Direct electrochemical flow analysis system for
simultaneous monitoring of total ammonia and
nitrite in seawater
E.A. Moschou
a, U. Azpiroz Lasarte
a, M. Fouskaki
a,
N.A. Chaniotakis
a,* , N. Papandroulakis
b, P. Divanach
baDepartment of Chemistry,Laboratory of Analytical Chemistry,Uni6ersity of Crete, Iraklion71 409Crete,Greece
bDepartment of Aquaculture,Institute of Marine Biology of Crete,Iraklion71003Crete,Greece
Received 2 October 1999; accepted 22 February 2000
Abstract
The design and the performance of a new portable flow analysis (FA) system for the continuous, fast and accurate measurement of total ammonia and nitrite content in non-filtered seawater samples is demonstrated. The complete system has been optimized to operate within the ammonia and nitrite concentration range of 0.05 – 10 ppm. The system offers good reproducibility (B5%) and stability (B0.02 ppm/h) at constant temperature, while the analysis time is in the order of 1.5 – 4 min depending on the sample analyzed. The analysis results of seawater samples obtained with the FA system were compared to that obtained with the standard colorimetric method and establish the suitability of the analyzer for the precise and continuous measurements of untreated samples for both in field and laboratory applications. In addition, its small size and weight offer the advantage of portability, while its datalogging capabilities also allow for independent ammonia and nitrite monitoring. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Ammonia; Nitrite; Monitoring; Seawater; Electrochemical portable monitor
www.elsevier.nl/locate/aqua-online
* Corresponding author. Tel.: +30-81-393618; fax:+30-81-393601. E-mail address:[email protected] (N.A. Chaniotakis)
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 theNitrosomonasbacteria 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 mg/l) 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).
Existing methods for the analysis of ammonia in seawater samples include colorimetry, ion chromatography, cathodic stripping voltammetry, mass spectrome-try and fluorimespectrome-try. 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 mg/l with sample rate of 16 samples/h. 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 min/sample. 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 15N – 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 witho-phthaldialdehyde and sulfite (Ke´rouel and Aminot, 1997). The range of detection is 0.5 – 250mM and the analysis time is 3 min/sample.
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-naphthyl)ethylenediamine 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 – 150mM and sampling rate of 45 samples/h. 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 min/sample. 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 NO2− while the nitrite peak in the electropherogram appears in the time period of 13 min.
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 NH3 and NO2− 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
2.1.Reagents
For all experiments, deionized water (Barnstead NAN-O-Pure) and chemicals puriss or p.a. grade were used. Ammonia 100 ppm and nitrite 200 ppm stock solutions were prepared from NH4Cl and KNO2 (Merck, Darmstadt, Germany), respectively.
The ammonia and nitrite standard solutions used were made of artificial seawater samples according to Moschou et al. (1998) with the appropriate amounts of the NH3, 100 ppm and KNO2, 200 ppm stock solutions, NaHCO3 (Merck), NaCl (Merck), MgCl2(H2O)6 (Fluka, Ronkonkoma, NY) and NaOH (Merck). All stan-dard solutions were prepared daily.
The ethylenediaminetetraacetic acid (EDTA) reagent was prepared from the disodium salt of EDTA (Serva, Haupauge, NY) and the NaOH reagent from sodium hydroxide pellets (Merck). The H2SO4/K2SO4 reagent was prepared from H2SO4 and K2SO4 (Fluka).
The internal filling solution of the NH3-ISE contained NH4Cl and NaCl (Merck), while the internal filling solution of the NO2-ISE contained KNO2 (Merck) and KCl (Fluka).
2.2.Apparatus
and finally two reaction coils spiral-shaped (0.030 in. i.d., 50 cm length) and one reaction coil (0.30 in. i.d., 100 cm length) for the NH3 and the NO2 compart-ment, respectively. All other tubing, connectors and tees were also PEEK (PolyEtherEtherKetone) for minimum ammonia loss. The potentiometric flow-through cells were constructed of DERLIN® Acetal Resin (Goodfellow Cam-bridge Ltd., CamCam-bridge, UK). The Severinghaus-type ammonia and nitrite ISEs were constructed in our laboratory and are commercially available. The potential was monitored using two model 290-A pH/mV meters (ORION Research, Bev-erly, USA), with datalogging capabilities, operating at 9 V DC. When extensive data analysis was required, a personal computer equipped with a 16 Bit A/D converter to collect the data, controlled by home-made software written in Basic. The whole analytical system was embodied in a case made of stainless steel and polyethylene with dimensions 44.5 cm length, 25 cm width and 26 cm height, with the designation of portability. The reagent vials and the sample-reagent tubing are the only parts of the apparatus outside the case. All laboratory experiments were performed at 2491°C.
