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Automated fluorometric method for determination of boron in waters, detergents and sewage effluents
Water Research Pergamon Press 1972. Vol. 6, pp. 1475-1485. Printed in Great Britain
AUTOMATED FLUOROMETRIC METHOD FOR DETERMINATION OF BORON IN WATERS, DETERGENTS AND SEWAGE EFFLUENTS BADAR K. AFGHAN, PETER D. GOULDEN and JAMES F. RYAN Water Quality Division, Department of the Environment, Canada Centre for Inland Waters, Burlington, Canada (Received 1 October 1971)
Abstract--An automated method for the determination of boron in natural waters, detergents and sewageeffluents is described. The method is based on the reaction of 4'-chloro-2-hydroxy-4methoxybenzophenone (CHMB) with boron to produce fluorescent species, in a 90 ~ sulfuric acid medium. The method has been made specificto remove any interferences from all major and minor ions and other organic compounds normally present in water. The method is capable of measuring different chemical forms of boron such as boric acid, borax, sodium perborate and tetraphenyl boron. The method analyses 10 samples per hour, in the 5-100 p.g 1-1 boron range. The rate of sample analysis can be increased to 20 per hour at higher concentration ranges. The limit of detection is 1 leg 1-1 boron. INTRODUCTION THE WAVER Quality Division laboratories are engaged in a program of monitoring water quality across Canada by a national water quality network. Boron is one of the parameters that it is desired to measure. WAGGOTT (1969) has already discussed the potential problem of increasing boron concentration in natural waters in the British Isles. Although, at present, small amounts of boron are found in most waters in Canada, additional quantities may be introduced by industrial and domestic wastes. Standard methods for the examination of water and wastewater (1971) recommend the use of curcumin, carmine and potentiometric titration methods for boron. Spectrophotometric procedures using 1-1'-dianthrimide (ELLIS et al., 1949; LEVlNSON, 1971) are also used for boron determination. Fluorometric procedures, which are capable of greater sensitivity, have also been reported in the literature (WHITE and HOFFMAN, 1957; MON-NIER and MARCANTONATOS, 1966) but have not attained widespread use in water, detergent and effluent analysis. All these manual methods are either time consuming or require careful control of experimental conditions to obtain precise and accurate results and therefore are not suitable for monitoring purposes where a large number of samples have to be analyzed. There have been a few automated methods for the determination of boron reported (JAMESand KING, 1966; HULTHE et al., 1970), but unfortunately no detailed study has ever been carried out to compare various procedures and automate the most sensitive and simple procedure for this determination. In the Water Quality Division laboratories all the above procedures were evaluated in terms of sensitivity, selectivity, reproducibility, ease of automation and economy. The fluorometric procedure which utilizes 4'-chloro-2-hydroxy-4-methoxybenzophenone (CHMB) (MONNIERand MARCANTONATOS,1966) was found to be the most suitable among all the spectrophotometric, potentiometric and fluorometric procedures discussed above. Hence this method was automated. The method is based upon the reaction of boron with C H M B in a 90 ~ sulfuric acid medium and the measurement of the resultant fluorescence. The m a x i m u m excitation is from 340 to 380 nm and the maximum fluorescence emission is from 460 to 1475
1476
BADAR K. AFGHAN, PETER D. GOULDEN and JAMESF. RYAN
480 nm. The method has been shown to determine different forms of boron such as boric acid, borax, sodium perborate and tetraphenyl boron. The method has been made free of interferences from all major and minor ions and other compounds normally found in water. It was found that organic matter in some natural waters and sewage effluents produce high background fluorescence which was eliminated by photooxidation of the sample prior to the development of fluorescence. The method is capable of analyzing samples at the rate of 10 samples h - ~ in the 5-100 ~g 1-1range. Concentration as low as 1 t~g 1-1 can easily be detected. The rate of sample analysis can be increased to 20 h-a at higher concentration ranges. In the Water Quality Division laboratories the above method has been successfully employed for the analysis of total boron in filtered natural water .samples across Canada and for the analysis of boron in sea water, detergents and sewage effluents for over 18 months. EXPERIMENTAL
Manifold The manifold used is shown in FIG. 1. All the manifold pump tubes and transmission
tubes were acidflex. Tubing for the sample aspiration and for filling the wash receptable were ordinary standard tubing. For sleeving purposes, green acidflex tubing was employed. To obtain reproducible results it was found necessary to arrange all the large tubes on one side of the pump, descending to small tubes on the other side. The sample was aspirated at a rate of 0.32 ml min-1 and was mixed with concentrated sulfuric acid at a rate of 3.42 rrd rain- 1. The acidified sample was segmented, with 1.60
ml min-1 of air and irradiated for approximately 10 min in a u.v. reactor. The air flow through the photolysis equipment was adjusted so that the solution leaving the CAM - 10 SAMPLESPER HOUR 1:2 SAMPLETO WASH RATIO
RECEPTACLE
TUBE SEE (INCHES) DEIONIZEDWATER 0.01%C.H.M.B. SAMPLE AIR SULFURK3ACID D-o k___
D-O [ ~
0.040 0.035°
SMC O0
o.o o
o.o8~, tQQQQI 0.081" o°°8" o81. 0°~5 ~~ - ' o-o O.V.~ACTOR I 1
I PUMPI
WASTE ~
RECORDER
9
HEATING BATH 50.55oC
PULSESUPPRESSORS 1o0.025 INCH I.D.ACIDFLEX MANIFOLDTUBE 2-0.020 ORANGE-YELLOW 3-0.005 ORANGE-BLACK
"acidflex
SMC 105-0084
FLUORIMETER II PRIMARY-355nm SECONDARYo 46Ohm SAMPLEAPERTURE- 4 REFERENCEAPERTURE- 2
FIG. 1. Manifold for proposed method.
DMC 105-0085
~
HAAKE-FK CONSTANT TEMPERATURE CRCULATOR 20°C
Determination of Boron in Waters, Detergents and Sewage Effluents
1477
reactor was 42 4- I°C. After irradiation the sample was preheated to an optimum temperature of fluorescence development by passing through a 3-mm i.d. single mixing coil in a Technicon oil bath which was maintained at 50-55°C. The reagent was then added while the sample remained in the bath and was passed through a series of two 3-mm i.d. single mixing coils. After development of fluorescence the solution was passed through a 3-mm i.d. double mixing coil in a Haake constant temperature water bath at 20°C prior to entering the fluorometric flow cell. The solution was drawn through the flow cell at a rate of 3.42 ml rain -1, and was excited using an 85 W u.v. lamp in conjunction with a 355-nm narrow pass primary filter. A 460-nm narrow pass secondary filter was used to measure the resulting fluorescence intensity.
