sustainability
Article Quantitative Analysis and Correction of Temperature Effects on Fluorescent Tracer Concentration Measurement
Zhihong Zhang 1,2, Heping Zhu 2,* and Huseyin Guler 3 1 College of Agriculture and Food, Kunming University of Science and Technology, Kunming 650500, China; [email protected] 2 USDA-ARS Application Technology Research Unit, Wooster, OH 44691, USA 3 Department of Agricultural Engineering and Technology, Ege University, 35040 Izmir, Turkey; [email protected] * Correspondence: [email protected]; Tel.: +1-330-263-3871
Received: 20 March 2020; Accepted: 27 May 2020; Published: 2 June 2020
Abstract: To ensure an accurate evaluation of pesticide spray application efficiency and pesticide mixture uniformity, reliable and accurate measurements of fluorescence concentrations in spray solutions are critical. The objectives of this research were to examine the effects of solution temperature on measured concentrations of fluorescent tracers as the simulated pesticides and to develop models to correct the deviation of measurements caused by temperature variations. Fluorescent tracers (Brilliant Sulfaflavine (BSF), Eosin, Fluorescein sodium salt) were selected for tests with the solution temperatures ranging from 10.0 ◦C to 45.0 ◦C. The results showed that the measured concentrations of BSF decreased as the solution temperature increased, and the decrement rate was high at the beginning and then slowed down and tended to become constant. In contrast, the concentrations of Eosin decreased slowly at the beginning and then noticeably increased as temperatures increased. On the other hand, the concentrations of Fluorescein sodium salt had little variations with its solution temperature. To ensure the measurement accuracy, correction models were developed using the response surface methodology to numerically correct the measured concentration errors due to variations with the solution temperature. Corrected concentrations using the models agreed well with the actual concentrations, and the overall relative errors were reduced from 42.36% to 2.91% for BSF, 11.72% to 1.55% for Eosin, and 2.68% to 1.17% for Fluorescein sodium salt. Thus, this approach can be used to improve pesticide sprayer performances by accurately quantifying droplet deposits on target crops and off-target areas.
Keywords: agricultural sprayer; spray deposition and drift; fluorescent tracer; pesticides; concentration measurement; mixing uniformity; correction model; BSF; Eosin; Fluorescein sodium salt
1. Introduction Applications of pesticide have ensured a noticeable increase in crop yields and high-quality food production [1–4]. However, excessive pesticide use due to improper spray applications has caused great concerns on risks to health and damage to the environment and sensitive ecosystems. To reduce pesticide waste, the performances of agricultural sprayers need to be evaluated adequately and accurately [5,6]. Fluorescent tracers have been widely used to simulate pesticides in field tests, including the determination of pesticide spray distribution, deposition, and drift [7–15], and the assistance of sprayer design and improvement [16–20]. This is because fluorescent tracers are relatively highly sensitive, economical, practical, and non-poisonous compared to the analysis of active ingredients in pesticides [21–24]. During the field spray tests, fluorescent tracer is usually
Sustainability 2020, 12, 4501; doi:10.3390/su12114501 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 4501 2 of 15 mixed in water first, and then the spray solution is discharged as spray droplets in a cloud to the plants. Some droplets deposit in the target area while some reach the ground or drift in the air. Collecting spray samples from target and off-target areas and quantifying the fluorescent tracers on the samples can help determine sprayer performances. The quantification of fluorescent tracers is processed by washing tracers off the samples with a given amount of distilled water, and then using a fluorometer to measure the fluorescent concentration in the washed solution. Because the fluorescent tracer concentration in the spray solution is a pre-determined constant, the amount of spray deposits on the samples can be calculated from the amount of fluorescent tracers measured. For techniques associated with injection spray, the mixing of pesticide and diluent in the line to the nozzles should be adequate to make sure mixtures are uniform. Hence, quantifying mixing uniformity has been identified as an important subject for evaluating sprayer injection systems [25–27]. One effective method for performance evaluation utilizes fluorescent tracers which are injected into the carrier stream. The resulting concentrations of mixture are then estimated based on fluorescent analysis by fluorometers [28,29]. However, there are rising concerns about the measurement accuracy due to the stability of fluorescence during experiments. Various factors can cause a severe inaccuracy of fluorescence analysis. Considerable investigations have been reported on degradation by solar radiation and photodegradation of commonly used fluorescent tracers, which can result in a severe deviation of the test results [30–32]. The fluorescent strength of some fluorescent tracers behaved differently in alkaline or acidic solutions. For example, the fluorescence of Brilliant Sulfaflavine (BSF) and Eosin remained nearly constant over the solution pH range of 6.9–10.4; however, Fluorescein had much higher fluorescent sensitivity than BSF and Eosin [33,34]. The variation of solution temperature is another critical factor that can influence fluorescent measurement accuracy. For this reason, some fluorometer manufacturers suggest to keep solutions at stable ambient temperatures, but little information is available on how the temperature can affect fluorometer measurements. For example, Trilogy Laboratory Fluorometer specifies that the operating temperature for its fluorometer should be kept in the range of 15–40 ◦C, and recalibration is needed if the ambient temperature changes by 10 C[35]. In an actual experimental scenario, it is found that the effects of temperature on the ± ◦ deviation of fluorescent analysis varies with the type of fluorescent substances. For some types of fluorescent tracers, temperature effects on the concentration measurement have been underestimated. Taking BSF as an example, a slight variation in the solution temperature can lead to significant measurement errors by a fluorometer. However, there are few reports available about the effect of temperature on the measured concentration of fluorescent tracers commonly used in the evaluation of pesticide spray application efficiency and spray mixture uniformity. Evaluating the effects of temperature on the measurement accuracy of fluorescent tracers is of importance, because it is difficult to maintain a constant solution temperature under certain analytical conditions. For example, some tests must be conducted with field in-situ measurements of spray deposition. For the field tests, the instruments generally operate in a wide range of outdoor temperatures, and the solution temperature also varies with the environment. Similarly, for indoor laboratory experiments, samples for measurements also have the temperature variation problem, which can be significantly lower or higher than the desired temperature. Therefore, the influence of the variation of temperature on the accuracy of fluorescent tracer analysis should be determined before and after the tests. Furthermore, it is not only important to know the temperature dependence of the fluorescent tracers, but also to propose a method that can increase the accuracy of fluorescent tracer measurements. The behaviors of florescent tracers under various temperatures are predictable, and they have been generally used for thermometry purposes, such as non-contact measurement of fluid temperature at the microscale with high accuracy [36–41]. Therefore, it is also possible to develop reliable correction models [42] which can be used to compensate the deviation of measurement results caused by the solution’s temperature. Response surface analysis is a set of mathematical techniques. By graphing Sustainability 2020, 12, 4501 3 of 15 the results of polynomial regression analyses in a three-dimensional space, response surface analysis can provide relationships between predictor variables and an outcome variable [43,44]. In this study, response surface methodology was adopted to establish the relationship among measured concentration, temperature, and nominal concentration to correct the measurement errors. The objectives of this research were to examine the effect of the changes of solution temperatures on the measured concentrations of fluorescent tracers, and to develop models to compensate the deviation of measurement results caused by temperature changes. The effort of this research was to minimize analytical errors in the concentration measurement of fluorescent tracers, in order to increase the accuracy of accessing pesticide spray mixture uniformity and quantifying spray deposition and drift.
2. Materials and Methods
2.1. Fluorescent Tracers Used Three typical fluorescent tracers were selected for the tests: BSF (MP Biomedicals, Inc., Aurora, OH, USA), Eosin (Acros Organics, Thermo Fisher Scientific, Pittsburgh, PA, USA), and Fluorescein sodium salt (Sigma-Aldrich, Inc., Saint Louis, MO, USA). BSF and Eosin were the yellowish free acid dyes. Fluorescein sodium salt contained 70% dye content and 30% sodium salt. The excitation and emission wavelengths, Chemical Abstracts Service (CAS) registry number, molecular weight, chemical formula, and fluorescence quantum yield of these tracers, indicated by the manufacturers, are listed in Table1.
Table 1. Fluorescent tracers used in the tests.
