Colorimetric Fiber Optic Based Probe for Real-Time Monitoring of Dissolved CO 2 in Aquaculture Systems

: Dissolved carbon dioxide (dCO 2 ) evaluation is very important in many different fields. In this work, a new, integrated, colorimetric-optical fiber-based system for dCO 2 monitoring in aquaculture industry was developed. The sensing chemistry is based on colorimetric changes of the used indicator—poly p-nitrophenol (pNPh)—in contact with CO 2 . Preliminary tests were done in a laboratory environment (calibration) and in a laboratory Recirculating Aquaculture System (RAS) with controlled CO 2 injection. The results have shown the suitability of the new sensor for assessing dCO 2 dynamics in RAS and its fast detection of low dCO 2 concentrations in an appropriate operation range.


Introduction
Dissolved carbon dioxide (dCO2) evaluation is very important in many different fields as ocean, river and lake monitoring, food industry control (e.g., aquaculture) and clinical analysis. So far, there are some different methods for CO2 measurements, including gas chromatography, colorimetry, amperometry, potentiometry, UV/VIS spectrophotometry and IR spectrometry [1]. These methods are time-consuming, expensive and usually not suitable for real-time monitoring of dCO2. In the last years, several authors developed optical fiber-based technology for real time detection and quantification of dissolved carbon dioxide [2][3][4][5]. However, some complications appeared when measuring in the field due to the carbonate system complexity [2]. In this work, a new and integrated colorimetric-optical fiber-based system combined with a sensing membrane was developed for dCO2 monitoring in aquaculture industry. The sensing chemistry of the membrane is based on colorimetric changes of the used indicator-p-nitrophenol (pNPh)-in contact with CO2. To increase the sensitivity of the sensing membrane, the indicator was polymerized (poly pNPh) and its derivatives used to make the sensing cocktail. The poly pNPh is kept deprotonated in the sensing membrane by action of a quaternary ammonium hydroxide (QA-OH). In that form, the hydroxyl group is involved in protolithic reactions by action of the hydrogen carbonate formed by the equilibrium H2O-CO2. In the presence of carbon dioxide, the sensing layer changes its optical properties (absorption and refractive index), which changes the posterior optical response. Laboratory tests were made to ensure the suitability of the new sensor for assessing dCO2 dynamics and its detection of low dCO2 concentrations, important for aquaculture operations.

Chemical Reagents
The sensing membrane was developed from a cocktail with the following chemicals

Sensing Chemistry and Sensing Layer Preparation.
The sensing chemistry is based on the acid-basic equilibrium of p-nitrophenol (pNPh) derivatives, a well-known colorimetric indicator for titrations with a pKa of around 7.2, that is converted into the anionic form by addition of a quaternary ammonium hydroxide. The sensing layers were attained by dissolution of an amount of poly pNPh in MeOH:H2O mixture and mixed with the QA-OH. After total mixing it was added the Hydrogel D4 solution (10% in Ethanol, 96%) and stirred till obtaining a homogeneous solution. The resulting cocktail was spread on a Mylar foil by spin coating and allowed to dry. After that, the sensing layer was protected with a thin layer of a gas permeable silicone. Furthermore, the total isolation of the sensing layer avoids the direct contact of the sensing film with the tested media making it impossible that the sensor responds to a media pH variation, allowing just the gas passage through it till the detection layer. In its turn, the CO2 will react chemically with the ion pair formed by the polymer with the QA-OH (QA + pNPh − ·xH2O) as shown in equation 1. That reaction will decrease the internal pH of the membrane become it more colorless. The resulting membrane was attached to an optical fiber probe in transmission mode illuminated with integrated dual wavelength LED combined with collimating lenses to improve the light signal. The signal is received by a micro detector and analyzed by a computer software specially developed for this purpose. Figure 1 shows the preparation scheme of the sensing membrane and the optical fiber probe configuration.

Sensor Calibration
For a more precise dCO2 evaluation it was made a chemical calibration using standard solutions with known concentrations of CO2. The formation of carbon dioxide results from the reaction of sodium carbonate in a citric acid solution as shown in Equation (2). The sensor calibration was made in a range between 1 and 25 mg·L −1 (Figure 2a). Furthermore, for a linear correlation, the graph in Figure 2b is presented in the form of logarithmic of the CO2 concentration (log[CO2]) versus the registered absorbance at each CO2 concentration. The calibration values obtained from data calibration are presented in Table 1.

Sensor Validation
Tests were made in a laboratory aquaculture system (V = 1 m 3 of dechlorinated freshwater), with a culture tank and water filtering systems (Figure 3a). This system recreates the environment of a Recirculating Aquaculture System (RAS) existent in several aquaculture facilities.
Gaseous carbon dioxide was injected in the tank to simulate the presence of a certain biomass of fish, controlled by a flow controller (7.5 mL·min −1 ) and the experience was repeated twice in the same day. Each CO2 injection lasted 1 h and the registered sensor data is plotted in Figure 3b.
The performance of the new dCO2 sensor is almost the same after two injections, showing up to approximately 14.5 mg·L −1 (see Figure 3b). Furthermore, the carbonate hardness (KH, Aquapex kit test) and the pH of the water (after full injection) were also accessed and from these parameters the dCO2 was calculated to be in the range between 14.3 and 18.0 mg·L −1 ( Table 2).

Conclusions
In conclusion it is possible to say that the new colorimetric fiber optic based probe is appropriate to work in low CO2 levels situations. These tests have shown the suitability of the sensor and it fast and precise detection in close to real applications. The dissolved carbon dioxide showed by the colorimetric sensor is in the range of concentrations estimated by the pH, carbonate hardness and temperature. It is secure to say that the sensor needs a complementary work on the sensing scheme but also in the sensor body to optimize its functionality and avoid the effect of the external interferences present in the real environment.