Apparatus All AutoAnalyzer equipment used was standard Technicon modules. A Haake Model F K constant temperature circulator was used to maintain a temperature of 20°C during fluorescence measurement in the flow cell. The u.v.-reactor was made as shown in FIG. 2. A 550-W photochemical lamp was placed inside the fused quartz lamp protection jacket which is plugged at the bottom with 1-in. asbestos tape and
a
c
Ill
4 in.
/
b J
\
d
FIG. 2. Ultraviolet-irradiator (a) chimney, (b) quartz coil, (c) ultraviolet-lamp, (d) cooling fan.
mounted axially in the centre of the galvanized iron cylinder body in the reactor. A quartz coil was placed in such a way that the u.v.-lamp could irradiate all the sample in the coil. The quartz coil was made of Purci1453 quality fused silica tubing approximately 10 mm long, 3 mm i.d., 0.6 mm wall thickness and the coil diameter is approximately 5 in.
1478
BADARK. AFGHANPETER , D. GOULDENand JAMESF. RYAN
Sample preparation For water samples and detergent solutions 2-3 drops of 30 ~ hydrogen peroxide was added to each of 50 ml of samples prior to analysis. This is added to assure oxidizing conditions. Water samples were filtered through a 0.45/zm membrane filter prior to the addition of hydrogen peroxide. For the determination of boron in detergents 100 mg of detergent was dissolved in 1 1. of water and this solution, diluted if necessary, was analyzed in a similar way to natural water samples. For the analysis of sewage effluents the suspended material in the sample was first removed by centrifugation at 15,000 rev/min. The resultant solution was filtered through a 0.45-Fm membrane filter and treated in a manner similar to that used for water samples. Procedure The manifold AutoAnalyzer equipment was connected as shown in FIG. 1. This involves two steps (1) oxidation of any organic material (II) development of fluorescence to determine boron. The sample tray was filled and each sample withdrawn was first oxidized by u.v.irradiation to remove any background fluorescence and then analyzed for boron content, as shown in FIG. 1. RESULTS Calibration curves and related accuracy studies The calibration curves were run in the ranges of 5-50/zg 1-l, 10--100/zg 1-1 and 0.1-0.4 mg 1-1 by changing the sensitivity and full scaler response of the fluorometer. All calibration curves gave straight lines passing through the origin, using the manifold illustrated in FIG. 1. The lower detection limit of the method was found to be approxi100
100 ppb 90 -80
80ppb
70 60ppb
60 50 40 ppb 40 30 20 10
~
20ppb
/2 i
I
f
I
t
1
FIG. 3. Typicalcalibrationcurve.
f
!
Determination of Boron in Waters, Detergents and Sewage Effluents
1479
mately 1/zg 1-1. The typical calibration curve most frequently used is shown in FI6. 3. Identical calibration curves were also obtained in natural waters, synthetic lake water, solutions containing effluent samples and detergents. The precision of the methods was evaluated by determining the per cent standard deviation of a series of 50 solutions each containing 100/~g 1-1 of boron. The coefficient of variation was found to be 1.87 per cent. Replicate analysis of several samples from across Canada gave values within the standard deviation mentioned above. Occasionally, variation of fluorescence intensity using the same standard solution was experienced, due to either changes in the internal diameter of the flow tube or variation of sulfuric acid strength from bottle to bottle. Therefore, it was found necessary to run a few standards daily, to obtain accurate results. Recovery of boron in different forms such as boric acid, borax, sodium perborate and tetraphenyl boron was also confirmed using the above procedure. Results shown in TABLE 1 indicate that all the above forms of boron react similarly with C H M B to produce fluorescence and hence this procedure can be used to determine total boron in any sample. TABLE 1. RESULTS OF ANALYSIS OF DIFFERENT BORON COMPOUNDS IN VARIOUS SAMPLE TYPES
Type of sample
Chemicalform added
Amount added (mg l- 1) 1.0 1.0 1.0 1.0
Amount found* (mg 1-1)
Deionized water Boric acid Borax Tetraphenyl boron Perborate
0.05, 0.1, 0.05, 0.1, 0.05, 0.1, 0.05, 0.1,
0.045, 0.1, 1.01 0.055, 0.98, 1.00 0.05, 0.105, 1.00 0.05, 0.105, 1.00
Detergent
Boric acid Borax Tetraphenyl boron Perborate
0.1, 0.1, 0.1, 0.I,
1.0, 5.0 1.0, 5.0 1.0, 5.0 1.0, 5.0
0.105, 0.95, 0.105, 0.10,
1.05, 1.00, 1.05, 1.00,
5.01 5.04 5.00 5.05
Sewage effluent
Boric acid Borax Tetraphenyl boron Perborate
0.5, 0.5, 0.5, 0.5,
1.0, 5.0 1.0, 5.0 1.0, 5.0 1.0, 5.0
0.505, 0.485, 0.500, 0.505,
1.00, 1.01, 1.04, 1.00,
5.05 4.95 5.01 5.03
* All the values are the averages of 10 determinations. Several samples were analyzed using the proposed method, and results were compared with those obtained using the potentiometric method and the spectrophotometric method using curcumin described in the Official Standard Methods for Examination of Water and Wastewater (1971). The results are shown in TABLE 2.
Analytical applications TABLE3 shows the results obtained by spiking some natural water samples, detergent solutions and sewage effluents.
Boron concentrations in natural waters across Canada Typical results showing the levels of boron found in water samples from the Maritime Provinces, Ontario and from Saskatchewan are shown in TABLE 3. Also shown
BADAR K. AFGHAN, PETER D. GOULDENand JAMESF. RYAN
1480
TABLE2. GXPARIS~N OF DIFFERENT METHODS USEDTO DETERMINE BORON Total amount found* (mg 1-V
Amount found (mg 1-Y
Amount added (mg 1-V
Potentiometric method Curcumin method Proposed fluorometric method
0.62 0.60
1.0 1.0
1.65 1.60
0.67
1.0
1.70
Lake water 2
Potentiometric method Curcumin method Fluorometric method
3.65 3.50 3.60
5.0 5.0 5.0
8.60 8.50 8.65
Lake water 3
Potentiometric method Curcumin method Fluorometric method
0.70 0.73 0.70
1.0 1.0 1.0
1.75 1.75 1.65
Lake water 4
Potentiometric method Curcumin method Fluorometric method
0.48 0.55 0.50
0.5 0.5 0.5
1.05 1.oo 1.05
Procedure used
Type of sample Lake water 1
* The results reported are the average of 10 replicates analyzed using different procedures. The numbers indicate different lake water samples taken at different locations each with different concentration of organic matter and major ions.