Emission Molecular Fluorescence Tracers Excitation (nm) CAS Number Formula Fluorescence Quantum Yield (nm) Weight
BSF 460 500 2391-30-2 404.4 C19H13N2NaO5S 0.27
Eosin 525 545 15086-94-9 647.9 C20H8Br4O5 0.63
Fluorescein sodium salt 460 515 518-47-8 376.27 C20H10Na2O5 0.97
2.2. Fluorescent Tracer Solutions Pilot work is necessary to identify the linear range for each type of tested fluorescent tracers. In this study, the linear range for each tested fluorescent tracer was identified by preparing sample solutions at different concentrations. Then, linearity was checked by measuring sample solutions from minimum detectable concentrations. If the sample was still in the linear range, the reading would increase in direct proportion to the concentration. If the reading did not increase in direct proportion to the concentration, or if the reading decreased instead, this meant “signal quenching” had begun and the sample was beyond the linear range of the fluorophore. The pilot work of this study identified 1 1 that the linear range was between 5.00 and 30.00 mg L− for BSF, 37.50 and 600.00 µg L− for Eosin, 1 and 4.88 and 156.25 µg L− for Fluorescein sodium salt. For each type of tested fluorescent tracer and at each concentration, initial solutions were prepared and then diluted to the desired concentrations. A digital scale with readability to 0.01 g was used to weigh each tracer. A graduated cylinder class with 1.0 mL subdivision and 0.5 mL tolerance was used ± to measure the volume of purified distilled water. The concentrations of tracers in the solutions were 1 1 5.00, 10.00, 15.00, 20.00, 25.00, and 30.00 mg L− for BSF, 37.50, 75.00, 150.00, 300.00, and 600.00 µg L− 1 for Eosin, and 4.88, 9.77, 19.53, 39.06, 78.12, and 156.25 µg L− for Fluorescein sodium salt, respectively. For each concentration, five samples were prepared for five replications and each sample was stored in a 3.5 mL transparent cuvette.
2.3. Solution Temperatures Fluorescence intensities were measured with a portable fluorometer (Turner Designs, Inc., San Jose, CA, USA). This fluorometer used fluorescence optical modules which contained the necessary light Sustainability 2020, 12, x FOR PEER REVIEW 4 of 16 Sustainability 2020, 12, 4501 4 of 15
Fluorescence intensities were measured with a portable fluorometer (Turner Designs, Inc., San Jose,source CA, and USA). pre-set This filters fluorometer for therequired used fluorescence application. optical Thetests modules were which conducted contained with the the fluorometer necessary lightlocated source inside and a laboratorypre-set filters with for a constant the required ambient application. temperature. The The tests laboratory were conducted was equipped with withthe fluorometeran automatic located controller inside and a alaboratory Circular Chart with Recordera constant (model: ambient DR5000, temperature. Future DesignThe laboratory Controls, was Inc., equippedBridgeview, with IL, an USA) automatic to control controller the ambient and a temperature Circular Chart continuously. Recorder During(model: theDR5000, measurement, Future Designif the cuvetteControls, was Inc., kept Bridgeview, inside the fluorometer IL, USA) to for control too long, the the ambient fluorometer temperature could raise continuously. the solution Duringtemperature the measurement, significantly. if Therefore,the cuvette cuvettes was kept that inside contained the fluorometer solution samples for too long, were the inserted fluorometer into the couldfluorometer raise the for onlysolution about temperature 6 s. Within thesignificantl 6 s time duration,y. Therefore, the increasecuvettes of th solutionat contained temperature solution was samplesless than were 0.3 ◦ C.inserted into the fluorometer for only about 6 s. Within the 6 s time duration, the increaseBefore of solution each test, temperature a digital scale was (model:less than Practum1102-1S, 0.3 °C. Sartorius, Goettingen, Germany) with readabilityBefore each to 0.01 test, g wasa digital used scale to weight (model: each Practum1 tracer. The102-1S, ratio Sartorius, between Goettingen, the fluorescent Germany) tracer weight with readabilityand the distilled to 0.01 g water was used volume to weight was determined each tracer. to The represent ratio between the actual the fluorescent concentration tracer or nominalweight andconcentration the distilled of water the solution. volume was determined to represent the actual concentration or nominal concentrationAll tests of were the conducted solution. with the ambient temperature ranging from 19.0 ◦C to 21.0 ◦C. As shown in FigureAll tests1, temperatures were conducted of tracer with solution the ambient samples temperature were adjusted ranging with thefrom following 19.0 °C step to 21.0 for analysis.°C. As shownThe fluorometer in Figure 1, cuvettes temperatures containing of tracer fluorescent solution tracer samples solution were samples adjusted were with submerged the following into thestep water for analysis.bath container. The fluorometer Cold water cuvettes and warm containing water were fluo alternativelyrescent tracer applied solution to adjustsamples the were temperature submerged until intothe the desired water value bath was container. reached. Cold A stirring water and rod warm was used water to speedwere alternatively up heat exchange applied and to temperature adjust the temperatureuniformity distribution.until the desired Probe value digital was thermometers reached. A stirring (PDT650, rod was UEi used Test Instruments, to speed up heat Beaverton, exchange OR, andUSA) temperature were used to uniformity monitor the tracerdistribution. solution temperaturesProbe digital in thethermometers cuvettes and (PDT650, the water temperatureUEi Test Instruments,in the container Beaverton, consistently. OR, USA) The were samples used were to mo keptnitor in thethe containertracer solution for at temperatures least 10 min, in for the the cuvettestemperature and the reading water of temperature the thermometer in the placed container in the consistently. cuvette to reach The ansamples interval were of 0.5kept Cin of the the ± ◦ containerdesired temperature. for at least 10 min, for the temperature reading of the thermometer placed in the cuvette to reach an interval of ±0.5 °C of the desired temperature.
FigureFigure 1. 1. SchematicSchematic view view of of the the water water bath bath container container and and the the other other apparatus apparatus used used to to control control the the temperaturestemperatures of of fluorescent fluorescent tracer tracer solutions. solutions. Th Thee temperatures temperatures of of the the fluorescent fluorescent samples samples were were maintainedmaintained by by immersing immersing the the cuvettes cuvettes in in a amanua manuallylly temperature-controlled temperature-controlled water water bath bath container. container.
SinceSince the teststests in thisin this study study were conductedwere conducted in an indoor in laboratory,an indoor the laboratory, factor of photodegradation the factor of photodegradationcaused by solar exposure caused wasby excluded.solar exposure To avoid was other excluded. potential To sources avoid ofother degradation, potential forsources each typeof degradation,of fluorescent for tracer each andtype under of fluorescent each concentration tracer and condition, under each the concentration time elapsed condition, between the the sample time elapsedpreparation between and measurementthe sample preparation were 4 h. This and interval measur wasement used were for weighing4 h. This theinterval tracers, was mixing used withfor weighingdistilled water,the tracers, diluting, mixing adjusting with the temperature,distilled water, and diluting, measuring adjustin with theg fluorometer.the temperature, Within and such measuringa short time with duration, the fluorometer. readings wereWithin stable. such Moreover,a short time the duration, measurements readings of were fluorescent stable. Moreover, intensity in thethis measurements study have been of fluorescent based on the intensity assumption in this that study for have the same been concentrationbased on the assumption of fluorescent that tracer for thesolution same concentration with the same of temperature fluorescent andtracer same soluti pHon value, with readingsthe same fromtemperature the same and fluorometer same pH value, should readingsbe constant. from the same pH valuefluorometer of the should purified be distilled constant. water the pH used value in thisof the study purified was distilled 6.4. To water avoid solution samples contacting with the ambient air containing CO and to keep the solution pH condition used in this study was 6.4. To avoid solution samples contacting2 with the ambient air containing CO2 andconsistent, to keep thethe cuvettessolution containingpH condition solution consistent samples, the were cuvettes sealed containing with air-tightened solution samples caps. After were the sealedsolution with samples air-tightened in the cuvettes caps. After were sealedthe soluti withon air-tightened samples in caps,the theircuvettes pH valuewere couldsealed be with kept air-tightenedconstant. To caps, avoid their contaminations pH value causedcould be by openingkept constant. and closing To avoid the cuvettes contaminations filled with caused fluorescent by openingtracer solutions, and closing five the additional cuvettes filled cuvettes with filled fluore onlyscent with tracer distilled solutions, water five were additional used to verifycuvettes the filled only with distilled water were used to verify the solution temperatures. The five cuvettes were Sustainability 2020, 12, 4501 5 of 15 solution temperatures. The five cuvettes were randomly distributed inside the water container. The temperatures in these five cuvettes were measured within the error range of 0.5 ◦C. In this study, solution temperatures of 10.0 to 45.0 ◦C with 5.0 ◦C intervals were selected. The fluorometer manufacturer recommended that the ambient temperature should be within 15.0–40.0 ◦C during operation [35]. For some tests, the solution temperature could be higher or lower than the ambient temperature. Therefore, temperatures with 5.0 C above and below the ± ◦ fluorometer manufacturer’s recommended temperature range (10.0 and 45.0 ◦C) were used along with the recommended range.
