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Wat. Res. Vol. 29, No. 12, pp. 274(>2750, 1995 Copyright ~ 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/95 $9.50 + 0.00

D E T E R M I N A T I O N OF A M M O N I U M IN SEAWATER BASED ON THE I N D O P H E N O L REACTION WITH O - P H E N Y L P H E N O L (OPP)


JOTA KANDA*@ Laboratory of Environmental Sciences, Faculty of Liberal Arts, Shizuoka University, 836 Ohya, Shizuoka-shi, Shizuoka 422, Japan (First received June 1994; accepted in revised form May 1995) Abstract--A method for the determination of ammonium in seawater is described. The method is based on the indophenol (or the Berthelot) reaction with o-phenylphenol [(1,1'-biphenyl)-2-ol, abbreviated as OPP] as an alternative of phenol. Unlike phenol, OPP is available as stable flaky crystal with no caustic odor. This nature of OPP can eliminate much of the inconvenience associated with the use of phenol. The sensitivity of detection is comparable to that of the common phenol-based methods. The indophenolic compound obtained from the reaction with OPP is more hydrophobic than that from the reaction with phenol. Thus the indophenolic compound can be extracted directly from the alkaline reaction solution of the present method either with organic solvent or with reversed-phase column. The extracted indophenol can be used to recover ammonium nitrogen for the determination of 15-N isotope ratio. The experimental procedure of the extraction of indophenolic compound is relatively simple compared to the other existing methods. Key words--ammonium determination, seawater, indophenol reaction, Berthelot reaction, o-phenylphenol, 15-N isotope determination

INTRODUCTION The i n d o p h e n o l reaction, or the Berthelot reaction, has been c o m m o n l y used for the d e t e r m i n a t i o n o f a m m o n i u m nitrogen in natural waters. The reaction occurs with a m m o n i a and a phenolic c o m p o u n d u n d e r suitable oxidizing conditions and yields a blue indophenolic dye (Searle, 1984). Use o f a p e n t a c y a n o ferrate coupling reagent ( H a r f m a n n and Crouch, 1989) such as sodium nitroprusside greatly i m p r o v e d the sensitivity of a m m o n i u m detection. A wide variety of m e t h o d s using different phenolic c o m p o u n d s and o t h e r reagents have been published so far (Searle, 1984). Irrespective of different c o m b i n a t i o n of reagents, however, the sensitivity of detection is similar a m o n g different m e t h o d s of a m m o n i u m determin a t i o n by the i n d o p h e n o l reaction with a p e n t a c y a n o ferrate coupling reagent. A m o l a r absorptivity of a b o u t 2 l0 41 mol ~cm ~ for a m m o n i u m is commonly achieved u n d e r an o p t i m u m condition (Searle, 1984). The i n d o p h e n o l reaction using sodium nitroprusside was first i n t r o d u c e d for the d e t e r m i n a t i o n o f a m m o n i u m in seawater by Sagi (1966). Since then, m e t h o d s involving the reaction of phenol, sodium hypochlorite a n d sodium nitroprusside u n d e r an al*Author to whom all correspondence should be addressed [Fax: (81) 054 237 9184].

kaline condition became p o p u l a r in seawater analysis (Searle, 1984). In these methods, a complexing agent such as trisodium citrate (Sol6rzano, 1969) and E D T A ( M a n a b e , 1969) is used in order to prevent the precipitation o f insoluble hydroxides in seawater u n d e r the alkaline condition of reaction. In practical use, however, phenol has some objectionable features; it has caustic o d o r a n d toxicity a n d is in transition between solid and liquid phase at room temperature. Use of an alternative c o m p o u n d may eliminate m u c h of the inconvenience associated with the use of phenol. Y a m a g u c h i and M a c h i d a (1968) d e m o n s t r a t e d that o - p h e n y l p h e n o l [(1, l'-biphenyl)-2-ol, an a b b r e v i a t i o n of O P P will be used in this paper] can be used as an alternative of phenol in the indophenol reaction. The original m e t h o d of Y a m a g u c h i and M a c h i d a (1968) was intended for clinical analysis. They added d i m e t h y l f o r m a m i d e as a solvent for O P P and the a m o u n t of the a d d e d solvent was as m u c h as 57% of the final volume of the reaction solution. The m e t h o d has not been widely used presumably because the addition o f the large a m o u n t of the organic solvent is t r o u b l e s o m e for practical use, and more importantly, the addition of the solvent resulted in a high b l a n k a b s o r b a n c e of a b o u t 0.35 in 10 m m light path. In this paper, I examined the use of O P P as an alternative o f phenol for the determination of amm o n i u m in seawater. A preliminary e x a m i n a t i o n