TABLE3. ANALYSISOF BORONIN DIFFERENT SAMPLESUSINGPROPOSEDPROCEDURE
Type of sample
Amount found (mg 1-Y
Amount added (mg 1-l)
Total amount found* (mg 1-Y
Lake water 1 Lake water 2 Lake water 3
0.005 0.030 0.7
0.05, 0.05, 0.50,
Snow sample 1 Snow sample 2 Snow sample 3
0.001 0.005 0.012
0.010, 0.025 0.010, 0.025 0.010, 0.025
0.009, 0.026 0.018, 0.032 0.020, 0.036
Raw sewage 1 Raw sewage 2
0.14 0.13
0.10, 0.10,
0.50 0.50
0.25, 0.21,
Final sewage effluent 1 Final sewage effluent 2
0.095 0.095
0.1, 0.1,
0.5 0.5
0.20, 0.590 0.195, 0.605
0.004 % Detergent solution 1 0.004% Detergent solution 2 0.004% Detergent solution 3
0.04 2.24 5.08
0.05, 1.0 1.0, 2.5 5.0, 10.0
Sea water 1 Sea water 2 Sea water 3
3.7 4.3 4.0
1.0, 1.0, 1.0,
* Total amount found is the average of five determinations
0.10 0.10 1.00
5.0 5.0 5.0
on each sample.
0.050, 0.100 0.075, 0.135 1.20, 1.705
0.65 0.64
0.085, 1.04 3.25, 4.73 10.05, 15.00 4.65, 5.25, 5.05,
8.75 9.30 9.02
Determination of Boron in Waters, Detergentsand SewageEffluents
1481
are the levels found in snow samples taken in the Ottawa area. These results show the need of a method capable of detecting 1/xg 1-1 monitoring Canadian waters. The levels of boron found in waters in England have been described by WAGOTT (1969) who found average levels of about 1 mg 1- ~ in river water. This high level is due to large amounts of sodium perborate used in laundry detergents in that country. In Canada there is very little usage of perborate in laundry detergents since Canadian housewives do not normally soak or boil their laundry, the two conditions under which perborate bleach is effective. DISCUSSION
Effect of temperature and time In preliminary studies using the manual method it was found that variation in temperature and time of fluorescence development had a pronounced effect on the intensity of fluorescence produced by the reaction of boron and CHMB. Therefore, the effect of temperature and time was studied in detail. Initially, the manifold for automated determination was designed so that it duplicated the chemistry of the manual procedure. The manifold used was essentially the same as in FIr. 1 except u.v. irradiation set-up. For these experiments 0.1 mg 1- ~ of boron solution as boric acid was sampled for 10 rain to give steady state values at various temperatures and time intervals. Sulfuric acid was mixed with the sample and the resultant mixture was cooled using a jacketed mixing coil with cold tap water before entering the oil bath. Special precautions were taken to ensure that there was no change in temperature when the reagent was mixed with the acidified sample, since the exothermic reaction between sulfuric acid and water would raise the temperature. The reagent, containing the same ratio of sulfuric acid to water, and the acidified sample were then preheated to a desired temperature in the oil bath before mixing. After attaining the desired temperature the acidified sample and the reagent were made to react by passing through a single and double mixing coil. The solutions were then cooled to 20°C before entering the flow cell for fluorescence measurements. The optimum temperature for the development of fluorescence was found to be between 50-55°C illustrated in Fir. 4. The time required for the maximum fluorescence development was investigated by changing the mixing coils and adding suitable delay coils in the oil bath which was maintained at 50-55°C. It was found that there was no significant variation of fluorescence intensity with time. Therefore, only two single mixing coils were used for further studies as illustrated in FIG. 1.
Effect of sulfuric acid-water ratio The manifold employed to determine the effect of sulfuric acid concentration on the fluorescence intensity of the boron-CHMB reaction was essentially the same as that used to investigate the effect of temperature and time. A 0.1 mg 1- x boron solution was sampled for 10 rain and the steady state values were compared using various ratios of water to sulfuric acid. To obtain various water to sulfuric acid ratios, the tubing delivering the sulfuric acid was changed to deliver calculated smaller volumes of the acid with the addition of extra deionized water pump tubes so that the total volume of acid and water delivered by the manifold was the same. By this arrangement it was possible to change the ratio
1482
BADAR K. AFGHAN, PETER D. GOULDEN and JAMES F. RYAN
100
80
60
40
20
I 20
l 40
I 60
I 8O
I 100
! 120
OIL BATH TEMPERATURE °C
F]o. 4. Effectof temperature on fluorescence. of water to sulfuric acid without changing the concentration of the sample in the sulfuric acid-sample mixture. Different reagent solutions were also substituted to contain suitable sulfuric acid to water ratios in each case to eliminate any temperature changes due to the exothermic reaction between water and sulfuric acid. The results shown in FIG. 5, indicate a rapid decrease in fluorescence intensity as the sulfuric acid concentration decreases much below 90 per cent. Because of this, 90 Yo sulfuric acid medium was used for further studies.
Interferences Major ions normally found in most fresh waters and seawater did not interfere using the above procedure. The above procedure was also evaluated for possible interferences from trace metals normally present in natural waters using concentrations at least 10--20 times in excess of those normally present. The metal ions investigated were antimony, arsenic, cadmium, chromium, cobalt, iron, indium, nickel, silver, vanadium and zinc. Vanadium gave the only interference, a positive interference where present in excess of 2.5 mg 1-1 over 0.1 mg 1-1 of boron. This interference can be eliminated by the use of ion exchange (MARTIN and HAVES, 1952). However, it is seldom that vanadium concentrations exceed these levels in samples from natural waters and any effluents. All the synthetic sewage components at 150 mg 1-1 levels did not interfere with the above procedure. The different synthetic sewage components included glucose, nutrient broth, beef extract, dipotassium hydrogen phosphate, peptone, urea and sodium chloride. The organic matter normally found in some natural waters and in sewage effluents gave a high background fluorescence. In order to determine boron in these samples it was necessary to first obtain the levels of background fluorescence by replacing the
Determination of Boron in Waters, Detergentsand SewageEffluents
1483
100
e) 8O z
z
6o
kl-
~ 4o
w
0
20
90
80
70
60
PER CENT(W~) SULFURICACID
FxG.