2.4. Model Development Response surface methodology was used to establish the relationships between the measured concentration, temperature, and corrected concentration. A statistical analysis and polynomial regression with response surface analysis was performed using the function Curve Fitting Toolbox™ in the Matlab software (ver. 8.5.1.281278 (R 2015a) Service Pack 1, MathWorks, Inc., Natick, MA, USA). The Curve Fitting application was used to fit response surfaces to fluorometer data to determine the effects of temperature on the measured concentrations. In the response to the surface analysis approach, a polynomial regression with the third order functions was processed. This model was used for prediction of the corrected concentrations. The Fitting Tool was applied and the individual model could be interpreted as a surface fitting function of the experimental data. The general form of a complete third-degree polynomial regression model in two variables (x, y) was expressed as:
2 2 3 2 2 3 f (x, y) = p00 + p10x + p01 y + p20x + p11xy + p02 y + p30x + p21x y + p12xy + p03 y (1) where p00, p10, p01, p20, p11, p02, p30, p21, p12, and p03 are constants, x represents the solution temperature, and y represents measured concentration. The constants of the equation were determined by the least square method. After fitting the models, surfaces and contour plots were constructed to predict the relationship between the independent variables and determine coefficient R2.R2 measured the successful level of the fit in explaining the variation of the data. An R2 closer to 1 indicated that a greater proportion of variance was accounted by the model and a better fitting result was reached. The residuals were the differences between the calculated and experimental results for a determined set of conditions. A satisfactory mathematical model fitted to experimental data must present low residual values. Therefore, residual plots of the fitted models were also constructed.
3. Results
3.1. Linear Range and Calibration Model The fluorescent tracer calibration curves in Figure2 were obtained with an ambient temperature ranging from 19.0 ◦C to 21.0 ◦C. The same ambient temperature range was used for establishing the correction model. The temperature of the solution samples in the cuvette for calibration was 20.0 0.5 C. ± ◦ In order to achieve a fluorescent intensity that fell within the range of the fluorometer used, fluorescent tests were limited to the linear range of the response. As shown in Figure2a–c, the linear 1 1 range of concentrations used in this study was 5.00 to 30.00 mg L− for BSF, 37.50 to 600.00 µg L− for 1 Eosin, and 4.88 to 156.25 µg L− for Fluorescein sodium salt, respectively. Sustainability 2020, 12, 4501 6 of 15 Sustainability 2020, 12, x FOR PEER REVIEW 6 of 16 ) ) )
-1 160.00 -1 30.00 (a) 600.00 (b) -1 (c)
450.00 120.00 20.00 300.00 80.00 10.00 150.00 40.00
0.00 0.00 Concentration (μg L
Concentration (μg L (μg Concentration 0.00 Concentration (mg L (mg Concentration 0.0 5.0x104 1.0x105 1.5x105 2.0x105 0.0 1.0x102 2.0x102 3.0x102 0.0 5.0x103 1.0x104 1.5x104 2.0x104 Fluorescent intensity (RFU) Fluorescent intensity (RFU) Fluorescent intensity (RFU)
Figure 2. Calibration curves of three different fluorescent tracers: (a) BSF; (b) Eosin; (c) Fluorescein Figure 2. Calibration curves of three different fluorescent tracers: (a) BSF; (b) Eosin; (c) Fluorescein sodium salt. RFU represents Relative Fluorescence Units. sodium salt. RFU represents Relative Fluorescence Units. The tracer concentration of the solution as a function of fluorescence intensity was calibrated The tracer concentration of the solution as a function of fluorescence intensity was calibrated before the tests. The calibration equation for the tracer concentration and fluorescence intensity was: before the tests. The calibration equation for the tracer concentration and fluorescence intensity was: BSFBSF tracer: tracer: 4 2 y = 0.305 + 1.681 10− x, R = 0.999 (2) = 0.305 + 1.681 ×× 10 , = 0.999 (2) Eosin: Eosin: y = 0.798 + 2.427x, R2 = 0.999 (3) − = −0.798 + 2.427 , = 0.999 (3) Fluorescein sodium salt: Fluorescein sodium salt: y = 0.031 + 0.008x, R2 = 0.999 (4) where y is the tracer concentration in = the 0.031 solution + 0.008 , and x is= the 0.999 fluorescence intensity. (4) where y is the tracer concentration in the solution and x is the fluorescence intensity. 3.2. Effect of Temperature on Measured Concentration of Fluorescent Tracers 3.2. Effect of Temperature on Measured Concentration of Fluorescent Tracers 3.2.1. Estimation of the Accuracy of the Fluorometer 3.2.1.Because Estimation random of the Accuracy errors could of the beFluorometer caused by tiny fluctuations in temperatures due to sample-to-sampleBecause random variations—for errors could example, be caused because by tiny of temperature fluctuations variationsin temperatures in the laboratorydue to environment,sample-to-sample the solution variations—for samples example, or the fluorometer because of itself—measuringtemperature variations the solution in the samplelaboratory even withenvironment, the same concentrationthe solution samples by a fluorometer or the fluorome couldter result itself—measuring in slightly di thefferent solution readings sample each even time. Thesewith the errors same could concentration be reduced by by a fluorometer taking the averagecould result of repeated in slightly measurements. different readings To compareeach time. the fluorometerThese errors uncertainty could be reduced regarding by thetaking observed the average sample-to-sample of repeated variations,measurements. overall To averagecompare valuesthe forfluorometer the coefficient uncertainty of variation regarding (CV) underthe observed tested nominalsample-to-sample concentrations variations, were overall calculated. average For values BSF with 1 nominalfor the coefficient concentrations of variation ranging (CV) from under 5.00 to tested 30.00 nominal mg L− , theconcentrations overall average were CV calculated. values were For BSF 1.01%, 0.97%,with nominal 1.16%, 1.29%, concentrations 1.66%, and ranging 1.51%; from for Eosin5.00 to with 30.00 nominal mg L−1, the concentrations overall average ranging CV values from 37.50were to 1 600.001.01%,µ g0.97%, L− , the 1.16%, overall 1.29%, average 1.66%, CV and values 1.51%; were for 3.54%, Eosin 1.70%, with 1.50%,nominal 0.88%, concentrations and 0.74%; ranging for Fluorescein from −1 1 sodium37.50 to salt 600.00 with µg nominal L , the overall concentrations average CV ranging values from were 4.88 3.54%, to 156.25 1.70%,µ 1.50%,g L− , the0.88%, overall and 0.74%; average for CV valuesFluorescein were 7.82%,sodium 3.33%, salt with 1.08%, nominal 1.34%, concentrations 1.48%, and 0.69%, ranging respectively. from 4.88 to It 156.25 was found µg L−1 that, the BSFoverall had a slightlyaverage higher CV values fluorometer were 7.82%, measurement 3.33%, 1.08%, uncertainty 1.34%, 1.48%, at higher and nominal 0.69%, respectively. concentrations. It was Conversely, found Eosinthat andBSF Fluoresceinhad a slightly Sodium higher Salt had fluorometer higher fluorometer measurement uncertainty uncertainty at lower at concentrations.higher nominal concentrations. Conversely, Eosin and Fluorescein Sodium Salt had higher fluorometer uncertainty 3.2.2.at lower BSF concentrations.