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Ammonium determination with OPP suggested that the organic solvent used by Yamaguchi and M a c h i d a (1968) to dissolve O P P was not essential and that the indophenolic c o m p o u n d formed from O P P was h y d r o p h o b i c enough to be extracted directly into hexanol under the alkaline condition o f the color-forming reaction. The latter feature could potentially be useful for the purpose o f further increasing the sensitivity o f a m m o n i u m detection and also o f recovering a m m o n i u m nitrogen for the d e t e r m i n a t i o n o f 15-N isotope ratio.

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MATERIALS AND METHODS All the reagents used were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Sodium hydroxide was of the grade for amino acid analysis. Trisodium citrate dihydrate, sodium nitroprusside [sodium nitrosylpentacyanoferrate (III) dihydrate], OPP, methanol, and ammonium sulfate were of the analytical reagent grade. Sodium hypochlorite solution was of Wako's practical grade, which nominally contains 5~i% of active chlorine. Standard solutions of ammonium were prepared with ammonium sulfate which was dried for 24 h at 60C before weighing. Ammonium sulfate labelled with 15-N (99.8 at.%, Shoko Co. Ltd, Tokyo) was also used for 15-N recovery experiment. For standard solution of ammonium in seawater, stored seawater which was originally taken from surface subtropical Pacific was used. Fresh deionized water which was supplied directly from the outlet of an ionexchange column (G-10, Organo Inc., Tokyo) was used for the standard solution of ammonium in deionized water. All the glassware were rinsed with 1 N HCI prior to use and then thoroughly rinsed with the fresh deionized water as described above. Reagent solutions were prepared as follows. (I) Sodium citrate solution; trisodium citrate dihydrate (400g) was dissolved in deionized water and the final volume was made up to 1000ml. The solution is stable for months in a refrigerator. (2) OPP solution: sodium hydroxide (2 g) and 4 g of OPP was dissolved in deionized water successively, and the final volume was made up to 100 ml. The solution is usable for at least 2 weeks if kept in a refrigerator. The solution shows a slight brown color during the prolonged storage, but it does not affect the analysis. (3) Sodium hypochlorite solution: the commercial solution of sodium hypochlorite (4 ml) was diluted with deionized water and the final volume was made up to 100 ml. The diluted solution is stable for at least a week if kept in a refrigerator. The content of active chlorine in the original solution should be checked periodically by iodometric titration (Kempers and Kok, 1989). (4) Sodium nitroprusside solution: sodium nitroprusside (0.05g) was added to 100ml of sodium hydroxide solution (3 N). The solution must be prepared daily. The analysis was conducted according to the following procedure unless otherwise stated. Sample water (5 ml) was taken in a 10-ml screw capped test tube. To this, 1 ml of Reagent (1), 0.1 ml of (2), and 0.05 ml of (3) were added successively. The test tube was swirled between each addition of reagent. Between 2 and 5 min after the addition of Reagent (3), 0.I ml of Reagent (4) was added and swirled. The tubes were placed in a water bath of 40C for 15 min, and absorbance was measured at 670nm. A Shimadzu UV-160A spectrophotometer (Shimadzu Inc., Kyoto, Japan) with a 10mm light path was used. When contamination of ammonium in trisodium citrate reagent is a problem, Reagent (1) and (2) can be combined and boiled to purge ammonia. In this case, to 1000 ml of Reagent (1) and 100ml of Reagent (2), approx. 500 ml of deionized water was added, and then the solution was boiled until the