5. Effectof sulfuricacid concentrationon fluorescence.
reagent with concentrated sulfuric acid. This procedure was generally satisfactory but sometimes resulted in difficulties when small concentrations of boron were determined in samples which contained relatively high background fluorescence. It was therefore necessary to find a suitable method of removing the background fluorescence prior to the development of fluorescence due to boron and CHMB. Various chemical oxidizing agents and u.v.-irradiation were tried and it was found that u.v.-irradiation/ oxidation of the sample (AFGHANet al., 1970) prior to mixing with the reagent was the most suitable technique to eliminate interferences from organic matter. Water samples from Saskatchewan which typically gave maximum background fluorescence were chosen to determine the efficiency of u.v.-irradiation to remove this interference. Fifty-ml aliquots of these samples were transferred to quartz tubes and two drops of 30 ~o hydrogen peroxide were added to each quartz tube. The solutions were irradiated for 5, 10, 15, 30 and 60 min. At each interval background fluorescence of the aliquots was measured. FIGURE6 shows the effect of u.v.-irradiation on decrease in background fluorescence of the samples. TABLE4 shows the results obtained for a few Saskatchewan Water Samples using the background subtraction technique and u.v.-irradiation for 1 h followed by direct determination of boron. After confirming the manual u.v.-irradiation technique for the removal of organic interference, the possibility was investigated of automating this technique prior to the development of fluorescence for the determination of boron. In the automation of u.v.-irradiation it was found that by passing the sample through a quartz coil, rather than using quartz tubes, only 5-10 min irradiation time was required to completely remove background fluorescence due to organic compounds in all the samples analyzed in our laboratories. Therefore automated u.v.-irradiation of the sample was incorporated in the manifold which is shown in FIG. 1. The use of u.v.-irradiation was found to be the best solution to background fluorescence in a monitoring program and in none of the natural samples that have been WATER 6 / | 2 - - E
1484
BADAR K. AFGHAN, PETER D. GOULDEN a n d JAMES F. RYAN 100
o
80
z
o o tj
<= 6o O9 I-Z
40
o
20
®
o
13
I 10
I 20
I 30
TIME OF PHOTOLYStS,
I 40
I 50
I 60
rnin
FIG. 6. Effect of irradiation on background fluorescence.
examined was there found background fluorescence after the irradiation However, this technique has the demand for specialized equipment and in laboratories where it is not available the procedure of subtracting the separately-determined background will give satisfactory results for most natural water samples, although it is not as convenient. In some samples, such as sewage effluents, the background is too high for a satisfactory subtraction determination and in these cases it has been found that the interfering material can be removed by treatment with ion exchange resin. A variety of resins were investigated using undiluted effluent from a sewage plant as the test TABLE 4. COMPARISON OF RESULTS USING BACKGROUND-SUBTRACTION METHOD AND U.V. IRRADIATION
Sample
Result by background subtraction (mg 1- t boron)
Result after u.v.-irradiation (mg 1-1 boron)
1 2 3 4 5 6 7 8 9 10
0.70 0.70 0.08 0.33 0.02 0.29 0.14 0.04 0.04 0.05
0.69 0.71 0.10 0.31 0.03 0.27 0.14 O.O3 0.03 0.04
Determination of Boron in Waters, Detergents and Sewage Effluents
1485
m e d i u m . O f the resins examined " A m b e r l i t e A L 2 " was f o u n d to give the best r e m o v a l w i t h o u t r e m o v i n g a n y b o r o n . The procedure that has been f o u n d satisfactory is as follows: 100 ml of sample are stirred with 25 ml of 50 per cent Amberlite AL2 in xylene for 1 h. The phases are allowed to separate for 30 m i n a n d the aqueous phase is then analyzed as above. It should be stressed that even when u.v.-irradiation is used to destroy the backg r o u n d fluorescence it is necessary to check o n the completeness of removal o f the b a c k g r o u n d periodically or when samples of a completely u n k n o w n character are analyzed. This is done by replacing the C H M B reagent solution with sulfuric acid solution of the same concentration. Acknowledgements The authors thank Mr. S. GORURof the Department of the Environment and Mr. A. HOUCK of Green Creek Sewage Plant, Ottawa, for providing us with sewage samples. Technical assistance of Mr. E. ARNOLDis also appreciated for carrying out the experimental work to optimise ion exchange process for the removal of organic matter from sewage samples.
REFERENCES AFGHANB. K., GOULDENP. D. and RYANJ. F. (1970) Use of ultraviolet irradiation in the determination of nutrients in water with special reference to nitrogen; Technical Bulletin No. 40, Inland Waters Branch, Dept. of Energy, Mines and Resources, Ottawa, Canada, 1971. ELLIS G. H., ZOOKE. G. and BAr,InCH O. (1949) Colorimetric determination of boron using 1,1dianthrimide. Anal. Chem. 21, 1345-1348. HULTHE P., UPPSTROML. and OSTLINGG. (1970)An automated procedure for the determination of boron in sea water. Anal. Chem. Acta 51, 31-37. JAMESH. and KING G. H. (1966) A new automated determination of boron in the presence of nitrate. Automation in Analytical Chemistry, Paris, France, 123-127. LEVINSONA. A. (1971) An improved dianthrimide technique for the determination of boron in river waters. Water Research 5, 41-42. MARTINJ. R. and HAYESJ. R. (1952) Application of ion exchange to determination of boron. Anal. Chem. 24, 182-185. MONNIER D. and MARCANTONATOSM. (1966) Dosage direct de traces de bore dans l'acier par la methode fluorimetrique a 1-hydroxy-2-metho oxy-4-chloro-4'-benzophenone. Anal. Chem. Acta 36, 360-365. Standard Methods for the Examination of Water and Wastewater (1971) 13th edn. pp. 69-75. Am. Publ. Hlth Ass. WAGGOTA. (1969) An investigation of the potential problem of increasing boron concentrations in rivers and water courses. Water Research 3, 749-765. WHITEC. E. and HOFFMAND. E. (1957) Characteristics of boron-benzoincomplex--improved fluorometric determination of boron. Anal. Chem. 29, 1105-1108.