As shown in Fig. 1, for the measurement of the ammonia concentration, the sample solution is first mixed with the EDTA reagent within the FA system for the complexation of the cationic species such as Ca2+, Mg2+ and Ba2+. These substances act as interferences because they complex with NH3 thus reducing the actual free concentration of the ammonia that is measured. Then the ammonia
Fig. 1. Schematic diagram of the FA system used for the continuous monitoring of NH3 and NO2
seawater samples. S, sample; A, EDTA reagent; B, NaOH reagent; C, H2SO4/K2SO4 reagent; RC,
sample solution is mixed with the basic NaOH reagent for the rise of the sample’s pH above 12, ensuring the complete conversion of ammonium ions into the gaseous form of ammonia, which is measured by the NH3-ISE. For the measurement of nitrite, the sample is mixed with the H2SO4/K2SO4 reagent for the production of the measured gaseous NOx (O’Reilly et al., 1991). The length of the reaction coils are such that ensure the completion of all reactions taking place before the solution reaches the ISEs for the detection of NH3 and NO2−.
The validation of the system’s accurate measurement of ammonia and nitrite has been held by recovery studies of seawater samples with low NH3 and NO2−. Recovery studies on filtered and non-filtered spiked seawater samples have been held in order to validate that no sample pretreatment is required prior to the analysis of seawater samples by the presented system. Furthermore, comparison of the system’s performance to that of the well-established colorimetric tech-niques is being presented. The colorimetric indophenol blue method for the ammonia determination is based on the Berthelot reaction between ammonia, phenol and hypochlorite leading to the formation of an indophenol dye (Aminot et al., 1997a). The measurement of the absorbance of the sample at 630 nm is analogous to the NH3 content of the sample. The colorimetric method for the nitrite measurement is based on the Griess method where the reaction between nitrite and NED reagent for the production of an azo dye is taking place (Daniel et al., 1995). The measurement of the absorbance of the sample at 540 nm is analogous to the NO2− content of the sample. All the samples measured with both colorimetric techniques were filtered prior to the analysis. On the contrary, the samples without pretreatment were analyzed by the presented FA system. The seawater samples analyzed were obtained from the intensive hatch-ery unit of the Aquaculture Department of the Institute of Marine Biology of Crete. The rearing tanks (500 l), from which samples were taken, were function-ing as closed recirculatfunction-ing system containfunction-ing on average 70 larvae/l, of 4 – 12 mm in length. The rearing medium also included five – ten zooplanktonic organisms/
ml (rotifers and Artemia nauplii); and 0.5 – 1×106 cells
/ml of the phytoplankton species Chlorella minutissima(cell diameter 1 – 2 mm).
2.3.Analysis procedure
The reagents of EDTA, NaOH and H2SO4/K2SO4 and the standard solution containing 2.5 ppm NH3 and 2.5 ppm NO2− are inserted in the system for a period of 15 min for the system to be equilibrated. Then the calibration curve is held by inserting each of the following three standards containing 0.8, 2.5 and 5.0 ppm of both NH3 and NO2− for a period of 3.5 min. Each time the actual concentration of NH3 and NO2
−
of each standard is being set. Then distilled water is introduced in the system for 3 min to avoid sample contamination. Subsequently, the unknown sample solutions are introduced to the system for the simultaneous measurement of the NH3 and the NO2
− concentration with an
Fig. 2. Analysis of artificial seawater standard solutions with the ammonia and nitrite levels indicated.
3. Results and discussion
3.1.E6aluation of the FA system’s performance
The potential chart recordings of the FA system in operation when samples of different ammonia and nitrite concentrations are analyzed are presented in Fig. 2. In Fig. 2A the system’s response to the ammonia levels in standard solutions is presented, while in Fig. 2B the response of the system to the nitrite content of the standard solutions is demonstrated. The range of detection presented is between 0.1 and 10 ppm of NH3and NO2
−. Furthermore, in Fig. 2A and B the response of the
system to the primary species, NH3 and NO2
− in the presence of NO
2
− and NH
3, respectively as interfering species is demonstrated. The measured concentration of 0.5 ppm NH3by the presented system in the presence of 5 ppm NO2
−in the sample
monitoring of an artificial seawater sample containing 2.5 ppm NH3 and 2.5 ppm NO2
−is presented. The signal drift of the system is 0.02 mV
/min for the monitoring of both ammonia and nitrite substances for the time of 3 h indicating the measurement of 2.590.12 ppm/h NH3 and 2.590.17 ppm/h NO2.
