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A simplified hydrazine-reduction method for determining high concentrations of nitrate in recirculated seawater
Aquaculture, 21 (1980) 281-286 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
281
A SIMPLIFIED HYDRAZINE-REDUCTION METHOD FOR DETERMINING HIGH CONCENTRATIONS OF NITRATE IN RECIRCULATED SEAWATER’
CAROL E. BOWER and THOMAS HOLM-HANSEN Sea Research Foundation, CT 06107 (U.S.A.) (Accepted
Institute
for Aquarium
Studies,
P.O. Box 7-495, West Hartford,
20 December 1979)
ABSTRACT Bower, C.E. and Helm-Hansen, T., 1980. A simplified hydrazine-reduction method for determining high concentrations of nitrate in recirculated seawater. Aquaculture, 21: 281-286. A manual method is described in which nitrate is reduced to nitrite in 2 h by hydrazine in the presence of copper; the reducing medium is buffered to pH 10 with cyclohexylaminopropane sulfonic acid and sodium hydroxide. No special apparatus is required, and reduction is not affected by normal laboratory lighting or changes in ambient temperature within the range of 23 to 27’C. Absorbances are linear for concentrations from 1 to 20 mg NO,-N/l. The standard deviations for samples containing 5 and 20 mg NO,-N/l were 0.1 and 0.2, respectively.
INTRODUCTION
Recirculated seawater in culture systems with biological filtration is characterized by a high concentration of nitrate. After several months, levels from about 8 to 100 mg NO,-N/I are common in aquarium water (Saeki, 1962), and concentrations as high as 309 mg NO,-N/l have been reported (Oliver, 1957). Although many marine animals are tolerant of high nitrate concentrations (De Graaf, 1964; Hirayama, 1966; Kuwatani et al., 1969; Epifanio and Srna, 1975), the accumulation of nitrate in recirculated water appears to be accompanied by other, generally detrimental chemical changes (King, 1973). Nitrate can, therefore, serve as a useful indicator of overall water quality and a standard against which the need for water replacement can be gauged. In the standard assay for nitrate in seawater (Strickland and Parsons, 1977), nitrate is reduced to nitrite by passage through a column containing copperized-cadmium filings, and the resulting nitrite is determined calorimetrically. ’ Contribution
No. 10, Sea Research Foundation,
0044-8486/80/0000-0000/$02.50
0 1980
Inc.
Elsevier Scientific Publishing Company
282
We found this method impractical for monitoring nitrate accumulation in culture water chiefly because of the expense and complexity of preparing and maintaining the reduction column. In an earlier method (Mullin and Riley, 1955) that was modified by Strickland and Parsons (1960) and subsequently adapted for high nitrate determinations by King and Spotte (1974), copper was used to catalyze the reduction of nitrate by hydrazine in a solution buffered with phenol and sodium hydroxide. Reduction had to be carried out in a constant temperature bath in the dark, and required 20 h for completion. In routine use of this method, we encountered high reagent blanks (ca. 0.03 to 0.04 absorbance units), which probably resulted from oxidation of the phenol reagent. The procedure given here is a modification of earlier hydrazine-reduction methods for determining high concentrations of nitrate in seawater. A more stable organic acid has been substituted for phenol in the buffer solution, reduction time has been decreased to 2 h, and determinations can be made at ambient temperatures under’artificial light. MATERIALS
AND METHODS
Reagents All chemicals were analytical reagent grade. Solutions were prepared with tap water passed through a mixed-resin deionizing column (Crystalab DI-3) immediately before use. Buffer solution. 60 g cyclohexylaminopropane sulfonic acid (CAPS, Sigma Chemical Co., St. Louis, MO) + 3.720 g sodium hydroxide in 1 1 of water. Stable. Hydrazine sulfate solution. 7.250 g/l. Stable for at least 1 month. Copper sulfate solution. 0.100 g/l. Stable. Reducer solution. Equal volumes of hydrazine sulfate and copper sulfate solutions, used within 1 h of mixing. Acetone. Sulfunilumide solution. 10 g/l in 10% concentrated hydrochloric acid. Stable. NED solution. 1 g N-l- naphthylethylenediamine dihydrochloride/l. Stable for at least 1 month. Sodium nitrate standard solutions. Stock solution (100 mg NO,-N/l) 0.607 g NaNOJ. Working standard solution (10 mg NO,-N/l) - 5 ml stock solution diluted to 50 ml; prepared fresh daily. The buffer, hydrazine sulfate, and NED solutions were stored in amber glass bottles in a refrigerator. Procedure Samples consisted of 1 ml seawater containing 1 to 20 mg NO,-N/l or 0.5 ml seawater containing > 20 mg NO,-N/l, diluted to 50 ml with deionized water. Calibration standards were 1 ml working standard solution in seawater or deionized water, diluted to 50 ml with deionized water. Blanks consisted of 50 ml deionized water or 1 ml seawater diluted to 50 ml with deionized