AUTOMATED FLUOROMETRIC METHOD FOR DETERMINATION OF BORON IN WATERS, DETERGENTS AND SEWAGE EFFLUENTS BADAR K. AFGHAN, PETER D. GOULDEN and JAMES F. RYAN Water Quality Division, Department of the Environment, Canada Centre for Inland Waters, Burlington, Canada (Received 1 October 1971)
Abstract--An automated method for the determination of boron in natural waters, detergents and sewageeffluents is described. The method is based on the reaction of 4'-chloro-2-hydroxy-4methoxybenzophenone (CHMB) with boron to produce fluorescent species, in a 90 ~ sulfuric acid medium. The method has been made specificto remove any interferences from all major and minor ions and other organic compounds normally present in water. The method is capable of measuring different chemical forms of boron such as boric acid, borax, sodium perborate and tetraphenyl boron. The method analyses 10 samples per hour, in the 5-100 p.g 1-1 boron range. The rate of sample analysis can be increased to 20 per hour at higher concentration ranges. The limit of detection is 1 leg 1-1 boron. INTRODUCTION THE WAVER Quality Division laboratories are engaged in a program of monitoring water quality across Canada by a national water quality network. Boron is one of the parameters that it is desired to measure. WAGGOTT (1969) has already discussed the potential problem of increasing boron concentration in natural waters in the British Isles. Although, at present, small amounts of boron are found in most waters in Canada, additional quantities may be introduced by industrial and domestic wastes. Standard methods for the examination of water and wastewater (1971) recommend the use of curcumin, carmine and potentiometric titration methods for boron. Spectrophotometric procedures using 1-1'-dianthrimide (ELLIS et al., 1949; LEVlNSON, 1971) are also used for boron determination. Fluorometric procedures, which are capable of greater sensitivity, have also been reported in the literature (WHITE and HOFFMAN, 1957; MON-NIER and MARCANTONATOS, 1966) but have not attained widespread use in water, detergent and effluent analysis. All these manual methods are either time consuming or require careful control of experimental conditions to obtain precise and accurate results and therefore are not suitable for monitoring purposes where a large number of samples have to be analyzed. There have been a few automated methods for the determination of boron reported (JAMESand KING, 1966; HULTHE et al., 1970), but unfortunately no detailed study has ever been carried out to compare various procedures and automate the most sensitive and simple procedure for this determination. In the Water Quality Division laboratories all the above procedures were evaluated in terms of sensitivity, selectivity, reproducibility, ease of automation and economy. The fluorometric procedure which utilizes 4'-chloro-2-hydroxy-4-methoxybenzophenone (CHMB) (MONNIERand MARCANTONATOS,1966) was found to be the most suitable among all the spectrophotometric, potentiometric and fluorometric procedures discussed above. Hence this method was automated. The method is based upon the reaction of boron with C H M B in a 90 ~ sulfuric acid medium and the measurement of the resultant fluorescence. The m a x i m u m excitation is from 340 to 380 nm and the maximum fluorescence emission is from 460 to 1475
1476
BADAR K. AFGHAN, PETER D. GOULDEN and JAMESF. RYAN
480 nm. The method has been shown to determine different forms of boron such as boric acid, borax, sodium perborate and tetraphenyl boron. The method has been made free of interferences from all major and minor ions and other compounds normally found in water. It was found that organic matter in some natural waters and sewage effluents produce high background fluorescence which was eliminated by photooxidation of the sample prior to the development of fluorescence. The method is capable of analyzing samples at the rate of 10 samples h - ~ in the 5-100 ~g 1-1range. Concentration as low as 1 t~g 1-1 can easily be detected. The rate of sample analysis can be increased to 20 h-a at higher concentration ranges. In the Water Quality Division laboratories the above method has been successfully employed for the analysis of total boron in filtered natural water .samples across Canada and for the analysis of boron in sea water, detergents and sewage effluents for over 18 months. EXPERIMENTAL
Manifold The manifold used is shown in FIG. 1. All the manifold pump tubes and transmission
tubes were acidflex. Tubing for the sample aspiration and for filling the wash receptable were ordinary standard tubing. For sleeving purposes, green acidflex tubing was employed. To obtain reproducible results it was found necessary to arrange all the large tubes on one side of the pump, descending to small tubes on the other side. The sample was aspirated at a rate of 0.32 ml min-1 and was mixed with concentrated sulfuric acid at a rate of 3.42 rrd rain- 1. The acidified sample was segmented, with 1.60
ml min-1 of air and irradiated for approximately 10 min in a u.v. reactor. The air flow through the photolysis equipment was adjusted so that the solution leaving the CAM - 10 SAMPLESPER HOUR 1:2 SAMPLETO WASH RATIO
RECEPTACLE
TUBE SEE (INCHES) DEIONIZEDWATER 0.01%C.H.M.B. SAMPLE AIR SULFURK3ACID D-o k___
D-O [ ~
0.040 0.035°
SMC O0
o.o o
o.o8~, tQQQQI 0.081" o°°8" o81. 0°~5 ~~ - ' o-o O.V.~ACTOR I 1
I PUMPI
WASTE ~
RECORDER
9
HEATING BATH 50.55oC
PULSESUPPRESSORS 1o0.025 INCH I.D.ACIDFLEX MANIFOLDTUBE 2-0.020 ORANGE-YELLOW 3-0.005 ORANGE-BLACK
"acidflex
SMC 105-0084
FLUORIMETER II PRIMARY-355nm SECONDARYo 46Ohm SAMPLEAPERTURE- 4 REFERENCEAPERTURE- 2
FIG. 1. Manifold for proposed method.
DMC 105-0085
~
HAAKE-FK CONSTANT TEMPERATURE CRCULATOR 20°C
Determination of Boron in Waters, Detergents and Sewage Effluents
1477
reactor was 42 4- I°C. After irradiation the sample was preheated to an optimum temperature of fluorescence development by passing through a 3-mm i.d. single mixing coil in a Technicon oil bath which was maintained at 50-55°C. The reagent was then added while the sample remained in the bath and was passed through a series of two 3-mm i.d. single mixing coils. After development of fluorescence the solution was passed through a 3-mm i.d. double mixing coil in a Haake constant temperature water bath at 20°C prior to entering the fluorometric flow cell. The solution was drawn through the flow cell at a rate of 3.42 ml rain -1, and was excited using an 85 W u.v. lamp in conjunction with a 355-nm narrow pass primary filter. A 460-nm narrow pass secondary filter was used to measure the resulting fluorescence intensity.