final volume becomes 1000 ml. The combined reagent (1 ml) can be used instead of Reagent (1) and (2), and the reagent is stable at least for 2 weeks if kept in a refrigerator. Indophenolic compound was extracted by prepacked disposable columns of reversed phase octadecylsilane beads (Bakerbond Spe Octadecyl, J. T. Baker Inc., Phillipsburg, N.J., U.S.A.) following the procedure of Brzezinski (1988). To seawater samples of 800 ml, proportional amounts of above describe reagents were added. After the completion of color formation, the reaction mixture was passed through the octadecyl columns (6 ml volume) at a flow rate of 9mlmin -~. Sodium hydroxide solution (0.0675N, 10ml) was passed through the pigment-retained column. The retained blue pigment was then eluted with methanol. The eluted pigment was evaporated and absorbed onto a precombusted Whatman GF/F filter (25 mm dia.). The filter was dried at 60C, and the nitrogen content and the 15-N isotope ratio of the filters were determined with a Europa Scientific ANCA-MS system (Europa Scientific Ltd, Crewe, U.K.). RESULTS The absorption spectra o f the indophenolic comp o u n d formed with the above procedure for a seawater sample is s h o w n in Fig. 1. The absorption m a x i m u m is at 670 nm. After reagents were a d d e d to a seawater sample containing 10 ~ M o f a m m o n i u m nitrogen, the absorbance at 670 n m was continuously m o n i t o r e d at 40C in a temperature-controlled cell ( 1 0 m m light path) o f the spectrophotometer. A reaction time o f 15rain was a d o p t e d for routine analysis (Fig. 2), showing that the absorbance was c o n s t a n t for at least 90 min. The blue pigment in the solution was stable for days in the test tubes. The absorbance values obtained with a replicate o f 5 samples for each concentration o f seawater standard solution were summarized in Table 1. Linear relationship was observed up to 20/~ M, and the slope o f a linear regression o f the data (0-20/~M) is 2.00 1041mo1-1 cm -~ (corresponding to a molar absorptivity o f 2.50 1041mol -~ cm ~ for final concentrations o f a m m o n i u m nitrogen in the reaction solution). A correlation coefficient o f 0.9998 was obtained and the s t a n d a r d deviation o f a b s o r p t i o n readings from the regression was 2.032 10 3

0.25

0.20 ~ 0.15
.~ ~ 0.10

0.05
0.0( 4,,.kO 5~ 5~ 6~0 6~ 7(~0 7..~ Wavelength (nrn) 8(

Fig. 1. Absorbance spectrum of the indophenolic compound obtained with OPP which was taken with the standard seawater solution containing 10#M ammonium.

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0.25

J. K a n d a
0.2, =

~E 0.20r'~ ~.o 0.15-,~

E -

0.2( E

0.15-

8 tin .~

0.10

0.10

:;~ 0.05

<

0.05-

0.00

1000

2000

3000

4000

5000

6000

0.00

20

40

60

80

100

Time (s)
Fig. 2. The d e v e l o p m e n t o f the blue pigment with time at 40~C. The a b s o r b a n c e at 670 n m was c o n t i n u o u s l y monitored as described in the text.

Portion of Seawater (%)

Fig. 3. The absorbance at 670 nm for 10 lzM ammonium standard solution and prepared with a series of diluted
seawater. Triplicate m e a s u r e m e n t s at each seawater dilution were conducted.

(n = 35). The latter value corresponds to an ammonium concentration of 0.10#M. Figure 3 shows the absorbance for 10#M ammonium in standard solutions prepared with a series of diluted seawater with the fresh deionized water. The absorbance is practically constant in the range of 100% and 43%, but decreases with decreasing salinity in the range of 43% and 0%. The absorbance with 0% seawater (i.e. deionized water) corresponded 83% of the absorbance with 100% seawater. Appropriate reduction of sodium hydroxide yields higher absorbance of deionized water (data not shown). Hence the lower absorbance observed for the deiom ized water reflects the increase of pH in the reaction system with decreasing salinity (Nimura, 1973). Some amino acids are known to interfere with formation of the indophenolic dye (Searle, 1984). The absorbance values of standard seawater solutions (0#M and 10/~M of ammonium nitrogen) in the presence of and in the absence of these compounds were compared (Table 2). Most of the compounds listed in Table 2 at the concentration of 1 mM do not significantly interfere with the color formation. However, the presence of cystine, histidine, methionine, tryptophane and phenylalanine resulted in a decrease in absorbance of 7 34%. Suppression of color formation by these compounds is commonly found in phenol based methods (Searle, 1984). Reaction with
Table I. Reproducibility of analysis with a replicate o1"5 samples of seawater standard in the range of 0-50 l~M of ammonia Concentration #M 0.0 2.5 5.0 7.5 10.0 15.0 20.0 30.0 40.0 50.0 Absorbance at 670 nm Mean (n = 5) SD u 0.012 0.059 0.1 I 0 0.160 0.211 0.313 0.410 0.602 0.788 0.958 0.0000 0.0000 0.0005 0.0016 0.0005 0.0008 0.0036 0.0026 0.0073 0.0065 RSD" (%) 0.00 0.00 0.45 1.00 0.23 0.26 0.89 0.43 0.93 0.67