283
water. Artificial seawater (Instant Ocean@, Aquarium Systems, Mentor, OH, S = 30%0) was used throughout. Glassware was washed with detergent and rinsed copiously with deionized water before use. All determinations were carried out in triplicate, unless otherwise noted, using 125-ml Erlenmeyer flasks held at ambient temperature (23 to 27°C). Lighting was from four 40-W “cool white” fluorescent lamps at a distance of 2 m (ca. 10 c/ft*). To each sample, standard solution and blank, 2 ml buffer solution, then 1 ml reducer solution were dispensed from graduated pipettes. Contents were mixed and the flasks were stoppered. After 2 h of reduction, 2 ml acetone was added to the series of flasks in rapid succession, to stop reduction by destroying excess hydrazine. After 2 min, 1 ml sulfanilamide solution and 1 ml NED solution were added sequentially, with 2 to 8 min allowed between additions. After 15 min but before 2 h, absorbances were read against deionized water at 543 nm with a Turner Model 330 spectrophotometer (AMSCO Instrument Co., Carpenteria, CA), using lo-cm length cuvettes. Absorbances were converted to concentrations of N03-N in mg/l by using the equation NOJ-N = (Es - Eb)F
(1) in which E, is the mean sample absorbance, Eb is the mean blank absorbance, and F is a conversion factor representing the quotient of the concentration of the calibration standard (10 mg/l) divided by the mean corrected absorbance of the standard (Estandard - Eb). When nitrate standards were prepared with seawater, their corrected absorbances were obtained by subtracting the mean absorbance of seawater blanks. RESULTS
AND DISCUSSION
Reagent concentrations Maximum absorbances were obtained with a reduction pH of 10 f 0.05, achieved with 3.5 X 10m3M sodium hydroxide and 0.01 M CAPS (pK, = 10.4) in the reducing medium. Erratic absorbances occurred at higher and lower pH values, and low recoveries resulted when the concentration of CAPS was decreased. The concentrations of hydrazine sulfate and copper sulfate recommended by Strickland and Parsons (1960) were optimal Reduction
time
When nitrate is reduced by hydrazine, an 85% yield of nitrite should result after 2 h (Mullin and Riley, 1955). Based on the absorbances of our reduced-nitrate samples and those of samples spiked with equivalent concentrations of nitrite-nitrogen, only 38% reduction occurs in 2 h. We found that a 2-h reduction period was sufficient to give reliable determinations. After only 1 h of reduction, standard deviations were greater, and the sensitivity of the test was lower.
Light and temperature We could detect no differences between the absorbances of samples reduced in the dark and under artificial light. Changes of 2°C in ambient temperature during the reduction period had no effect on absorbances or recoveries. Inhibition of reduction in seawater Known concentrations of nitrate in seawater, diluted with deionized water and treated by the standard procedure, consistently absorbed about 5% lower than those in 100% deionized water. The phenomenon occurred regardless of whether natural or artificial seawater was used. At the salinity of our diluted samples (0.6°/oo), there should be no salt effect on reduction (Mullin and Riley, 1955). Although the concentration of magnesium in our samples might inhibit reduction by precipitating as magnesium hydroxide at pH 10 (Mullin and Riley, 1955), we could not relate discrepant absorbances to the presence of magnesium, alone, nor could any other cause be isolated. It is therefore necessary that the absorbances of deionized water blanks and calibration standards be multiplied by 0.95, before using them to calculate conversion factors for seawater samples. Calibration curve A calibration curve was prepared with known concentrations of nitrate in seawater diluted 1: 50 with deionized water and treated as described (Fig. 1). Beer’s law was obeyed in the concentration range from 1 to 20 mg NO,-N/l. Calibration curves were constant to within 5% over a one-month period, and were not affected by the preparation of fresh reagents.
mg NO,-N/I
Fig. 1. Calibration curve for nitrate determination by the 2-h hydrazine-reduction Concentrations refer to mg NO,-N/l in the original l-ml seawater samples.
method.