Apparatus All AutoAnalyzer equipment used was standard Technicon modules. A Haake Model F K constant temperature circulator was used to maintain a temperature of 20°C during fluorescence measurement in the flow cell. The u.v.-reactor was made as shown in FIG. 2. A 550-W photochemical lamp was placed inside the fused quartz lamp protection jacket which is plugged at the bottom with 1-in. asbestos tape and
a
c
Ill
4 in.
/
b J
\
d
FIG. 2. Ultraviolet-irradiator (a) chimney, (b) quartz coil, (c) ultraviolet-lamp, (d) cooling fan.
mounted axially in the centre of the galvanized iron cylinder body in the reactor. A quartz coil was placed in such a way that the u.v.-lamp could irradiate all the sample in the coil. The quartz coil was made of Purci1453 quality fused silica tubing approximately 10 mm long, 3 mm i.d., 0.6 mm wall thickness and the coil diameter is approximately 5 in.
1478
BADARK. AFGHANPETER , D. GOULDENand JAMESF. RYAN
Sample preparation For water samples and detergent solutions 2-3 drops of 30 ~ hydrogen peroxide was added to each of 50 ml of samples prior to analysis. This is added to assure oxidizing conditions. Water samples were filtered through a 0.45/zm membrane filter prior to the addition of hydrogen peroxide. For the determination of boron in detergents 100 mg of detergent was dissolved in 1 1. of water and this solution, diluted if necessary, was analyzed in a similar way to natural water samples. For the analysis of sewage effluents the suspended material in the sample was first removed by centrifugation at 15,000 rev/min. The resultant solution was filtered through a 0.45-Fm membrane filter and treated in a manner similar to that used for water samples. Procedure The manifold AutoAnalyzer equipment was connected as shown in FIG. 1. This involves two steps (1) oxidation of any organic material (II) development of fluorescence to determine boron. The sample tray was filled and each sample withdrawn was first oxidized by u.v.irradiation to remove any background fluorescence and then analyzed for boron content, as shown in FIG. 1. RESULTS Calibration curves and related accuracy studies The calibration curves were run in the ranges of 5-50/zg 1-l, 10--100/zg 1-1 and 0.1-0.4 mg 1-1 by changing the sensitivity and full scaler response of the fluorometer. All calibration curves gave straight lines passing through the origin, using the manifold illustrated in FIG. 1. The lower detection limit of the method was found to be approxi100
100 ppb 90 -80
80ppb
70 60ppb
60 50 40 ppb 40 30 20 10
~
20ppb
/2 i
I
f
I
t
1
FIG. 3. Typicalcalibrationcurve.
f
!
Determination of Boron in Waters, Detergents and Sewage Effluents
1479
mately 1/zg 1-1. The typical calibration curve most frequently used is shown in FI6. 3. Identical calibration curves were also obtained in natural waters, synthetic lake water, solutions containing effluent samples and detergents. The precision of the methods was evaluated by determining the per cent standard deviation of a series of 50 solutions each containing 100/~g 1-1 of boron. The coefficient of variation was found to be 1.87 per cent. Replicate analysis of several samples from across Canada gave values within the standard deviation mentioned above. Occasionally, variation of fluorescence intensity using the same standard solution was experienced, due to either changes in the internal diameter of the flow tube or variation of sulfuric acid strength from bottle to bottle. Therefore, it was found necessary to run a few standards daily, to obtain accurate results. Recovery of boron in different forms such as boric acid, borax, sodium perborate and tetraphenyl boron was also confirmed using the above procedure. Results shown in TABLE 1 indicate that all the above forms of boron react similarly with C H M B to produce fluorescence and hence this procedure can be used to determine total boron in any sample. TABLE 1. RESULTS OF ANALYSIS OF DIFFERENT BORON COMPOUNDS IN VARIOUS SAMPLE TYPES
Type of sample
Chemicalform added
Amount added (mg l- 1) 1.0 1.0 1.0 1.0
Amount found* (mg 1-1)
Deionized water Boric acid Borax Tetraphenyl boron Perborate
0.05, 0.1, 0.05, 0.1, 0.05, 0.1, 0.05, 0.1,
0.045, 0.1, 1.01 0.055, 0.98, 1.00 0.05, 0.105, 1.00 0.05, 0.105, 1.00
Detergent
Boric acid Borax Tetraphenyl boron Perborate
0.1, 0.1, 0.1, 0.I,
1.0, 5.0 1.0, 5.0 1.0, 5.0 1.0, 5.0
0.105, 0.95, 0.105, 0.10,
1.05, 1.00, 1.05, 1.00,
5.01 5.04 5.00 5.05
Sewage effluent
Boric acid Borax Tetraphenyl boron Perborate
0.5, 0.5, 0.5, 0.5,
1.0, 5.0 1.0, 5.0 1.0, 5.0 1.0, 5.0
0.505, 0.485, 0.500, 0.505,
1.00, 1.01, 1.04, 1.00,
5.05 4.95 5.01 5.03
* All the values are the averages of 10 determinations. Several samples were analyzed using the proposed method, and results were compared with those obtained using the potentiometric method and the spectrophotometric method using curcumin described in the Official Standard Methods for Examination of Water and Wastewater (1971). The results are shown in TABLE 2.
Analytical applications TABLE3 shows the results obtained by spiking some natural water samples, detergent solutions and sewage effluents.
Boron concentrations in natural waters across Canada Typical results showing the levels of boron found in water samples from the Maritime Provinces, Ontario and from Saskatchewan are shown in TABLE 3. Also shown
BADAR K. AFGHAN, PETER D. GOULDENand JAMESF. RYAN
1480
TABLE2. GXPARIS~N OF DIFFERENT METHODS USEDTO DETERMINE BORON Total amount found* (mg 1-V
Amount found (mg 1-Y
Amount added (mg 1-V
Potentiometric method Curcumin method Proposed fluorometric method
0.62 0.60
1.0 1.0
1.65 1.60
0.67
1.0
1.70
Lake water 2
Potentiometric method Curcumin method Fluorometric method
3.65 3.50 3.60
5.0 5.0 5.0
8.60 8.50 8.65
Lake water 3
Potentiometric method Curcumin method Fluorometric method
0.70 0.73 0.70
1.0 1.0 1.0
1.75 1.75 1.65
Lake water 4
Potentiometric method Curcumin method Fluorometric method
0.48 0.55 0.50
0.5 0.5 0.5
1.05 1.oo 1.05
Procedure used
Type of sample Lake water 1
* The results reported are the average of 10 replicates analyzed using different procedures. The numbers indicate different lake water samples taken at different locations each with different concentration of organic matter and major ions.