3.2.2.Figure BSF 3 shows the measured concentration of BSF solution with the variation of temperature from 10.0 to 45.0 ◦C. The measured concentration was obviously affected by the solution’s temperature. It wasFigure found 3 that shows the measuredthe measured concentration concentration decreased of BSF solution as the solution with the temperature variation of increasedtemperature at all from 10.0 to 45.0 °C. The measured concentration was obviously affected by the solution’s nominal concentrations. As the temperature was increased from 10.0 to 35.0 ◦C, a sharp drop in the measuredtemperature. concentration It was found occurred that the over measured this temperature concentration range. decreased As the temperatureas the solution increased temperature further, increased at all nominal concentrations. As the temperature was increased from 10.0 to 35.0 °C, a the decrease rate slowed down, and the measured concentration tended to be consistent and stable. sharp drop in the measured concentration occurred over this temperature range. As the temperature For example, at a nominal concentration of 20 mg L 1, the measured concentration was 36.41, 26.59, increased further, the decrease rate slowed down, −and the measured concentration tended to be 20.45, 15.90, 12.83, 10.61, 8.92, and 7.78 mg L 1 when the solution temperature was 10.0, 15.0, 20.0, 25.0, consistent and stable. For example, at a− nominal concentration of 20 mg L−1, the measured 30.0, 35.0, 40.0, and 45.0 ◦C, respectively. Sustainability 2020, 12, x FOR PEER REVIEW 7 of 16
Sustainability 2020, 12, 4501 7 of 15 concentration was 36.41, 26.59, 20.45, 15.90, 12.83, 10.61, 8.92, and 7.78 mg L−1 when the solution temperature was 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 °C, respectively. )
-1 80.00 5.00 mg L-1 10.00 mg L-1 70.00 15.00 mg L-1 20.00 mg L-1 60.00 25.00 mg L-1 30.00 mg L-1 Data point 50.00 40.00 30.00 20.00 10.00 0.00 Measured Concentration (mg L (mg Concentration Measured 5.010.015.020.025.030.035.040.045.050.0 Temperature (ºC)
Figure 3. TheThe effect effect of of temperature temperature on on the measured concentration of BSF solutions at nominal 1 concentrations of of 5.00, 10.00, 15.00, 20.00, 25.00, and 30.0030.00 mgmg LL−−1 (error(error bars bars represent the doubled standard deviation ( 2σ)). standard deviation (±2± σ)). BSF should be considered as highly sensitive to temperatures, because a slight variation in solution BSF should be considered as highly sensitive to temperatures, because a slight variation in temperatures could lead to a significant measurement error by the fluorometer. For instance, for the solution temperatures could lead to a significant measurement error by the fluorometer. For BSF solution of 30.00 mg L 1, the measured concentration decreased over 2.5 times (from 75.40 to instance, for the BSF solution− of 30.00 mg L−1, the measured concentration decreased over 2.5 times 29.90 mg L 1) when the solution temperature increased from 10.0 to 20.0 C. (from 75.40 to− 29.90 mg L−1) when the solution temperature increased from◦ 10.0 to 20.0 °C. For different nominal concentrations, the measured concentrations did not display a constant For different nominal concentrations, the measured concentrations did not display a constant decrease trend with increased solution temperatures. As the temperature increased, the decrease of decrease trend with increased solution temperatures. As the temperature increased, the decrease of measured concentrations was found to be even more remarkable for samples with higher nominal measured concentrations was found to be even more remarkable for samples with higher nominal concentrations. For example, at a nominal concentration of 5.00 mg L 1, the measured concentration concentrations. For example, at a nominal concentration of 5.00 mg L−−1, the measured concentration was 7.65, 6.02, 4.95, 4.18, 3.50, 3.04, 2.66, and 2.37 mg L 1 when the solution temperature was 10.0, 15.0, was 7.65, 6.02, 4.95, 4.18, 3.50, 3.04, 2.66, and 2.37 mg− L−1 when the solution temperature was 10.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 C, respectively; however, at a nominal concentration of 30.00 mg L-1, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and◦ 45.0 °C, respectively; however, at a nominal concentration of the measured concentration was 75.44, 43.57, 29.88, 22.62, 17.61, 14.56, 11.90, and 9.92 mg L 1. 30.00 mg L-1, the measured concentration was 75.44, 43.57, 29.88, 22.62, 17.61, 14.56, 11.90, and− 9.92 These results revealed that relatively higher nominal concentrations could aggravate a negative mg L−1. influence of the solution temperature on the measured concentrations and could reduce the detection These results revealed that relatively higher nominal concentrations could aggravate a negative accuracy. For the BSF solution, over the temperatures tested (10.0 to 45.0 C), the relative error influence of the solution temperature on the measured concentrations and◦ could reduce the of measured concentrations to nominal concentrations fell in the range of 1.04–53.08% (average detection accuracy. For the BSF solution, over the temperatures tested (10.0 to 45.0 °C), the relative 32.44%), 1.28–60.38% (average 35.83%), 0.93–72.59% (average 39.17%), 2.23–82.07% (average 42.14%), error of measured concentrations to nominal concentrations fell in the range of 1.04–53.08% (average 0.66–120.84% (average 49.39%), and 0.39–151.46% (average 55.21%) for the nominal concentrations of 32.44%), 1.28–60.38% (average 35.83%), 0.93–72.59% (average 39.17%), 2.23–82.07% (average 42.14%), 5.00, 10.00, 15.00, 20.00, 25.00, and 30.00 mg L 1, respectively. 0.66–120.84% (average 49.39%), and 0.39–151.46%− (average 55.21%) for the nominal concentrations of Table2 shows the mean measured concentration and CV of the BSF solutions across the eight 5.00, 10.00, 15.00, 20.00, 25.00, and 30.00 mg L−1, respectively. temperatures at six different nominal concentrations. The CV increased from 42.44% to 78.18% as the Table 2 shows the mean measured concentration and CV of the BSF solutions across the eight nominal concentration increased from 5.00 to 30.00 mg L 1. The results of the statistical analysis also temperatures at six different nominal concentrations. The− CV increased from 42.44% to 78.18% as the indicated that for all the nominal concentrations (5.00, 10.00, 15.00, 20.00, 25.00, and 30.00 mg L 1), nominal concentration increased from 5.00 to 30.00 mg L−1. The results of the statistical analysis also− the means of measured concentrations were significantly different (p < 0.01) for the BSF solution under indicated that for all the nominal concentrations (5.00, 10.00, 15.00, 20.00, 25.00, and 30.00 mg L−1), the tested solution temperature conditions (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 C). The solution means of measured concentrations were significantly different (p < 0.01) for the BSF◦ solution under temperature influenced the measured concentrations more significantly when the measurement was tested solution temperature conditions (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 °C). The conducted at relatively higher nominal concentrations. solution temperature influenced the measured concentrations more significantly when the measurement was conducted at relatively higher nominal concentrations.
Table 2. Mean measured concentrations for BSF, Eosin and Fluorescein sodium salt solutions at different nominal concentrations across eight solution temperatures (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 °C).