Comparison with another hydrazine method The concentration of nitrate in four established seawater aquariums was determined by a 20-h hydrazine-reduction method (King and Spotte, 1974) and by the 2-h method. As shown in Table I, there was excellent agreement between the two methods. TABLE I Comparison of recoveries of nitrate from aquarium seawater by the 2-h reduction method and an earlier, 20-h reduction methoda Sample no.b
1 2 3 4
mg NO,-N recovered (SD) 2-h reduction
20-h reduction
66.3 21.4 38.4 41.6
67.4 21.8 37.8 41.2
(0) (0.1) (0.5) (0.4)
(2.0) (0.5) (0.4) (0.7)
Difference (%)
1.7 1.9 1.6 1.0
aprocedure from King and Spotte (1974). “N= 3.
Accuracy and reproducibility The accuracy of the 2-h reduction method was determined by analyzing triplicate samples of seven known nitrate concentrations in seawater, and then calculating the percent recoveries. Results are given in Table II. The standard deviation was 0.10 (2.0%) for 5 mg NO,-N/l and 0.2 (1.0%) for 20 mg NO,-N/l, indicating excellent reproducibility. The absorbances of reagent blanks were always less than 0.01. TABLE II Cont. NO,-N (mg/I)a
Recoveryb nig NO,-N/I (SD)
Percent
1 5 10 15 20 25 30
1.0 (0.1) 5.1 (0.1) 10.0 (0.3) 14.6 (0.2) 19.1(0.2) 23.6 (0.4) 27.0 (0)
100 102 100 97 96 94 90
Woncentration bN=3.
in the original 1-mI sample.
286 ACKNOWLEDGMENTS
We are grateful to Dr. James W. Atz and Stephen Spotte for reading and criticizing the manuscript. REFERENCES De Graaf, F., 1964. Maintenance problems in large public aquaria. Arch. Neerl. Zool., 16: 142-143. Epifanio, C.E. and Srna, R.F., 1975. Toxicity of ammonia, nitrite ion, nitrate ion, and orthophosphate to Mercenaria mercenaria and Crassostrea virginica. Mar. Biol., 33: 241-246. Hirayama, K., 1966. Influence of nitrate accumulated in culturing water on Octopus vulgaris. Bull. Jap. Sot. Sci. Fish., 32: 105-111. King, J.M., 1973. Recirculating system culture methods for marine organisms. SEA Scope, 3(l): 1;6-8. King, J.M. and Spotte, S., 1974. Marine Aquariums in the Research Laboratory. Aquarium Systems, Inc., Eastlake, Ohio, 39 pp. Kuwatani, Y., Nishii, T. and Isogai, F., 1969. Effects of nitrate in culture water on the growth of the Japanese pearl oyster. Bull. Natl. Pearl Res. Lab,, 14: 1735-1747. Mullin, J.B. and Riley, J.P., 1955. The spectrophotometric determination of nitrate in natural waters, with particular reference to sea-water. Anal. Chim. Acta, 12: 464-480. Oliver, J.H., 1957. The chemical composition of the sea water in the aquarium. Proc. Zool. Sot. London, 129: 137-145. Saeki, A., 1962. The composition and some chemical control of the sea water of the closed circulating aquarium. Bull. Mar. Biol. Stn. Asamushi., Tokohu Univ., 1: 99-104. Strickland, J.D.H. and Parsons, T.R., 1960. A manual of sea water analysis. Fish. Res. Board Can. Bull., No. 125: 61-69. Strickland, J.D.H. and Parsons, T.R., 1977. A practical handbook of seawater analysis, 2nd ed. Fish. Res. Board Can. Bull., No. 167: 71-76.
