1. Introduction
The increase of environmental ammonia level as a by-product of the nitrogen cycle has toxic effects in marine water systems. Ammonia is the first indicator of
contamination, which unless minimised can lead to direct toxicity, high bacterial counts, oxygen depletion, fish disease, and mortality. As proteins and amino acids
are metabolized, there is a direct excretion of ammonia from the living organisms to their surroundings. Ammonia is the principal nitrogen waste product of fish
Russo, 1985. Under normal conditions, it is oxidized by nitrifying bacteria to nitrite and nitrate. However, in heavily stocked ponds or in recirculating culture
systems with inadequate filtration systems, ammonia concentration may increase to high levels Wise et al., 1989. The increase of ammonia levels in marine aquacul-
ture facilities can occur rapidly, since the process is related to the fish feeding rates, external water supply and other parameters such as temperature, pH or salinity.
Once generated, the removal of ammonia is dependent on natural processes, mainly on nitrification where ammonia is oxidized to nitrite by the Nitrosomonas bacteria
and then to nitrate by the Nitrobacter bacteria Walker, 1975. Environmental nitrite may also reach high concentrations in fish culture, primarily because of
inhibited conversion of nitrite to nitrate due to the lack of the required nitrifying- bacteria population. Although less toxic than ammonia, nitrite enters the circula-
tory system of fish via the gills Perrone and Meade, 1977 oxidizing the hemoglobin to methemoglobin, which is incapable of binding and transporting
oxygen Brown and McLeay, 1975; Wise and Tomasso, 1989.
In aquaculture operations, fish are reared at high densities and the increase of environmental ammonia and nitrite level lead to undesired results such as reduction
of growth level, changes in metabolic rates, perturbation of protein processing, virus development and diminished survival rate Colt et al., 1981; Hanson and
Grizzle, 1985; Wise et al., 1989. The toxic effect of ammonia has been demon- strated for several aquatic species Goldman and Azov, 1982; Person Le Ruyet et
al., 1995; Abraham et al., 1996. The effects are more important during the rearing of the early developmental stages of the individuals when low concentrations 0.01
mgl of unionised ammonia can result to mortalities and pathological disturbances of the young larvae Wajsbrot et al., 1993; Guillen et al. 1994 as well as depressed
growth rates Guillen et al., 1993. The most documented effect of fishes exposure to nitrite is the oxidation of hemoglobin to methemoglobin, which has a lower
affinity for the binding and transport of oxygen Brown and McLeay, 1975; Weirich et al., 1993. High levels of nitrite for a long period of time are required for toxicity,
in the contrary to the relatively lower levels of ammonia required for a short period of time in order to lead to toxic effects Wheaton et al., 1991.
It is thus evident that in any system either a pond, a coastal area, or a recirculating system, water quality is of major concern, and in order to sustain and
expand life a healthy environment is mandatory. Management of an aquaculture system must thus begin with the continuous and precise analysis of the ammonia
and nitrite levels, since their concentration for normal fish growth must be kept below harmful values.
Existing methods for the analysis of ammonia in seawater samples include colorimetry, ion chromatography, cathodic stripping voltammetry, mass spectrome-
try and fluorimetry. The colorimetric indophenol blue method is based on the Berthelot reaction between ammonia, phenol and hypochlorite leading to the
formation of an indophenol dye Aminot et al., 1997a. In the sequential injection analysis-colorimetry for the determination of ammonia content in seawater samples
Van Staden and Taljaard, 1997 the range of detection is 0.36 – 50 mgl with sample rate of 16 samplesh. One of the major drawbacks of the colorimetric method is the
required sample pretreatment which includes the mandatory filtration step. This step is the major obstacle in using the colorimetric method for the continuous
monitoring of ammonia in aquaculture seawater samples. In FIA flow injection analysis-ion chromatography ammonia is transmembrane diffused into an acidic
media and determined as solvated ammonium Gibb et al., 1995 with a range of detection from 0 to 17 ppb and an analysis time of 15 minsample. In FIA-cathodic
stripping voltammetry, ammonia reacts with formaldehyde to form the determined methylenimine Harbin and van den Berg, 1993. The range of detection is 0.17 – 51
ppb while the analysis time is in the order of 20 – 35 min depending on the sample analyzed. In mass spectrometry, the
15
N – NH
4 +
is determined in large sample volume 4 l by the diffusion method with the range of detection 0.5 – 10 mM
Holmes et al., 1998. Finally, in FIA-fluorimetry, ammonia is determined after derivatization with o-phthaldialdehyde and sulfite Ke´rouel and Aminot, 1997. The
range of detection is 0.5 – 250 mM and the analysis time is 3 minsample.
Nitrite in seawater can be determined by various colorimetric methods in FIA systems as well as by capillary zone electrophoresis. The reference colorimetric
method with Griess reagent for the determination of nitrite includes the formation of an azo dye by the reaction of nitrite with the N-1-naphthylethylenediamine
dihydrochloride NED, a light-sensitive reagent. Daniel et al. 1995 presented an FIA system for the automated colorimetric determination of nitrite in seawater with
range of detection of 0.5 – 150 mM and sampling rate of 45 samplesh. Furthermore, another photometric-FIA system has been presented by Ensafi and Kazemzadeh
1999 using the catalytic effect of nitrite on the oxidation of gallocyanine by bromate in acidic media. The method presents a linear range of detection of
0.010 – 2.5 ppm with an analysis time of 3 minsample. Finally, capillary zone electrophoresis has been used for the analysis of nitrite in seawater samples using
artificial seawater as the carrier solution Fukushi et al., 1999. The range of detection is 0.07 – 2 ppm NO
2 −
while the nitrite peak in the electropherogram appears in the time period of 13 min.
Most of the above techniques can offer low detection limits of ammonia or nitrite measurement in seawater samples with high salinity and large amounts of sus-
pended matter. But when measuring aquaculture samples with relatively high concentration of ammonia and nitrite, dilution of the sample must take place due
to the relatively low upper limit that most of the above techniques present. In addition, due to the important role of ammonia and nitrite in fish farming, the
simultaneous measurement of both substances is of great importance. In this paper, a direct electrochemical ammonia and nitrite monitor is presented for the simulta-
neous measurement of ammonia and nitrite in seawater and aquacultural samples. The ammonia and the nitrite detectors used are Ion-Selective Electrodes Severing-
haus-type Electrodes, Collison et al., 1989 with the well-known fast response time and good reproducibility in conjunction with the capabilities of the flow analysis
FA manifold. The samples are directly inserted into the system with no sample pretreatment indicating a simple analysis procedure. Furthermore, the signal of
each electrode is recorded in a different mV-meter with data logging with the capability of concentration reading after the calibration curve is performed. There-
fore, the presented system is fully automated and capable of the direct, continuous and simultaneous measurement of NH
3
and NO
2 −
levels in seawater and aquacul- ture samples. Furthermore, due to the simple analysis procedure and the robust,
small and light system hardware, the system is portable. Therefore, it can be used for field measurements, which is important for both the ammonia and nitrite
measurement that otherwise a sample pretreatment for the stabilization of the analyte concentrations is required Aminot and Ke´rouel, 1997b.
2. Materials and methods
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