hypochlorite and/or nucleophilic addition to quinone chloroamine are postulated for the mechanism of such suppression (Ngo el al., 1982; Searle, 1984), and the degree of suppression depends on concentration of these compounds (Nimura, 19731. The concentration of these compounds in natural seawater is lower by at least a factor of 1000. Hence the suppression by these compounds should not be significant in practical analysis of natural seawater. Amino acids and urea may also break down under the alkaline reaction condition and liberation of ammonium may positively interfere with the analysis (Searle, 1984). The ammonium liberated from the compounds
Table 2. Absorbance (670 nm) obtained with and without 111/~M ammonia in the presence of an amino acid or urea Compound ~ L-Alanine L-Arginine Glycine L-Leucine L-Threonine L-Serine L-Lysine L-lsoleucine k-Phenylalanine L-Valine L-Methionine L-Aspartic acid k-Histidine L-Glutamic acid L-Tyrosine L-Glutamine L-Asparagine [ -Tryptophan L-Cystine (0.1 raM) L-Proline c-Ornithine t.-CitrulIine Urea Control (no additive) Absorbance at 670 nm ~ Without NH~+ With 10/~M NH2 0.015 0.000 0.012 _+ 0.000 0.012 + 0.000 0.014 + 0.001 0.013 + 0.000 0.015 +_ 0.001 0.014 0.001 0.012 + 0.000 0.010 0.000 0.015 +_ 0.000 0.010 + 0.000 0.010 + 0.000 0.006 0.001 0.1112 0.000 0.011 + 0.001 0.012 + 0.000 0.012 0.000 0.010 + 0.000 0.005 + 0.000 0.012 _+ 0.000 0.010 _+ 0.000 0.011 + 0.000 0.010 + 0.000 0.010 _+ 0.000 0.224 + 0.002 0.226 + 0.003 0.222 + 0.000 0.227 0.000 0.223 0.001 0.223 + 0.002 0.231 + 0.000 0.221 0.001 0.203 + 0.001 0.232 + 0.000 0.194 + 0.001 0.223 + 0.000 0.182 + 0.000 0.223 0.001 0.216 + 0.001 0.227 * 0.000 0.217 ~ 0.002 0.204 0.001 11.144 + 0.001 0.212 _+ 0.000 0.225 + 0.1101 0.223 11.001 0.221 + 0.000 0.219 + 0.001 Yield c (%) 100 103 101 102 101 100 104 100 92 1f14 88 101 85 101 98 103 98 93 66 96 103 101 101 100

"Relative standard deviation. UStandard deviation.

"Concentration of compounds was I mM except for cystine (0.1 mM). bMean o f triplicate determination with + S D . ~Yield of absorbance with 10 ~ M a m m o n i u m (blank value corrected) relative to the absorbance of the control.

Ammonium determination with OPP


Table 3. The 15-N isotope ratio and total nitrogen amount ~ found in the extracted indophenolic compound 15-N isotope ratio (at.%) Theoretical Observed 5.339 5.337 5.350 5.354 5.390 5.358 0.020 Total N (/~mol) Theoretical Observed 8.0 3.62 3.18 3.17 3.15 3.28 0.20

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Mean SD

Mean SD

"Seawater samples (800 ml) which contains 15-N labelled ammonium sulfate equivalent to 10,uM of ammonium were used.