281
A SIMPLIFIED HYDRAZINE-REDUCTION METHOD FOR DETERMINING HIGH CONCENTRATIONS OF NITRATE IN RECIRCULATED SEAWATER’
CAROL E. BOWER and THOMAS HOLM-HANSEN Sea Research Foundation, CT 06107 (U.S.A.) (Accepted
Institute
for Aquarium
Studies,
P.O. Box 7-495, West Hartford,
20 December 1979)
ABSTRACT Bower, C.E. and Helm-Hansen, T., 1980. A simplified hydrazine-reduction method for determining high concentrations of nitrate in recirculated seawater. Aquaculture, 21: 281-286. A manual method is described in which nitrate is reduced to nitrite in 2 h by hydrazine in the presence of copper; the reducing medium is buffered to pH 10 with cyclohexylaminopropane sulfonic acid and sodium hydroxide. No special apparatus is required, and reduction is not affected by normal laboratory lighting or changes in ambient temperature within the range of 23 to 27’C. Absorbances are linear for concentrations from 1 to 20 mg NO,-N/l. The standard deviations for samples containing 5 and 20 mg NO,-N/l were 0.1 and 0.2, respectively.
INTRODUCTION
Recirculated seawater in culture systems with biological filtration is characterized by a high concentration of nitrate. After several months, levels from about 8 to 100 mg NO,-N/I are common in aquarium water (Saeki, 1962), and concentrations as high as 309 mg NO,-N/l have been reported (Oliver, 1957). Although many marine animals are tolerant of high nitrate concentrations (De Graaf, 1964; Hirayama, 1966; Kuwatani et al., 1969; Epifanio and Srna, 1975), the accumulation of nitrate in recirculated water appears to be accompanied by other, generally detrimental chemical changes (King, 1973). Nitrate can, therefore, serve as a useful indicator of overall water quality and a standard against which the need for water replacement can be gauged. In the standard assay for nitrate in seawater (Strickland and Parsons, 1977), nitrate is reduced to nitrite by passage through a column containing copperized-cadmium filings, and the resulting nitrite is determined calorimetrically. ’ Contribution
No. 10, Sea Research Foundation,
0044-8486/80/0000-0000/$02.50
0 1980
Inc.
Elsevier Scientific Publishing Company
282
We found this method impractical for monitoring nitrate accumulation in culture water chiefly because of the expense and complexity of preparing and maintaining the reduction column. In an earlier method (Mullin and Riley, 1955) that was modified by Strickland and Parsons (1960) and subsequently adapted for high nitrate determinations by King and Spotte (1974), copper was used to catalyze the reduction of nitrate by hydrazine in a solution buffered with phenol and sodium hydroxide. Reduction had to be carried out in a constant temperature bath in the dark, and required 20 h for completion. In routine use of this method, we encountered high reagent blanks (ca. 0.03 to 0.04 absorbance units), which probably resulted from oxidation of the phenol reagent. The procedure given here is a modification of earlier hydrazine-reduction methods for determining high concentrations of nitrate in seawater. A more stable organic acid has been substituted for phenol in the buffer solution, reduction time has been decreased to 2 h, and determinations can be made at ambient temperatures under’artificial light. MATERIALS
AND METHODS
Reagents All chemicals were analytical reagent grade. Solutions were prepared with tap water passed through a mixed-resin deionizing column (Crystalab DI-3) immediately before use. Buffer solution. 60 g cyclohexylaminopropane sulfonic acid (CAPS, Sigma Chemical Co., St. Louis, MO) + 3.720 g sodium hydroxide in 1 1 of water. Stable. Hydrazine sulfate solution. 7.250 g/l. Stable for at least 1 month. Copper sulfate solution. 0.100 g/l. Stable. Reducer solution. Equal volumes of hydrazine sulfate and copper sulfate solutions, used within 1 h of mixing. Acetone. Sulfunilumide solution. 10 g/l in 10% concentrated hydrochloric acid. Stable. NED solution. 1 g N-l- naphthylethylenediamine dihydrochloride/l. Stable for at least 1 month. Sodium nitrate standard solutions. Stock solution (100 mg NO,-N/l) 0.607 g NaNOJ. Working standard solution (10 mg NO,-N/l) - 5 ml stock solution diluted to 50 ml; prepared fresh daily. The buffer, hydrazine sulfate, and NED solutions were stored in amber glass bottles in a refrigerator. Procedure Samples consisted of 1 ml seawater containing 1 to 20 mg NO,-N/l or 0.5 ml seawater containing > 20 mg NO,-N/l, diluted to 50 ml with deionized water. Calibration standards were 1 ml working standard solution in seawater or deionized water, diluted to 50 ml with deionized water. Blanks consisted of 50 ml deionized water or 1 ml seawater diluted to 50 ml with deionized