3.2.Analytical results
In order to evaluate the performance of the FA system described, seawater samples containing ammonia and nitrite were measured and the values were compared to the results obtained using the well-established colorimetric methods. Initially, seawater sample with low ammonia and nitrite content was used to complete the recovery studies. The recovery studies were carried out by the additions of appropriate amounts of the stock ammonia and nitrite solutions to the filtered seawater sample with low ammonia and nitrite content. The filtration of the
Fig. 3. Calibration curve of the FA System towards the NH3and the NO2− content of the artificial
seawater standard solutions.
Table 1
Analysis time of artificial seawater samples varying the concentration of ammonia and nitrite under a single run
NH3(ppm) T(min) NO2 (ppm) t(min)
3.0
0.10 0.10 1.8
2.9 0.50
0.50 3.3
1.00
1.00 3.1 3.2
2.8
2.50 2.50 3.3
2.3 5.00
5.00 2.3
1.5 10.0 1.8
Table 2
Signal reproducibility of the FA systema
E2(mV)b E3(mV)c AverageE(mV)d E1(mV)
Sample type Concentration (ppm)
86.0 83.2
NH3 0.5 86.4 85.291.7
50.0 47.2
47.5 48.291.5
2.5
28.0 29.8 29.391.1
5.0 30.0
12.0 13.0
13.0 12.790.6
10
NO2 0.5 44.0 42.0 45.7 43.991.9
72.0 71.8
72.0 71.990.1
2.5
84.0
5.0 82.0 83.5 83.291.0
10 97.5 95.0 96.2 96.291.3
aThe time between each measurement of each sample is 1.5 h. bE
2 is the potentiometric response of the system 1.5 h afterE1. cE
3 is the potentiometric response of the system 3 h afterE1.
dAverageE(mV) is the average of the individual potentiometric responsesE
1,E2andE3.
Fig. 4. Signal stability of the FA system during the analysis of an artificial seawater sample containing 2.5 ppm NH3and 2.5 ppm NO2−. The signal drift of the FA System was measured to be 0.02 mV/min.
seawater samples was held for the ammonia and nitrite determination by the colorimetric technique and also for the comparison of the results obtained for the filtered and non-filtered seawater samples using the FA system.
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Table 3
Determination of ammonia in filtered and non-filtered seawater samples from rearing medium with FA system
% Non-filtered sample concentration (ppm) % R* Sample type Expected sample concentration Filtered sample concentration (ppm)
R* (ppm)
NH3 0.30 0.31 103 0.28 93
104
1.40 1.49 106 1.46
5.00 100
5.00 5.17 103
0.66 106
NO2 0.62 0.62 100
104
1.54 1.60 104 1.60
5.35 104
5.00 97
5.16
265
Determination of ammonia and nitrite (primary species) in non-filtered spiked seawater samples in the presence of nitrite and ammonia (interfering species)a
Interfering concentration (ppm) Resulting concentration Primary species Primary concentration (ppm) Interfering species type
type
The evaluation of the presented FA system was performed by the comparison of the determined NH3 and NO2
− levels in spiked seawater samples to that obtained
with the standard colorimetric methods. The photometric indophenol-blue method was used for the NH3 determination while the Griess method was held for the measurement of the NO2−content of the samples. The seawater samples containing 0.1 ppm NH3 and 0.04 ppm NO2 were filtered through a 0.4 mm filter in order to be used for the colorimetric technique. On the contrary, the samples analyzed with the FA system were not filtered. The seawater samples were spiked with different amounts of ammonia and/or nitrite and the analysis results are shown in Table 4. The samples containing ammonia and/or nitrite levels greater than 2 ppm could not be analyzed with the specific colorimetric methods due to the limitations in the range of detection. From Fig. 4 it can be seen that the presented FA system can be used for the accurate monitoring of non-filtered seawater samples with low and high ammonia and nitrite content.
4. Conclusions
The design and the performance of a portable FA system for the continuous, fast and accurate measurement of the ammonia and nitrite content in non-filtered samples difficult to analyze such as marine aquaculture rearing medium and effluents of waste water treatment plants, with large amounts of earth metal ions and suspended matter, is demonstrated. The complete system is capable of suitable operation within the ammonia and nitrite concentration range of 0.05 – 10 ppm. The system offers good reproducibility (B5%) and stability (B0.02 ppm/h) at constant temperature, while the analysis time is in the order of 1.5 – 4 min depending on the sample analyzed. The analysis results of seawater samples obtained with the FA system were compared to that obtained with the standard colorimetric method and is suitable for continuous measurements of untreated samples in both field and laboratory applications. In addition its small size and weight offer the advantage of portability, while its datalogging capabilities also allow for independent ammonia and nitrite monitoring. Application of the system in aquaculture facilities can provide improved culture management by reducing potential problems due to ammonia and nitrite toxicity.
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