is not visible, the tolerance would not be exceeded. The USGS, a large user of fluorescent-dye tracers, directed that the concentration should not exceed 10 ppb at the intake of a water supply (15). The visual and instrumental detectability of rhodamine WT, based on active ingredient, is about the same as rhodamine B (rhodamine WT is supplied as a 20% aqueous solution).
The Criteria and Standards Division (Office of Drinking Water) has reviewed the available data on chemistry and toxicity of Rhodamine dyes. We would not anticipate any adverse health effects resulting from the use of Rhodamine WT as a fluorescent tracer in water flow studies when used with the following guidelines.
- A maximum concentration of 100 micrograms/liter Rhodamine WT is recommended for addition to raw water in hydrological studies involving surface and ground waters.
- Dye concentration should be limited to 10 micrograms/liter in raw water when used as a tracer in or around drinking water intakes.
- Concentration in drinking water should not exceed 0.1 micrograms/liter. Studies which result in actual human exposure to the dye via drinking water must be brief and infrequent. This level is not acceptable for chronic human exposure.
- In all of the above cases, the actual concentration used should not exceed the amount required for reasonably certain detection of the dye as required to accomplish the intended purpose of the study.
The Criteria and Standards Division recommends that Rhodamine B not be used as a tracer dye in water flow studies.
This advisory supersedes all earlier advisories issued by EPA on the use of fluorescent dyes as tracers in water flow studies. This advisory is granted on a temporary basis only.
EPA is terminating its voluntary additives advisory program as announced in the Federal Register (53 FR, 25586, July 7, 1988). A copy of the Federal Register Notice is enclosed for your convenience. All EPA advisory opinions issued within the framework of the additives program will expire on April 7, 1990.
Our opinion concerning the safety of this tracer dye does not constitute an endorsement, nor does it relate to its effectiveness for the intended use. If this letter is to be used in any way, we require it to be quoted in its entirety.
Flow Measurements in Sanitary Sewers by Dye Dilution
Paragraph snippets.
Things We've Learned the Hard Way!
Dye should not be allowed to free fall for more than about one or two feet, or splashing and erratic results may occur.
If you are using a small injection system, place reservoir, pump, and battery in a box with a yoke on it, and suspend it by rope from street level. Tie a knot in the rope, and you can close the manhole for the duration of the test.
Do not attempt to use a long tube, at low flow rates from street level. The dye flow will surge, and the erratic suction on the pump exhaust line will cause erratic results.
In fact, long tubes are to be discouraged from another standpoint. Flexible vinyl tubing (e.g., Tygon©) is convenient, but it does adsorb dye reversibly. It does have a limited capacity for adsorbing dye and at the concentrations injected, equilibrium is rapidly achieved. A few feet is no problem. A hundred feet is unacceptable. Polyethylene, polypropylene, and unplasticized vinyl tubing do not adsorb, but are fairly rigid.
If you must inject from street level, use an appropriate length of PVC pipe with a tee on the top. Insert the tube from your injection pump well down the pipe or allow the stream from your dye injector to fall in the top, clearly missing the wall until well down. Run water from a hose through the tee. This is a particularly valuable technique for injecting dye through a pump directly into the bell housing of a pump, or for other situations where the desired point of injection is not directly below an opening. The PVC pipe is flexible and easily maneuvered. The top is easily secured to a ladder laid across the manhole, etc.
If the amount of water added in injecting the dye is substantial compared to the flow being measured, consider correcting for the added water. In infiltration studies, it will normally be a constant increase in baseline, and therefore can be ignored. In calibrating a flow meter, the added flow should be subtracted from the measured flow for comparison with an upstream flow meter. IF the flow meter is downstream, there will be no correction, as the flow meter is reading the total flow.
Where Should the Dye be Injected?
The dye injection and dye sampling points must be considered together. The important considerations are:
The dye must be injected sufficiently far from the sampling point so that it is completely mixed with the sewage before samples are taken. This may easily be checked. See SAMPLING -- Is Mixing Adequate? The more turbulent the flow, the better. A pass through a lift pump virtually assures mixing. Even under relatively smooth flow conditions, expect good mixing 100 to 200 pipe diameters down stream. Avoid situations where there is a reservoir between the injection and sampling points. It will increase the time required to make a test.
If flow increases downstream, dye dilution techniques measure the total flow at the point where the sample is taken! If there is infiltration or a branch entering between the injection and sampling point, you will be measuring the total flow at the point where the samples are taken. Exfiltration is a special situation. Assuming complete mixing prior to loss of flow, the flow measured is that prior to the diversion. To quantitate such situations requires moving the injection site as well as the sample site.
Convenience should be considered, particularly at the sampling point. Certainly, the first time around you will wish to take a fair number of samples.
Preparation of Standards for Dye Studies Using Rhodamine WT
Paragraph Snippets
MAKING STANDARD DILUTIONS
For a standard, you need any concentration no greater than the linear range of your tracer (for the rhodamine dyes, approximately 100-ppb (0.1-ppm; 100- micrograms/liter) active ingredient.