283
water. Artificial seawater (Instant Ocean@, Aquarium Systems, Mentor, OH, S = 30%0) was used throughout. Glassware was washed with detergent and rinsed copiously with deionized water before use. All determinations were carried out in triplicate, unless otherwise noted, using 125-ml Erlenmeyer flasks held at ambient temperature (23 to 27°C). Lighting was from four 40-W “cool white” fluorescent lamps at a distance of 2 m (ca. 10 c/ft*). To each sample, standard solution and blank, 2 ml buffer solution, then 1 ml reducer solution were dispensed from graduated pipettes. Contents were mixed and the flasks were stoppered. After 2 h of reduction, 2 ml acetone was added to the series of flasks in rapid succession, to stop reduction by destroying excess hydrazine. After 2 min, 1 ml sulfanilamide solution and 1 ml NED solution were added sequentially, with 2 to 8 min allowed between additions. After 15 min but before 2 h, absorbances were read against deionized water at 543 nm with a Turner Model 330 spectrophotometer (AMSCO Instrument Co., Carpenteria, CA), using lo-cm length cuvettes. Absorbances were converted to concentrations of N03-N in mg/l by using the equation NOJ-N = (Es - Eb)F
(1) in which E, is the mean sample absorbance, Eb is the mean blank absorbance, and F is a conversion factor representing the quotient of the concentration of the calibration standard (10 mg/l) divided by the mean corrected absorbance of the standard (Estandard - Eb). When nitrate standards were prepared with seawater, their corrected absorbances were obtained by subtracting the mean absorbance of seawater blanks. RESULTS
AND DISCUSSION
Reagent concentrations Maximum absorbances were obtained with a reduction pH of 10 f 0.05, achieved with 3.5 X 10m3M sodium hydroxide and 0.01 M CAPS (pK, = 10.4) in the reducing medium. Erratic absorbances occurred at higher and lower pH values, and low recoveries resulted when the concentration of CAPS was decreased. The concentrations of hydrazine sulfate and copper sulfate recommended by Strickland and Parsons (1960) were optimal Reduction
time
When nitrate is reduced by hydrazine, an 85% yield of nitrite should result after 2 h (Mullin and Riley, 1955). Based on the absorbances of our reduced-nitrate samples and those of samples spiked with equivalent concentrations of nitrite-nitrogen, only 38% reduction occurs in 2 h. We found that a 2-h reduction period was sufficient to give reliable determinations. After only 1 h of reduction, standard deviations were greater, and the sensitivity of the test was lower.
Light and temperature We could detect no differences between the absorbances of samples reduced in the dark and under artificial light. Changes of 2°C in ambient temperature during the reduction period had no effect on absorbances or recoveries. Inhibition of reduction in seawater Known concentrations of nitrate in seawater, diluted with deionized water and treated by the standard procedure, consistently absorbed about 5% lower than those in 100% deionized water. The phenomenon occurred regardless of whether natural or artificial seawater was used. At the salinity of our diluted samples (0.6°/oo), there should be no salt effect on reduction (Mullin and Riley, 1955). Although the concentration of magnesium in our samples might inhibit reduction by precipitating as magnesium hydroxide at pH 10 (Mullin and Riley, 1955), we could not relate discrepant absorbances to the presence of magnesium, alone, nor could any other cause be isolated. It is therefore necessary that the absorbances of deionized water blanks and calibration standards be multiplied by 0.95, before using them to calculate conversion factors for seawater samples. Calibration curve A calibration curve was prepared with known concentrations of nitrate in seawater diluted 1: 50 with deionized water and treated as described (Fig. 1). Beer’s law was obeyed in the concentration range from 1 to 20 mg NO,-N/l. Calibration curves were constant to within 5% over a one-month period, and were not affected by the preparation of fresh reagents.
mg NO,-N/I
Fig. 1. Calibration curve for nitrate determination by the 2-h hydrazine-reduction Concentrations refer to mg NO,-N/l in the original l-ml seawater samples.
method.