TABLE3. ANALYSISOF BORONIN DIFFERENT SAMPLESUSINGPROPOSEDPROCEDURE
Type of sample
Amount found (mg 1-Y
Amount added (mg 1-l)
Total amount found* (mg 1-Y
Lake water 1 Lake water 2 Lake water 3
0.005 0.030 0.7
0.05, 0.05, 0.50,
Snow sample 1 Snow sample 2 Snow sample 3
0.001 0.005 0.012
0.010, 0.025 0.010, 0.025 0.010, 0.025
0.009, 0.026 0.018, 0.032 0.020, 0.036
Raw sewage 1 Raw sewage 2
0.14 0.13
0.10, 0.10,
0.50 0.50
0.25, 0.21,
Final sewage effluent 1 Final sewage effluent 2
0.095 0.095
0.1, 0.1,
0.5 0.5
0.20, 0.590 0.195, 0.605
0.004 % Detergent solution 1 0.004% Detergent solution 2 0.004% Detergent solution 3
0.04 2.24 5.08
0.05, 1.0 1.0, 2.5 5.0, 10.0
Sea water 1 Sea water 2 Sea water 3
3.7 4.3 4.0
1.0, 1.0, 1.0,
* Total amount found is the average of five determinations
0.10 0.10 1.00
5.0 5.0 5.0
on each sample.
0.050, 0.100 0.075, 0.135 1.20, 1.705
0.65 0.64
0.085, 1.04 3.25, 4.73 10.05, 15.00 4.65, 5.25, 5.05,
8.75 9.30 9.02
Determination of Boron in Waters, Detergentsand SewageEffluents
1481
are the levels found in snow samples taken in the Ottawa area. These results show the need of a method capable of detecting 1/xg 1-1 monitoring Canadian waters. The levels of boron found in waters in England have been described by WAGOTT (1969) who found average levels of about 1 mg 1- ~ in river water. This high level is due to large amounts of sodium perborate used in laundry detergents in that country. In Canada there is very little usage of perborate in laundry detergents since Canadian housewives do not normally soak or boil their laundry, the two conditions under which perborate bleach is effective. DISCUSSION
Effect of temperature and time In preliminary studies using the manual method it was found that variation in temperature and time of fluorescence development had a pronounced effect on the intensity of fluorescence produced by the reaction of boron and CHMB. Therefore, the effect of temperature and time was studied in detail. Initially, the manifold for automated determination was designed so that it duplicated the chemistry of the manual procedure. The manifold used was essentially the same as in FIr. 1 except u.v. irradiation set-up. For these experiments 0.1 mg 1- ~ of boron solution as boric acid was sampled for 10 rain to give steady state values at various temperatures and time intervals. Sulfuric acid was mixed with the sample and the resultant mixture was cooled using a jacketed mixing coil with cold tap water before entering the oil bath. Special precautions were taken to ensure that there was no change in temperature when the reagent was mixed with the acidified sample, since the exothermic reaction between sulfuric acid and water would raise the temperature. The reagent, containing the same ratio of sulfuric acid to water, and the acidified sample were then preheated to a desired temperature in the oil bath before mixing. After attaining the desired temperature the acidified sample and the reagent were made to react by passing through a single and double mixing coil. The solutions were then cooled to 20°C before entering the flow cell for fluorescence measurements. The optimum temperature for the development of fluorescence was found to be between 50-55°C illustrated in Fir. 4. The time required for the maximum fluorescence development was investigated by changing the mixing coils and adding suitable delay coils in the oil bath which was maintained at 50-55°C. It was found that there was no significant variation of fluorescence intensity with time. Therefore, only two single mixing coils were used for further studies as illustrated in FIG. 1.
Effect of sulfuric acid-water ratio The manifold employed to determine the effect of sulfuric acid concentration on the fluorescence intensity of the boron-CHMB reaction was essentially the same as that used to investigate the effect of temperature and time. A 0.1 mg 1- x boron solution was sampled for 10 rain and the steady state values were compared using various ratios of water to sulfuric acid. To obtain various water to sulfuric acid ratios, the tubing delivering the sulfuric acid was changed to deliver calculated smaller volumes of the acid with the addition of extra deionized water pump tubes so that the total volume of acid and water delivered by the manifold was the same. By this arrangement it was possible to change the ratio
1482
BADAR K. AFGHAN, PETER D. GOULDEN and JAMES F. RYAN
100
80
60
40
20
I 20
l 40
I 60
I 8O
I 100
! 120
OIL BATH TEMPERATURE °C
F]o. 4. Effectof temperature on fluorescence. of water to sulfuric acid without changing the concentration of the sample in the sulfuric acid-sample mixture. Different reagent solutions were also substituted to contain suitable sulfuric acid to water ratios in each case to eliminate any temperature changes due to the exothermic reaction between water and sulfuric acid. The results shown in FIG. 5, indicate a rapid decrease in fluorescence intensity as the sulfuric acid concentration decreases much below 90 per cent. Because of this, 90 Yo sulfuric acid medium was used for further studies.
Interferences Major ions normally found in most fresh waters and seawater did not interfere using the above procedure. The above procedure was also evaluated for possible interferences from trace metals normally present in natural waters using concentrations at least 10--20 times in excess of those normally present. The metal ions investigated were antimony, arsenic, cadmium, chromium, cobalt, iron, indium, nickel, silver, vanadium and zinc. Vanadium gave the only interference, a positive interference where present in excess of 2.5 mg 1-1 over 0.1 mg 1-1 of boron. This interference can be eliminated by the use of ion exchange (MARTIN and HAVES, 1952). However, it is seldom that vanadium concentrations exceed these levels in samples from natural waters and any effluents. All the synthetic sewage components at 150 mg 1-1 levels did not interfere with the above procedure. The different synthetic sewage components included glucose, nutrient broth, beef extract, dipotassium hydrogen phosphate, peptone, urea and sodium chloride. The organic matter normally found in some natural waters and in sewage effluents gave a high background fluorescence. In order to determine boron in these samples it was necessary to first obtain the levels of background fluorescence by replacing the
Determination of Boron in Waters, Detergentsand SewageEffluents
1483
100
e) 8O z
z
6o
kl-
~ 4o
w
0
20
90
80
70
60
PER CENT(W~) SULFURICACID
FxG.