To obtain this concentration, you will make serial dilutions. By this, we mean you take your concentrated solution and make a dilution of it. You mix it thoroughly, then make a dilution of that, and so on, until the desired concentration is obtained.
We recommend preparing a higher concentration, i.e., around 0.1-ppm (100-ppb or 10^-7) active ingredient. At these high levels (high for fluorescence), contamination will be less of a problem. Contamination from dirt or other things is not a problem, but spurious tracer could be. In preparing the standards, you are handling the pure material and high concentrations, and it is safer to use the highest standard that is convenient.
Note that the dilutions you are after can be achieved in a variety of ways. The easiest way is with 1- and 10-ml pipettors, and a choice of 100-ml and 1-liter flasks. If intermediate concentrations are desired, use an adjustable pipettor, or pipette several shots into one flask, or use intermediate-size flasks (they are available in 200-, 250-, and 500-ml sizes). Generally, all you are after is some concentration not greater than 500 ppb (0.5 ppm)--or 100 ppb (0.1 ppm) if dealing in active ingredient. Since readings are proportional to concentration at or below this point, it is simply a question of convenience.
Don’t use all clean water. Your last dilution should always be done twice, once in distilled water, and once in the water in which the measurement will be made. This is because sometimes there are substances in the test water that interfere with the reading. This doesn’t happen often, but it can invalidate your readings if you don’t recognize it.
Your standard will be the dilution in the system water, but first you need to see that it reads the same as the dilution in distilled water -- or make sure you understand any difference.
A. To Prepare a 100-ppb (active ingredient) standard of rhodamine WT (20% solution):
First, prepare a 100-ppb standard of rhodamine WT (20% solution) PN 10-108 fold dilution by weight. (See section MEASURING BY WEIGHT OR VOLUME for an explanation.) Using an accurate laboratory scale, weigh 1 gram of dye directly into a 100-ml volumetric flask. The dye may be dripped into the flask with a pipette until 1 gram is obtained. Then dilute to the mark with distilled water. You now have a 10-g/liter (10 ppt, 10^-2) concentration of your tracer.
Note 1: You could obtain the same - concentration by weighing 10 g into a 1-liter flask, or 20 g into a 2-liter flask.
Note 2: If you intend to inject dye by volume, then pipette 1 ml of dye into a 100-ml volumetric flask and dilute to the mark with distilled water. Or measure 10 ml of dye into a 10-ml volumetric flask and rinse into a 1-liter flask. Then, dilute to the mark with distilled water. This will yield a 10-ml/liter (10-ppt, 10^-2) dilution. (Keep in mind the specific gravity factor. See MEASURING BY WEIGHT OR - VOLUME, above.)Next, pipette 1 ml (or weigh 1 gram) of the dilution in #1 (10^-2 or 10 ppt) into a clean 100- ml volumetric flask and dilute to the mark with distilled water. Mix thoroughly. You now have a 10^-4, or 100-ppm, dilution.
Now, pipette 5 ml (or weigh 5 grams) of the dilution in #2 (10^-4 or 100 ppm) into a clean 1- liter volumetric flask and fill to the mark with system water. Mix thoroughly. You now have a 10^-7 (or 100-ppb; 0.1-ppm) active ingredient standard.
Note: We measured 5 ml because rhodamine WT comes as a 20% solution (meaning 20% active ingredient). If you are not concerned with active ingredient, then diluting 1 ml 1000- fold yields a 100-ppb dilution of tracer (or 20- ppb active ingredient).Repeat step 3, using distilled water. Compare fluorometer readings of this dilution with that of #3.
Amount of Dye and Injection Rate
For the amount of dye to use and the injection rate, we must lay down some basic information. This can be used for Rhodamine WT, Fluorescein, or PTSA. In the following example, this uses Rhodamine WT.
Unit Conversions: 1 gal/day = 2.63 ml/min
Pure dye = 1,000,000,000 ppb (parts per billion)
Flow Equation: Q = qC/c
Where: q = flow of the plant
q = flow of the dye
C = concentration of the dye injected
c = concentration of the dye at the measurement point
Question: How much dye do I need for a 24-hour study of a 20 MGD (million gallons per day) plant?
Answer: Given that you can see 100 ppb of dye in the plant water, the flow equation tells us that: q = (100 ppb x 20,000,000 gal)/day 1,000,000,000 ppb
q = 2 gallons/day
Question: What should be my dye injection rate for this application? My pump has a range of 4.8 to 48 ml/min.
Answer: For a 20 MGD operation, we found that the injection rate was 2 gallons/day. Using the 2.63 ml/min = 1 gal/day relationship, we find that the final injection rate should be 2 x 2.63 or 5.26 ml/min. However, if we want to measure the flow in a 10 MGD operation, we will need to perform a 1:1 dilution of the Rhodamine WT dye and still inject 5.26 ml/min. because the calculated injection rate of 2.63 ml/min. falls outside of the range of the pump.
Note: Although the concentration of the pure dye is not 100% Rhodamine WT, we can still treat the concentration as 1,000,000,000 ppb because the diluted reading of 100 ppb is relative to the initial dye concentration. That is, any error introduced will be cancelled out. Only the ratio of C/c is important.