Comparison with another hydrazine method The concentration of nitrate in four established seawater aquariums was determined by a 20-h hydrazine-reduction method (King and Spotte, 1974) and by the 2-h method. As shown in Table I, there was excellent agreement between the two methods. TABLE I Comparison of recoveries of nitrate from aquarium seawater by the 2-h reduction method and an earlier, 20-h reduction methoda Sample no.b
1 2 3 4
mg NO,-N recovered (SD) 2-h reduction
20-h reduction
66.3 21.4 38.4 41.6
67.4 21.8 37.8 41.2
(0) (0.1) (0.5) (0.4)
(2.0) (0.5) (0.4) (0.7)
Difference (%)
1.7 1.9 1.6 1.0
aprocedure from King and Spotte (1974). “N= 3.
Accuracy and reproducibility The accuracy of the 2-h reduction method was determined by analyzing triplicate samples of seven known nitrate concentrations in seawater, and then calculating the percent recoveries. Results are given in Table II. The standard deviation was 0.10 (2.0%) for 5 mg NO,-N/l and 0.2 (1.0%) for 20 mg NO,-N/l, indicating excellent reproducibility. The absorbances of reagent blanks were always less than 0.01. TABLE II Cont. NO,-N (mg/I)a
Recoveryb nig NO,-N/I (SD)
Percent
1 5 10 15 20 25 30
1.0 (0.1) 5.1 (0.1) 10.0 (0.3) 14.6 (0.2) 19.1(0.2) 23.6 (0.4) 27.0 (0)
100 102 100 97 96 94 90
Woncentration bN=3.
in the original 1-mI sample.
286 ACKNOWLEDGMENTS
We are grateful to Dr. James W. Atz and Stephen Spotte for reading and criticizing the manuscript. REFERENCES De Graaf, F., 1964. Maintenance problems in large public aquaria. Arch. Neerl. Zool., 16: 142-143. Epifanio, C.E. and Srna, R.F., 1975. Toxicity of ammonia, nitrite ion, nitrate ion, and orthophosphate to Mercenaria mercenaria and Crassostrea virginica. Mar. Biol., 33: 241-246. Hirayama, K., 1966. Influence of nitrate accumulated in culturing water on Octopus vulgaris. Bull. Jap. Sot. Sci. Fish., 32: 105-111. King, J.M., 1973. Recirculating system culture methods for marine organisms. SEA Scope, 3(l): 1;6-8. King, J.M. and Spotte, S., 1974. Marine Aquariums in the Research Laboratory. Aquarium Systems, Inc., Eastlake, Ohio, 39 pp. Kuwatani, Y., Nishii, T. and Isogai, F., 1969. Effects of nitrate in culture water on the growth of the Japanese pearl oyster. Bull. Natl. Pearl Res. Lab,, 14: 1735-1747. Mullin, J.B. and Riley, J.P., 1955. The spectrophotometric determination of nitrate in natural waters, with particular reference to sea-water. Anal. Chim. Acta, 12: 464-480. Oliver, J.H., 1957. The chemical composition of the sea water in the aquarium. Proc. Zool. Sot. London, 129: 137-145. Saeki, A., 1962. The composition and some chemical control of the sea water of the closed circulating aquarium. Bull. Mar. Biol. Stn. Asamushi., Tokohu Univ., 1: 99-104. Strickland, J.D.H. and Parsons, T.R., 1960. A manual of sea water analysis. Fish. Res. Board Can. Bull., No. 125: 61-69. Strickland, J.D.H. and Parsons, T.R., 1977. A practical handbook of seawater analysis, 2nd ed. Fish. Res. Board Can. Bull., No. 167: 71-76.