during the reaction are judged to be negligible from the data shown in Table 2. Relatively high concentrations of liberated ammonium are detected for alanine, serine and valine, but the concentrations of liberated ammonium nitrogen are equivalent to only 2.3, 2.2 and 2.2% of the molar concentration of respective compounds. Note that these values may include ammonium contamination from the amino acid reagents. Table 3 shows the nitrogen amount and 15-N isotope ratio of the indophenolic compound extracted from 800 ml of seawater containing 10/~M of 15-N labelled ammonium. The 15-N isotope ratio of the ammonium was calculated to be 5.339 at.%. The 15-N at.% actually found in the pigment was quite close to the theoretical value. Hence nitrogen derived from other than the dissolved ammonium was not significant in total nitrogen recovered. The above described extraction procedure should be useful in 15-N determination in ammonium in seawater. Total nitrogen recovered was about 40% of the ammonium nitrogen in the solution. The reason for this low yield is not certain, but the recovery of indophenol was likely to be incomplete in experimental steps such as retention by the reversed phase column and absorption by glass fiber filters. This low yield should not be a problem in practice as long as the observed 15N a t . % of the recovered nitrogen reflects the 15N at.% of ammonium nitrogen.
DISCUSSION

In this study, several improvements were achieved over the original OPP method of Yamaguchi and Machida (1968). The addition of the substantial amount of organic solvent in the method of Yamaguchi and Machida (1968) was evaded, and the reaction was conducted in completely aqueous solution. The molar absorptivity obtained was quite close to a common maximum value obtained under optimized conditions with various phenolic compounds (Ivan6i6 and Degobbis, 1984; Catalano, 1987). The reproducibility (Table 1) was also improved over the result of Yamaguchi and Machida (1968). The blank absorbance was relatively low (Table 1) and was comparable to the lowest values reported in Ivan6i6 and Degobbis (1984) and Catalano (1987).

In seawater analysis, salinity content of the sample may sometimes affect the color development by the indophenol reaction (Sagi, 1966). This problem is actually associated with change of pH rather than salinity itself in the reaction solution (Nimura, 1973). In this method, a constant absorptivity was achieved over the range of 43 100% seawater (Fig. 3). This coverage is sufficient for most of the marine waters including estuary waters. The interference by amino acids and urea is also judged to be insignificant for natural seawater (Table 2). Because phenol itself and o-chlorophenol formed during the Berthelot reaction are caustic and poisonous, sodium salicylate was proposed to be an alternative of phenol in seawater analysis (Kempers and Kok, 1989). Since the reagent OPP is available as stable flaky crystal and it has no caustic odor and toxicity of phenol, OPP can be another safer alternative of phenol for the indophenol reaction. A number of methods has been proposed for extraction of indophenol derived from phenol. Moreno et al. (1986) concentrated indophenol directly under the alkaline reaction condition by passing through a hydrophobic beads (Amberlite XAD-7) column. Brzezinski (1988) reported a method for concentrating the indophenol by solvent extraction or by solid-phase extraction. In his method, the extraction of indophenol followed acidification of the reaction solution in order to increase hydrophobicity of indophenol. Yamamoto et al. (1990) developed a method for increasing hydrophobicity of indophenol by forming ionic paired complex with quaternary alkyl ammonium salts. The ionic paired complex can be extracted into various organic solvents. Except for the method of Moreno et al. (1986), some additional treatment of indophenol was necessary in order to increase the hydrophobicity of the indophenol. Use of phenolic compounds other than phenol is an alternative way for the extraction of indophenolic compounds. The indophenol reaction with thymol and ~-naphthol was shown to be suitable for solvent extraction, because the obtained indophenolic compound is more hydrophobic. The thymol method of Matsunaga et al. (1970) achieved a molar absorptivity of 8.2 103 lmol ~cm ~ for ammonium nitrogen without solvent extraction. Another method using c~-naphthol yielded similar sensitivity of ammonium detection corresponding to a molar absorptivity of about 1.1 1041 mol ~cm ~for ammonium (Morita and Kogure, 1963). While the sensitivity of these methods might be improved by further refinement of analytical procedure, the reported values are approximately half as much as those obtained from the common phenol methods and from the OPP method proposed in this study. The method developed in this study is one of the most simple among the various methods proposed for the extraction of indophenolic compound as well as for the recovery of ammonium nitrogen in isotope analysis (Dudeck et al., 1986). The 15-N isotope