Fluorescent Tracer BSF Nominal concentration (mg L−1) 5.00 10.00 15.00 20.00 25.00 30.00 Sustainability 2020, 12, 4501 8 of 15
Table 2. Mean measured concentrations for BSF, Eosin and Fluorescein sodium salt solutions at different nominal concentrations across eight solution temperatures (10.0, 15.0, 20.0, 25.0, 30.0, 35.0,
40.0, and 45.0 ◦C). Sustainability 2020, 12, x FOR PEER REVIEW 8 of 16 Fluorescent Tracer BSF 1 −1 MeasuredNominal concentrationconcentration (mg (mg L− ) L ) 5.004.3 10.008.52 15.0012.83 20.00 17.44 25.0022.74 28.19 30.00 Measured concentration(CV (%)) (mg L 1) 4.3(42.44) 8.52(47.58)12.83 (52.97)17.44 (56.90) (68.87) (78.18)28.19 − 22.74 (68.87) Fluorescent(CV (%)) tracer (42.44) (47.58) (52.97) (56.90) Eosin (78.18) Fluorescent tracer Eosin Nominal concentration (µg L−1) 37.50 75.00 150.00 300.00 600.00 1 Nominal concentration (µg L− ) 37.50 75.00 150.00 300.00 600.00 −1 Measured concentration (µg1 L ) 42.64 84.09 164.37 331.64 655.93 Measured concentration (µg L− ) 42.64 84.09 164.37 331.64 655.93 (CV(CV (%))(%)) (10.62)(10.62)(10.32) (10.32)(9.90) (9.90)(10.68) (10.68)(11.41) (11.41) FluorescentFluorescent tracer tracer Fluorescein Fluorescein sodium sodium salt salt 1 NominalNominal concentration concentration (µg (µg L− )L−1) 4.884.88 9.779.77 19.5319.53 39.06 39.06 78.12 78.12 156.25156.25 Measured concentration (µg L 1) 4.90 9.82 20.04 40.16 79.91 Measured concentration (µg− L−1) 4.90 9.82 20.04 40.16 79.91160.95 (1.79)160.95 (CV (%)) (1.10) (1.37) (1.54) (2.26) (1.64) (CV (%)) (1.10) (1.37) (1.54) (2.26) (1.64) (1.79) 3.2.3. Eosin 3.2.3. Eosin As with BSF, the measured concentration of Eosin was also affected by the solution’s temperature As with BSF, the measured concentration of Eosin was also affected by the solution’s (Figure4). However, the measured concentration of Eosin slightly decreased at first when the temperature (Figure 4). However, the measured concentration of Eosin slightly decreased at first temperature increased from 10.0 ◦C to 15.0 ◦C or 20.0 ◦C, and then increased smoothly as the when the temperature increased from 10.0 °C to 15.0 °C or 20.0 °C, and then increased smoothly as temperature increased further. Compared to BSF, the measured concentration of Eosin was more the temperature increased further. Compared to BSF, the measured concentration of Eosin was more temperature-independent at lower temperatures, but was more influenced by higher temperatures. temperature-independent at lower temperatures, but was1 more influenced by higher temperatures. For example, at a nominal concentration of 150.00 µg L− , the measured concentrations were 151.69, −1 For example, at a nominal concentration of 150.00 µg L , the 1measured concentrations were 151.69, 149.49, 148.76, 156.25, 163.93, 171.08, 179.66, and 194.09 µg L− when the solution temperatures were 149.49, 148.76, 156.25, 163.93, 171.08, 179.66, and 194.09 µg L−1 when the solution temperatures were 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 ◦C, respectively. 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 °C, respectively. 37.50 μg L-1 75.00 μg L-1 150.00 μg L-1 300.00 μg L-1 600.00 μg L-1 Data point ) -1 800.00 700.00 600.00 500.00 400.00 300.00 200.00 100.00 0.00 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 Measured Concentration (μg L Temperature (ºC) Figure 4. The effect of temperature on the measured concentration of Eosin solutions at nominal Figure 4. The effect of temperature on the measured concentration of Eosin solutions at nominal concentrations of 37.50, 75.00, 150.00, 300.00, and 600.00 µg L 1 (error bars represent the doubled concentrations of 37.50, 75.00, 150.00, 300.00, and 600.00 µg L−1 (error bars represent the doubled standard deviation ( 2σ)). standard deviation (±2± σ)). For the tested nominal concentrations, the influence of temperature on the measured concentrations For the tested nominal concentrations, the influence of temperature on the measured was lowest at the nominal concentration of 150.00 µg L 1. Relatively lower or higher nominal concentrations was lowest at the nominal concentration of −150.00 µg L−1. Relatively lower or higher concentrations were more severely influenced by temperature, and the measurement accuracy was nominal concentrations were more severely influenced by temperature, and the measurement lower. For the Eosin solution, over the tested temperature range from 10 to 45 C, the relative accuracy was lower. For the Eosin solution, over the tested temperature range from ◦10 to 45 °C, the error of measured concentrations to nominal concentrations fell in the range of 0.14–34.43% (average relative error of measured concentrations to nominal concentrations fell in the range of 0.14–34.43% 13.71%), 0.20–32.82% (average 12.58%), 0.34–29.40% (average 9.87%), 0.49–31.46% (average 11.15%), (average 13.71%), 0.20–32.82% (average 12.58%), 0.34–29.40% (average 9.87%), 0.49–31.46% (average 11.15%), and 0.43–30.64% (average 11.29%) for the nominal concentrations of 37.50, 75.00, 150.00, 300.00, and 600.00 µg L−1, respectively. The mean measured concentrations were affected by the solution temperature at different nominal concentrations. The CV decreased first from 10.62 to 9.90%, and then increased to 11.41% as the nominal concentration increased from 37.50 to 600.00 µg L−1 (Table 2). The results of the statistical analysis indicated that the differences in the means of measured concentrations were significant (p < Sustainability 2020, 12, 4501 9 of 15 and 0.43–30.64% (average 11.29%) for the nominal concentrations of 37.50, 75.00, 150.00, 300.00, 1 and 600.00 µg L− , respectively. The mean measured concentrations were affected by the solution temperature at different nominal concentrations. The CV decreased first from 10.62 to 9.90%, and then increased to 11.41% as the nominal 1 concentration increased from 37.50 to 600.00 µg L− (Table2). The results of the statistical analysis indicated that the differences in the means of measured concentrations were significant (p < 0.01) Sustainability 2020, 12, x FOR PEER REVIEW 9 of 16 for Eosin solutions under tested solution temperature conditions (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and0.01) 45.0 for◦C). Eosin solutions under tested solution temperature conditions (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 °C). 3.2.4. Fluorescein Sodium Salt 3.2.4. Fluorescein Sodium Salt Figure5 shows the e ffect of temperature on the measured concentration of Fluorescein sodium salt. DifferentlyFigure from 5 BSFshows and the Eosin, effect the of temperature measured concentrations on the measured of concentration Fluorescein sodium of Fluorescein salt did sodium not display a constantsalt. Differently increase from or decrease BSF and Eosin, trend the with measured increased concentrations or decreased of Fluoresc solutionein temperatures.sodium salt did not Instead, display a constant increase or decrease trend with increased or decreased solution temperatures. it fluctuated slightly with the temperatures tested. It was also found that among the three tracers the Instead, it fluctuated slightly with the temperatures tested. It was also found that among the three measured concentration of Fluorescein sodium salt was the least affected by solution temperature. tracers the measured concentration of Fluorescein sodium salt was the least affected by solution µ 1 Fortemperature. example, at For a nominal example, concentration at a nominal concentration of 39.06 g Lof− 39.06, the µg measured L−1, the measured concentrations concentrations were 38.45, 1 38.87,were 38.70, 38.45, 38.57, 38.87, 39.85, 38.70, 38.89, 38.57, 38.13, 39.85, and 38.89, 39.22 38.13,µg Land− when39.22 µg the L solution−1 when the temperatures solution temperatures were 10.0, 15.0, 20.0,were 25.0, 10.0, 30.0, 15.0, 35.0, 20.0, 40.0, 25.0, and 45.030.0, ◦35.0,C, respectively. 40.0, and 45.0 Especially, °C, respectively. low nominal Especially, concentrations low nominal led to a nearlyconcentrations constant measured led to a concentrationnearly constant over measured the tested concentration temperature over range. the tested At atemperature nominal concentration range. 1 −1 1 of 4.88At µag nominal L− , the concentration measured concentrations of 4.88 µg L , werethe measured 4.70, 5.50, concentrations 5.54, 5.15, 4.64, were 4.45, 4.70, 4.69, 5.50, and 5.54, 5.65 5.15,µ g L− 4.64, 4.45, 4.69, and 5.65 µg L−1 when the solution temperatures were 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, when the solution temperatures were 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 ◦C, respectively. 40.0, and 45.0 °C, respectively. 4.88 μg/l 9.77 μg/l 19.53 μg/l 39.06 μg/l 78.12 μg/l 156.25 μg/l ) -1 180.00 Data point 160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00