5. Effectof sulfuricacid concentrationon fluorescence.
reagent with concentrated sulfuric acid. This procedure was generally satisfactory but sometimes resulted in difficulties when small concentrations of boron were determined in samples which contained relatively high background fluorescence. It was therefore necessary to find a suitable method of removing the background fluorescence prior to the development of fluorescence due to boron and CHMB. Various chemical oxidizing agents and u.v.-irradiation were tried and it was found that u.v.-irradiation/ oxidation of the sample (AFGHANet al., 1970) prior to mixing with the reagent was the most suitable technique to eliminate interferences from organic matter. Water samples from Saskatchewan which typically gave maximum background fluorescence were chosen to determine the efficiency of u.v.-irradiation to remove this interference. Fifty-ml aliquots of these samples were transferred to quartz tubes and two drops of 30 ~o hydrogen peroxide were added to each quartz tube. The solutions were irradiated for 5, 10, 15, 30 and 60 min. At each interval background fluorescence of the aliquots was measured. FIGURE6 shows the effect of u.v.-irradiation on decrease in background fluorescence of the samples. TABLE4 shows the results obtained for a few Saskatchewan Water Samples using the background subtraction technique and u.v.-irradiation for 1 h followed by direct determination of boron. After confirming the manual u.v.-irradiation technique for the removal of organic interference, the possibility was investigated of automating this technique prior to the development of fluorescence for the determination of boron. In the automation of u.v.-irradiation it was found that by passing the sample through a quartz coil, rather than using quartz tubes, only 5-10 min irradiation time was required to completely remove background fluorescence due to organic compounds in all the samples analyzed in our laboratories. Therefore automated u.v.-irradiation of the sample was incorporated in the manifold which is shown in FIG. 1. The use of u.v.-irradiation was found to be the best solution to background fluorescence in a monitoring program and in none of the natural samples that have been WATER 6 / | 2 - - E
1484
BADAR K. AFGHAN, PETER D. GOULDEN a n d JAMES F. RYAN 100
o
80
z
o o tj
<= 6o O9 I-Z
40
o
20
®
o
13
I 10
I 20
I 30
TIME OF PHOTOLYStS,
I 40
I 50
I 60
rnin
FIG. 6. Effect of irradiation on background fluorescence.
examined was there found background fluorescence after the irradiation However, this technique has the demand for specialized equipment and in laboratories where it is not available the procedure of subtracting the separately-determined background will give satisfactory results for most natural water samples, although it is not as convenient. In some samples, such as sewage effluents, the background is too high for a satisfactory subtraction determination and in these cases it has been found that the interfering material can be removed by treatment with ion exchange resin. A variety of resins were investigated using undiluted effluent from a sewage plant as the test TABLE 4. COMPARISON OF RESULTS USING BACKGROUND-SUBTRACTION METHOD AND U.V. IRRADIATION
Sample
Result by background subtraction (mg 1- t boron)
Result after u.v.-irradiation (mg 1-1 boron)
1 2 3 4 5 6 7 8 9 10
0.70 0.70 0.08 0.33 0.02 0.29 0.14 0.04 0.04 0.05
0.69 0.71 0.10 0.31 0.03 0.27 0.14 O.O3 0.03 0.04
Determination of Boron in Waters, Detergents and Sewage Effluents
1485
m e d i u m . O f the resins examined " A m b e r l i t e A L 2 " was f o u n d to give the best r e m o v a l w i t h o u t r e m o v i n g a n y b o r o n . The procedure that has been f o u n d satisfactory is as follows: 100 ml of sample are stirred with 25 ml of 50 per cent Amberlite AL2 in xylene for 1 h. The phases are allowed to separate for 30 m i n a n d the aqueous phase is then analyzed as above. It should be stressed that even when u.v.-irradiation is used to destroy the backg r o u n d fluorescence it is necessary to check o n the completeness of removal o f the b a c k g r o u n d periodically or when samples of a completely u n k n o w n character are analyzed. This is done by replacing the C H M B reagent solution with sulfuric acid solution of the same concentration. Acknowledgements The authors thank Mr. S. GORURof the Department of the Environment and Mr. A. HOUCK of Green Creek Sewage Plant, Ottawa, for providing us with sewage samples. Technical assistance of Mr. E. ARNOLDis also appreciated for carrying out the experimental work to optimise ion exchange process for the removal of organic matter from sewage samples.
REFERENCES AFGHANB. K., GOULDENP. D. and RYANJ. F. (1970) Use of ultraviolet irradiation in the determination of nutrients in water with special reference to nitrogen; Technical Bulletin No. 40, Inland Waters Branch, Dept. of Energy, Mines and Resources, Ottawa, Canada, 1971. ELLIS G. H., ZOOKE. G. and BAr,InCH O. (1949) Colorimetric determination of boron using 1,1dianthrimide. Anal. Chem. 21, 1345-1348. HULTHE P., UPPSTROML. and OSTLINGG. (1970)An automated procedure for the determination of boron in sea water. Anal. Chem. Acta 51, 31-37. JAMESH. and KING G. H. (1966) A new automated determination of boron in the presence of nitrate. Automation in Analytical Chemistry, Paris, France, 123-127. LEVINSONA. A. (1971) An improved dianthrimide technique for the determination of boron in river waters. Water Research 5, 41-42. MARTINJ. R. and HAYESJ. R. (1952) Application of ion exchange to determination of boron. Anal. Chem. 24, 182-185. MONNIER D. and MARCANTONATOSM. (1966) Dosage direct de traces de bore dans l'acier par la methode fluorimetrique a 1-hydroxy-2-metho oxy-4-chloro-4'-benzophenone. Anal. Chem. Acta 36, 360-365. Standard Methods for the Examination of Water and Wastewater (1971) 13th edn. pp. 69-75. Am. Publ. Hlth Ass. WAGGOTA. (1969) An investigation of the potential problem of increasing boron concentrations in rivers and water courses. Water Research 3, 749-765. WHITEC. E. and HOFFMAND. E. (1957) Characteristics of boron-benzoincomplex--improved fluorometric determination of boron. Anal. Chem. 29, 1105-1108.