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
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 NH
4
Cl and KNO
2
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
NH
3
, 100 ppm and KNO
2
, 200 ppm stock solutions, NaHCO
3
Merck, NaCl Merck, MgCl
2
H
2
O
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 H
2
SO
4
K
2
SO
4
reagent was prepared from H
2
SO
4
and K
2
SO
4
Fluka. The internal filling solution of the NH
3
-ISE contained NH
4
Cl and NaCl Merck, while the internal filling solution of the NO
2
-ISE contained KNO
2
Merck and KCl Fluka.
2
.
2
. Apparatus The FA manifold used was the laboratory-built system shown in Fig. 1. It
consists of an ALITEA U4-T multichannel peristaltic pump Alitea AB, Stock- holm, Sweden for sample and reagent delivery operating between 9 and 16 V,
delivery tubing Alitea AB, R52125 i.d 1.60 ccM and Q76265 i.d. 0.030 in., a V-100 D diagonal flow selection valve UPCHURCH Scientific, Oak Harbor, WA
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 NH
3
and the NO
2
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., Cambridge, 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 pHmV 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 AD
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 24 9 1°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 Ca
2 +
, Mg
2 +
and Ba
2 +
. These substances act as interferences because they complex with NH
3
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 NH
3
and NO
2
seawater samples. S, sample; A, EDTA reagent; B, NaOH reagent; C, H
2
SO
4
K
2
SO
4
reagent; RC, reaction coil.
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 NH
3
-ISE. For the measurement of nitrite, the sample is mixed with the H
2
SO
4
K
2
SO
4
reagent for the production of the measured gaseous NO
x
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 NH
3
and NO
2 −
. The validation of the system’s accurate measurement of ammonia and nitrite
has been held by recovery studies of seawater samples with low NH
3
and NO
2 −
. 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 NH
3
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 NO
2 −
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 recirculating system containing on average 70 larvael, of 4 – 12 mm in length. The rearing medium also included five – ten zooplanktonic organisms
ml rotifers and Artemia nauplii; and 0.5 – 1 × 10
6
cellsml of the phytoplankton species Chlorella minutissima cell diameter 1 – 2 mm.
2
.
3
. Analysis procedure The reagents of EDTA, NaOH and H
2
SO
4
K
2
SO
4
and the standard solution containing 2.5 ppm NH
3
and 2.5 ppm NO
2 −
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 NH
3
and NO
2 −
for a period of 3.5 min. Each time the actual concentration of NH
3
and NO
2 −
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 NH
3
and the NO
2 −
concentration with an analysis time of 3 – 4 min.
Fig. 2. Analysis of artificial seawater standard solutions with the ammonia and nitrite levels indicated.
3. Results and discussion