Measured Concentration (μg L (μg Concentration Measured 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 ºC Temperature ( ) FigureFigure 5. The 5. eTheffect effect of temperature of temperature on the on measured the measured concentration concentration of Fluorescein of Fluorescein sodium sodium salt solutionssalt 1 at nominalsolutions concentrations at nominal concentrations of 4.88, 9.77, of 19.53, 4.88, 39.06,9.77, 19.53, 78.12, 39.06, and 156.2578.12, andµg L156.25− (error µg L bars−1 (error represent bars the doubledrepresent standard the doubled deviation standard ( 2σ )).deviation (±2σ)). ± TheThe least least affected affected tracer tracer by by the the variation variation of temperature temperature had had the the highest highest measurement measurement accuracy. accuracy. For theFor Fluoresceinthe Fluorescein sodium sodium salt salt solution, solution, over over the th temperaturee temperature range range from from 10.0 10.0 to 45.0to 45.0◦C, °C, the the relative errorrelative of the error measured of the measured concentrations concentrations to nominal to no concentrationsminal concentrations fell infell the in the range range of of 0.01 0.01 to to 1.82% (with1.82% an average (with an of average 0.95%), of 0.09 0.95%), to 3.02% 0.09 to (with 3.02% an (with average an average of 1.08%), of 1.08%), 0 to 4.91% 0 to (with4.91% an(with average an of 2.61%),average 0.23 of to 2.61%), 6.85% 0.23 (with to an6.85% average (with an of average 2.81%), of 0.04 2.81%), to 5.50% 0.04 to (with 5.50% an (with average an average of 2.30%), of 2.30%), and 0.02 and 0.02 to 5.27% (with an average of 3.01%) for nominal concentrations of 4.88, 9.77, 19.53, 39.06, to 5.27% (with an average of 3.01%) for nominal concentrations of 4.88, 9.77, 19.53, 39.06, 78.12, 78.12, and 156.25 µg L−1, respectively. µ 1 and 156.25The coefficientsg L− , respectively. of variation for the mean concentrations across the five temperatures at six differentThe coe ffinominalcients ofconcentrations variation for fluctuated the mean slightly concentrations between 1.10% across and the 2.26%—much five temperatures less than at six differentthose nominal of the BSF concentrations and Eosin solutions. fluctuated It was slightly noted between that for the 1.10% nominal and 2.26%—much concentrations less of 4.88 than and those of 1 the BSF9.77 and µg EosinL−1 there solutions. was no It wassignificant noted thatdifference for the (p nominal > 0.05) concentrationsbetween the means of 4.88 of and measured 9.77 µg L− thereconcentrations. was no significant Hence, di ffforerence these (p >two0.05) concentr betweenations, the meansthe measured of measured concentration concentrations. could be Hence, considered as agreeing satisfactorily with the nominal concentration. However, for the nominal concentrations of 19.53, 39.06, 78.12, and 156.25 µg L−1, the differences between means of measured concentration were significant (p < 0.01) under tested solution temperature conditions (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0 °C). Sustainability 2020, 12, 4501 10 of 15 for these two concentrations, the measured concentration could be considered as agreeing satisfactorily with the nominal concentration. However, for the nominal concentrations of 19.53, 39.06, 78.12, 1 and 156.25 µg L− , the differences between means of measured concentration were significant (p < 0.01) under tested solution temperature conditions (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, andSustainability 45.0 ◦C). 2020, 12, x FOR PEER REVIEW 10 of 16 Thus, the measured concentrations were strongly affected by the temperature of the fluorescent tracer solutions,Thus, the exceptmeasured for Fluoresceinconcentrations sodium were salt.strongly BSF affected was the by most the sensitivetemperature to temperature of the fluorescent changes tracer solutions, except for Fluorescein sodium salt. BSF was the most sensitive to temperature among the tested nominal concentrations, followed by Eosin. changes among the tested nominal concentrations, followed by Eosin. 3.3. Correction Models 3.3. Correction Models The experimental results showed that the variation in measured concentration with temperature The experimental results showed that the variation in measured concentration with depended on the type of tested florescent tracers; therefore, a specific response surface model was temperature depended on the type of tested florescent tracers; therefore, a specific response surface developed for a specific fluorescent tracer. model was developed for a specific fluorescent tracer. The surface response model for BSF was The surface response model for BSF was
2 2 2 (f ,( x), =y) 4.29= +4.29 0.438 + 0.438 + 1.088 x + 1.088 4.11y 4.11 10 10− xy 1.8381.838 10 10 −+7.09y − 5 3 − 3 ×2 − 4 ×2 + 107.09 10+− 1.904x + 1.904 10 10 +7.75 10− x y + 7.75 10−+xy 6.089 (5) × × × (5) + 106.089 10 (R 5 y=3 0.9957)R2 = 0.9957 × − for Eosin,for itEosin, was it was
f ( (x ,, y ) = 56.2556.25 ++ 5.94 5.94 +x + 1.122 1.122 y 0.1965 0.1965x2 4.9 104.9 10 3 xy 3.5673.567 1010 5 y2 − − − + 2.055 4 103 5.414− 5 102 − × 1.5746 10−2 ×+ 9.726 +2.055 10− x 5.414 10− x y 1.574 10− xy (6) (6) 10× (R −= 0.9997)× − × +9.726 10 8 y3 R2 = 0.9997 × − and forand Fluorescein for Fluorescein sodium so salt,dium it salt, was it was ( , ) = 5.935 + 1.146 + 0.86 – 4.927 10 +1.34 10 + 1.732 f (x, y) = 5.935 + 1.146x + 0.86y 4.927 10 2x2 + 1.34 10 3xy 10 + 6.204 10 2.109 10− 6.267 − 10 − 3 2 − 4 ×3 5 ×2 +1.732 10 y + 6.204 10 x 2.109 10 x y (7) (7) 6.468 × 10 − (R = 0.9996)× − − × − 6.267 10 6xy2 6.468 10 6 y3 R2 = 0.9996 − × − − × − The R2 values for the three models were 0.9957 for BSF, 0.9997 for Eosin, and 0.9996 for The R2 values for the three models were 0.9957 for BSF, 0.9997 for Eosin, and 0.9996 for Fluorescein Fluorescein sodium salt. A higher R2 indicated a more satisfactory adjustment of the cubic sodium salt. A higher R2 indicated a more satisfactory adjustment of the cubic polynomial model to polynomial model to the experimental data and a higher correlation between the measured and the thepredicted experimental values. data and a higher correlation between the measured and the predicted values. AA three-dimensional three-dimensional visualization visualization ofof thethe predictedpredicted models models could could be be obtained obtained by by the the response response surfacesurface plots plots shown shown in in Figure Figure6. 6.
FigureFigure 6. 6.Response Response surfacesurface graph for for BSF, BSF, Eosin Eosin and and Fluorescein Fluorescein sodium sodium salt: salt: (a) BSF; (a) BSF;(b) Eosin; (b) Eosin; (c) (c)Fluorescein Fluorescein sodium sodium salt. salt.
TheThe residual residual graphs graphs were were plotted plotted inin FigureFigure7 7a–ca–c.. TheThe low residuals residuals indicated indicated that that the the regression regression modelsmodels for for BSF, BSF, Eosin, Eosin, and and Fluorescein Fluorescein sodiumsodium salt tracers agreed agreed well well with with the the actual actual solution solution concentrations.concentrations. Thus, Thus, the the models models were were adequate adequate to to make make precise precise interpretations interpretations aboutabout thethe behaviorbehavior of fluorescentof fluorescent tracers tracers under under various various solution solution temperatures. temperatures.
Sustainability 2020, 12, x FOR PEER REVIEW 11 of 16
Sustainability 2020, 12, 4501 11 of 15 Sustainability 2020, 12, x FOR PEER REVIEW 11 of 16
Figure 7. Residual plots of the response surface graph: (a) BSF; (b) Eosin; (c) Fluorescein sodium salt.