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J. Kanda Berthelot reaction steps in the absence and presence of coupling reagents. Talama 36, 261 269. lvan~i~ I. and Degobbis D. (1984) An optimal manual procedure for ammonia analysis in natural waters by the indophenol blue method. War. Res. 18, 1143 1147. Kempers A. J. and Kok C. J. (1989) Re-examination of the determination of ammonium as the indophenol blue complex using salicylate. Anal. Chim. Acta 221, 147 155. Manabe T. (1969) New modification of Lubochinsky's indophenol method for direct microanalysis of ammonium-N in sea water. Bull. Japan. Soc. Scient. Fish. 35, 897-906. Matsunaga K., Sugino K. and Nishimura M. (1970) Spectrophotometric determination of ammonia with chloramine T and thymol. Japan Analyst 19, 1255 1258. Moreno P., Sfinchez E., Pons A. and Palou A. (1986) Ammonia and urea determination in water samples using Amberlite XAD-7 to concentrate indophenol. Anal. Chem. 58, 585 587. Morita Y. and Kogure Y. (1963) Spectrophotometric determination of nitrogen with hypochlorite and ~-naphthol. J. chem. Soc. Japan 84, 816 823. Ngo T. T., Phan A. P. H., Yam C. F. and Lenhoff H. M. (1982) Interference in determination of ammonia with the hypochlorite-alkaline phenol method of Berthelot. Anal. Chem. 54, 46~49. Nimura Y. (1973) A direct estimation of microgram amounts of ammonia in water without salt-error. Bull. Japan. Soc. Scient. Fish. 39, 1315- 1324. Sagi T. (1966) Determination of ammonia in sea water by the Indophenol method and its application to the coastal and off-shore waters. Oceanogr. Mag. 18, 43 51. Searle P. L. (1984) The Berthelot or Indophenol reaction and its use in the analytical chemistry of nitrogen. A review. Analyst 109, 549 568. Sol6rzano L. (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14, 799 801. Yamaguchi R. and Machida T. (1968) Colorimetric determination of ammonia. I. A new spectrophotometric method for the determination of ammonia by the use of o-phenylphenol. Yakugaku Zasshi 88, 1383 1387. Yamamoto N., Kasahara N. and Shirai T. (1990) Spectrophotometric determination of trace levels of atmospheric ammonia by conversion into indophenol and extraction with quaternary ammonium salts. Talanta 37, 1063 1065.

ratio o f the extracted indophenolic c o m p o u n d was confirmed to reflect the isotope ratio o f a m m o n i u m nitrogen in seawater (Table 3). The procedure described in this study can thus be used in tracer experiments for quantitative study o f nitrogen dynamics in marine environment. Such tracer experiments include the measurement o f dilution o f 15-N isotope in a m m o n i u m nitrogen in order to determine the rate o f a m m o n i u m regeneration by heterotrophic microorganisms (Dudeck et al., 1986). In conclusion, the m e t h o d developed in this study is a practical alternative o f the c o m m o n m e t h o d s for the determination o f a m m o n i u m in seawater. This m e t h o d will be applicable to determination of a m m o n i u m in various types o f aqueous solution although the specific reaction condition given in this paper may require some modification. The m e t h o d avoids much o f the inconvenience associated with the use o f phenol. It is especially useful when the determination o f a m m o n i u m should be accompanied with the determination o f 15-N isotope ratio o f amm o n i u m nitrogen, as is often in recent studies of nitrogen cycling in aquatic environment. Acknowledgements--This study was partly supported by Grants-in-aid No. 63790231, 04232206 and 04740355 from the Ministry of Eduction, Science and Culture, Japan.
REFERENCES

Brzezinski M. A. (1988) Vertical distribution of ammonium in stratified oligotrophic waters. Limnol. Oceanogr. 33,
117(~1182.

Catalano G. (1987) An improved method for the determination of ammonia in seawater. Mar. Chem. 20, 289-295. Dudek N., Brzezinski M. A. and Wheeler P. A. (1986) Recovery of ammonium nitrogen by solvent extraction for the determination of relative ~SN abundance in regeneration experiments. Mar. Chem. 18, 59~59. Harfmann R. G. and Crouch S. R. (1989) Kinetic study of