With the correction model, the analytical errors of corrected concentration and nominal
concentration were calculated and evaluated. The corrected concentrations for the BSF tracer are shownFigureFigure in 7.Figure Residual7. Residual 8a. The plots plots relative of of the the response responseerror of surface surfacecorrected graph: graph: concentrations ((aa)) BSF;BSF; ((b)) Eosin; to nominal ( c)) Fluorescein Fluorescein concentrations sodium sodium salt. salt. fell in the range of 0.03–25.93% (6.86% average), 0.88–3.24% (1.72% average), 0.46–5.28% (2.29% average), 0.15–6.82%WithWith the (2.80%the correction correction average), model, model, 0.06–2.54% the the analyticalanalytical (1.29% average), errorserrors ofof and correctedcorrected 0.08–4.55% concentrationconcentration (2.50% average) and and nominal nominalfor the concentrationnominalconcentration concentrations were were calculated calculated of 5.00, and 10.00,and evaluated. evaluated. 15.00, 20.00, TheThe 25.00, correctedcorrected and 30.00 concentrations mg L-1, respectively. for for the the BSF BSF tracer tracer are are shownshownThe in inFigurecoefficients Figure8a. 8a. The ofThe relativevariation relative error forerror the of of corrected mean corrected corr concentrations ectedconcentrations concentrations toto nominalnominal of the concentrationsconcentrations BSF solution fellacross fell in in theeightthe range rangetemperatures of of 0.03–25.93% 0.03–25.93% at six (6.86%different (6.86% average), average), nominal 0.88–3.24% 0.88–3.24%concentrations, (1.72%(1.72% based average),average), on Equation 0.46–5.28% (5), increased(2.29% (2.29% average), average), greatly 0.15–6.82%(Table0.15–6.82% 3) compared (2.80% (2.80% average), toaverage), the values 0.06–2.54% 0.06–2.54% in Table (1.29%(1.29% 2. The average), average),highest coefficients andand 0.08–4.55%0.08–4.55% of variation (2.50% (2.50% wereaverage) average) recorded for for the theas nominal concentrations of 5.00, 10.00, 15.00, 20.00, 25.00, and 30.00 mg L-11, respectively. nominal10.21% at concentrations the nominal concentration of 5.00, 10.00, of 15.00, 5 mg 20.00, L-1, while 25.00, the and rest 30.00 were mg lower L− , than respectively. 2.94%. The coefficients of variation for the mean corrected concentrations of the BSF solution across
-1 -1 eight temperatures 5.00 mg L-1 10.00 mg at L-1 six 15.00 different mg L-1 nominal concentr 37.5. μg L ations, 75.00 μg Lbased on Equation (5), 19.53 increasedμg/l 39.06 μg/l greatly ) 800.00 -1 -1 -1 -1 -1 -1 ) ) 20.00 mg L 25.00 mg L 30.00 mg L 150.00 μg L 300.00 μg L 78.12 μg/l 156.25 μg/l -1 (Table-1 3) compared to the values in Table 2. The highest coefficients 160.00of variation were recorded as 700.00 600.00 μg L-1 30.00 -1 140.00 10.21% at the nominal concentration600.00 of 5 mg L , while the rest were lower than 2.94%. 25.00 120.00 500.00 20.00 -1 -1 100.00 5.00 mg L-1 10.00 mg L-1 15.00 mg L-1 400.00 37.5. μg L 75.00 μg L 19.53 μg/l 39.06 μg/l ) 800.00 -1 -1 -1 -1 -1 -1 ) 80.00 15.00) 20.00 mg L 25.00 mg L 30.00 mg L 150.00 μg L 300.00 μg L 78.12 μg/l 156.25 μg/l
300.00 -1 -1 160.00 700.00 600.00 μg L-1 10.0030.00 60.00 200.00 140.00 600.00 40.00 5.0025.00 100.00 120.00 500.00 20.00 20.00 0.00 L Concentration(mg Corrected 0.00 100.00 CorrectedConcentration(mg L 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 400.005.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 CorrectedConcentration(mg L 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 15.00 Temperature (ºC) 80.00 300.00 Temperature (ºC) Temperature (ºC) 60.00 10.00 200.00 40.00 5.00 FigureFigure 8. 8.Corrected Corrected concentrations: concentrations:100.00 ((aa)) BSFBSF tracer;tracer; (b) Eosin; ( c) Fluorescein sodium sodium salt. salt. 20.00
0.00 L Concentration(mg Corrected 0.00 CorrectedConcentration(mg L 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 CorrectedConcentration(mg L 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 TheTable coe 3.ffi cientsMeanTemperature ofcorrectedvariation (ºC) concentrations for the mean for corrected BSF,Temperature Eosin concentrations (ºC and) Fluorescein of thesodium BSF Temperature solutionsalt solutions (ºC across) at eight temperaturesdifferent atnominal six diff concentrationserent nominal acro concentrations,ss eight solution based temperatures on Equation (10.0, (5), 15.0, increased 20.0, 25.0, greatly 30.0, 35.0, (Table 3) Figure 8. Corrected concentrations: (a) BSF tracer; (b) Eosin; (c) Fluorescein sodium salt. compared40.0, toand the 45.0 values °C). in Table2. The highest coe fficients of variation were recorded as 10.21% at the 1 nominal concentration of 5 mg L− , while the rest were lower than 2.94%. Table Fluorescent3. Mean corrected Tracer concentrations for BSF, Eosin and Fluorescein BSF sodium salt solutions at different nominal concentrations across eight solution temperatures (10.0, 15.0, 20.0, 25.0, 30.0, 35.0, TableNominal 3. Mean concentration corrected concentrations (mg L−1) forBSF, 5.00 Eosin10.00 and Fluorescein 15.00 sodium 20.00 salt solutions25.00 at different30.00 40.0, and 45.0 °C). nominalCorrected concentrations concentration across (mg eight L−1) solution 4.68 temperatures 10.09 (10.0,15.22 15.0, 20.0,20.39 25.0, 30.0, 25.04 35.0, 40.0, 29.52 and 45.0 ◦C). Fluorescent(CV (%)) Tracer (10.21) (1.76) (2.38) BSF (2.94) (1.62) (2.62) −1 NominalFluorescent concentrationFluorescent tracer Tracer(mg L ) 5.00 10.00 15.00 BSF Eosin 20.00 25.00 30.00 −1 1 NominalCorrected concentrationNominal concentration concentration (µg (mg (mgL −L1 L) −) ) 37.504.685.00 10.0075.0010.09 15.00150.0015.22 20.00 300.0020.39 25.00 30.00600.0025.04 29.52 1 Corrected(CV (%)) concentration (mg L− ) (10.21)4.68 10.09(1.76) 15.22(2.38)20.39 (2.94)25.04 29.52(1.62) (2.62) Measured concentration(CV (%)) (µg L−1) 37.53(10.21) (1.76)75.59 (2.38)148.96(2.94) 300.74(1.62) (2.62)599.89 Fluorescent tracer Eosin (CVFluorescent (%)) tracer(3.51) (2.87) (1.19) Eosin (0.94) (0.88) Nominal concentration (µg L−1) 1 37.50 75.00 150.00 300.00 600.00 FluorescentNominal concentration tracer (µg L− ) 37.50 75.00 Fluorescein 150.00 300.00 sodium 600.00 salt Measured concentration (µg− L1 1) 37.53 75.59 148.96 300.74 599.89 Measured concentration (µg L−1 −) 37.53 75.59 148.96 300.74 599.89 Nominal concentration(CV (%)) (µg L ) 4.88(3.51) (2.87)9.77 (1.19)19.53(0.94) 39.06(0.88) 78.12 156.25 (CV (%)) (3.51) (2.87) (1.19) (0.94) (0.88) Measured concentrationFluorescent tracer(µg L−1) 4.90 9.82 Fluorescein 20.04 sodium salt40.16 79.91 160.95 Fluorescent tracer 1 Fluorescein sodium salt Nominal(CV (%)) concentration (µg L− ) (1.10)4.88 (1.39) 9.77 19.53(1.53) 39.06 (2.26) 78.12 156.25(1.64) (1.79) −1 1 NominalMeasured concentration concentration (µg ( µLg L)− ) 4.884.90 9.829.77 20.0419.5340.16 39.0679.91 160.9578.12 156.25 (CV (%)) (1.10) (1.39) (1.53) (2.26) (1.64) (1.79) TheMeasured corrected concentration concentrations (µg forL−1 )Eosin 4.90 are shown 9.82 in Figure 20.04 8b. For the40.16 tested temperatures79.91 160.95 (10.0 to 45.0 °C), the relative(CV (%)) error of corrected concentr(1.10) ations(1.39) to nominal(1.53) concentrations(2.26) (1.64) fell in the(1.79) ranges of 0.63–4.93%The corrected (2.87% concentrations average), 0.10–4.43% for Eosin are(2.30% shown average), in Figure 0.34–2.23%8b. For the (1.09% tested average), temperatures 0.04–1.52% (10.0 to 45.0 ◦TheC), thecorrected relative concentrations error of corrected for Eosin concentrations are shown in to Figure nominal 8b. For concentrations the tested temperatures fell in the ranges (10.0 of 0.63–4.93%to 45.0 °C), the (2.87% relative average), error of 0.10–4.43% corrected concentr (2.30% average),ations to nominal 0.34–2.23% concentrations (1.09% average), fell in the 0.04–1.52% ranges (0.77%of 0.63–4.93% average), and(2.87% 0.03–1.39% average), (0.73% 0.10–4.43% average) (2.30% for average), the nominal 0.34–2.23% concentrations (1.09% average), of 37.50, 75.00,0.04–1.52% 150.00, 1 300.00, and 600.00 µg L− , respectively. Sustainability 2020, 12, 4501 12 of 15
The coefficients of variation for the mean corrected concentrations of Eosin decreased from 3.51% 1 to 0.88% as the nominal concentrations increased from 37.50 to 600.00 µg L− (Table3). For Fluorescein sodium salt (Figure8c), the di fferences between corrected concentrations and nominal concentrations at different temperatures were very small and could be negligible. The coefficients of variation for mean corrected concentrations across eight temperatures at four nominal concentrations fluctuated between 1.54% and 2.26% (Table3). Thus, it was unnecessary to apply the correction model for Fluorescein sodium salt. For the tracers of BSF, Eosin, and Fluorescein sodium salt with solution temperatures ranging from 10.0 to 45.0 ◦C, the above analyses showed that the relationships among the measured concentration, temperature, and corrected concentration were well correlated. The low relative errors and coefficients of variation with the correction models indicated that the variations in measured concentrations caused by the solution temperatures could be corrected accurately.
4. Discussion The measured concentration of different fluorescent tracers responded differently to their solution temperatures. To avoid this problem, pilot works should be essential to figure out the desired temperature range for different types of fluorescent tracers for ensuring the accurate measurement. For laboratory tests, it is necessary to follow the fluorometer manufacturer’s recommendations and control the ambient temperature within a specific range to reduce the analytical errors. If the tests require high-precision measurements for certain types of fluorescent tracers, pilot tests are necessary to identify desired temperature ranges that could provide measurement results with acceptable accuracy. If the temperature cannot be adjusted to a desired range, the temperature should be recorded along with the measured concentration, and the correction models should then be applied to correct the measured concentrations. For instruments that are deployed for in-situ field measurements, due to the difficulty of controlling the ambient temperature, stable tracers should be used. Otherwise, it is necessary to determine the relationship between the solution temperature and the tracer concentration prior to its use. To minimize analytical errors, the solution temperature should also be measured and recorded. After the experiments, correction models should be used to compensate the deviation of measured concentrations caused by the solution temperature. It should be noted that fluorometer manufacturers usually suggest a recalibration when new fluorescent tracers are used. Therefore, when the fluorescent tracer changes to a different provider, manufacture, or batch number, a recalibration is necessary. Accordingly, to ensure measurement precision and accuracy, correction models also need to be re-established by using the method presented in this study. Although the specific equations of the correction models obtained in this study may not be directly applied by other researchers, the methodology presented in this study could be effectively generalized and applied by other researchers to numerically correct the measured concentration errors due to variations in the solution temperature. This study also provided a technique for developing fluorometers equipped with a temperature compensation function. In this way, when the fluorometer measured the fluorescent intensity, the solution temperatures were also acquired at the same time to fit into the correction models. As a result, the error caused by the temperature variation could be compensated by using specific correction models embedded in the fluorometer for different tracers. Actually, the true concentration was not changed throughout the experiment. This is because the amount of fluorescent tracers (solute) in distilled water (solvent) was unchanged. However, during the reading, the variation of the measured concentrations was caused by the effects of the changes in the solution temperature on the fluorescent strengths. It has been found that the influence of temperature on the evolution of the fluorescence quantum yield is explained by the competition between the radiative relaxation mechanisms (fluorescence, phosphorescence) and the non-radiative relaxation mechanisms (inter-system crossing, internal conversion) [45]. For the future research, physical reasons Sustainability 2020, 12, 4501 13 of 15 for the temperature effect on the tracer concentration should be discovered, considering the following aspects: investigating the mechanisms of temperature effects based on spectroscopy, establishing models for the spectra of the dyes (both emission and absorption) as a function of the temperature, and analyzing the absorption of the incident excitation and fluorescence re-absorption. Because the temperature dependence of Eosin and Fluorescein disodium is driven by two mechanisms that are very different, one has a dependence on the quantum yield and the other on the absorption cross section. This has an influence on the interpretation of the results and should be explored and discussed in the future research.
5. Conclusions The effect of solution temperatures on the measured fluorescent concentration for fluorescent tracers of BSF, Eosin, and Fluorescein sodium salt was investigated. The following conclusions are highlighted. The measured concentrations of BSF, Eosin, and Fluorescein sodium salt were influenced by the solution temperature and the effect varied with the type of the tracer. Among the three fluorescent tracers, BSF was the most affected by the solution temperature, followed by Eosin, while the influence on Fluorescein sodium salt was negligible. The measured concentrations of BSF decreased as the solution temperature increased. The decrement rate was high at first, then slowed down, and then tended to become constant as the temperature increased. In contrast, the measured concentrations of Eosin decreased slowly at first and then increased noticeably as the temperatures increased. Third-order polynomial correction models were developed to minimize the error in the fluorescent analysis. These models, using the response surface methodology, enabled numerical corrections of the measured concentration errors due to variations of the solution temperatures. The corrected concentrations agreed well with the nominal concentrations. The overall relative errors were reduced from 42.36% to 2.91% for BSF, 11.72% to 1.55% for Eosin, and 2.68% to 1.17% for Fluorescein sodium salt. Therefore, the accuracy of the fluorescent concentration measurements should be examined with the solution temperatures to minimize analysis errors during the fluorescent tracer selection, for accurate quantifications of spray deposition on targets and off-target losses, and for obtaining spray mixture uniformity. It was necessary to adjust the fluorescent tracer solution temperature to a specific desired range for sample analyses. If the solution temperature adjustment is not feasible, the temperature should be recorded for each concentration measurement, and then the correction models should be applied to avoid measurement errors. As a result of this research, it is recommended that spray sample analyses use the correction models if the analyses must be processed in the field; otherwise, the samples should be calibrated and analyzed in laboratories with the same constant ambient temperature.
Author Contributions: Conceptualization, Z.Z., H.Z. and H.G.; methodology, Z.Z.; software, Z.Z.; validation, Z.Z., H.Z., and H.G.; investigation, Z.Z.; resources, H.Z.; data curation, Z.Z.; writing—original draft preparation, Z.Z.; writing—review and editing, H.G. and H.Z.; visualization, Z.Z.; supervision, H.Z.; project administration, H.Z.; funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the USDA-NIFA Specialty Crop Research Initiative, grant number 2015-51181-24253. Acknowledgments: The authors gratefully acknowledge Adam Clark, Barry Nudd, and Andy Doklovic for setting up the experimental instrumentations. Mention of company or trade names is for description only and does not imply endorsement by the USDA. The USDA is an equal opportunity provider and employer. Conflicts of Interest: The authors declare no conflict of interest.
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