PROTOTIPADO: Efectos de los caudales y la transmisión UV-T en un reactor con

para el tratamiento del agua del lastre

El prototipado tiene como objetivo abordar el problema real a una escala más económica y corregible. Todo esto para validar si las autoridades portuarias avalan el tratamiento, se identifican modificaciones que hay que realizarle e ir afrontando a los nuevos problemas que trae el aumentar los tamaños. Estos incluyen flujos, presiones, materiales, fuente de energía. La temperatura, la velocidad y la transmisión UV influyen directamente en las condiciones del proceso, siendo las condiciones de flujo la variable respuesta a estudiar. Teniendo en cuenta que el tratamiento se hará en puerto (Zona tropical), se fija la temperatura a la temperatura del puerto tentativo de instalación (Santa Marta), la transmisión UV se evalúa en tres niveles y finalmente se obtienen diferentes velocidades de flujo que indicaran la dosis de energía eléctrica (Electrical Energy Dose-EED) requerida en el tratamiento. Finalmente, toda invención debe protegerse por algún medio, el seleccionado hasta el momento ha sido el patentamiento y/o secreto industrial, por lo cual se procede a escribir la patente en los términos legales necesarios.

Como la radiación solar natural (SRAD) no tiene una buena eficiencia en inviabilizar organismos planctonicos y el reactor tipo placa plana es usado principalmente para esta, y, por otro lado, el reactor tipo anular (cilíndrico) se usa es para los procesos con UV, entonces el prototipo debe ser anular. Un reactor UV anular piloto a escala de laboratorio (Lab scale prototype) se usó inicialmente de manera individual para hallar cuales condiciones de flujo que debe tener para lograr la eficacia de menos de 10 org viables/mL de fitoplancton en el agua tratada. También se evaluó el efecto espacial que tiene la UV internamente en el reactor. Posteriormente se procedió a construir un prototipo a escala piloto (Pilot scale prototype, Figure 22) con una estructura acoplada y portátil para tener la posibilidad de llevarla a puerto o laboratorios para hacer testeos.

Artículo/Reporte: Flow rate and UV-T (UV transmission) effect in a UV reactor with Advanced Oxidation Process (UV/H2O2/TiO2) for a pilot ballast water treatment. En prensa (Revista por definir).

Patente: Sistema de Tratamiento de Amplio Espectro para el Agua de Lastre. Solicitada ante la SIC con Ref. Expediente N° NC2017/0009530.

Objetivo específico al que responde: Determinar el tamaño de mercado para la aplicación de la tecnología de tratamiento en puerto y las condiciones de un prototipo de fotorreactor (anular y/o placa) para la disminución de organismos planctónicos viables y patógenos.

Figure 22. Prototipo a escala laboratorio y a escala piloto para el tratamiento del agua de lastre.

ARTÍCULO: Electrical Energy Dose and UV-T effect in a UV reactor with Advanced Oxidation Process (UV/H2O2/TiO2) for treatment of ships’ ballast water at pilot scale

ABSTRACT

Ballast water treatments have been developing in recent years to reduce the viability of non-native marine organisms transported in ships’ ballast tanks, which pose potential problems to human health, economics and biodiversity. In Colombia, a country with high biodiversity, it is important to find suitable treatment solutions in port, capable to safely manage the volumes of ballast water discharged. For the purpose of designing and testing an adequate ballast water treatment prototype, this research evaluated a treatment solution based on Advanced Oxidation Processes (AOP), employing sterilization by UV/TiO2/H2O2, and hereby the effect of Electrical Energy Dose-EED, by varying flow rate and intensity of UV transmission (UVT) on the survival and activity of a microplankton species [10-50 µm diameter] amended by a culture of the alga Tetraselmis contracta in (A) laboratory-scale and (B) prototype-scale reactors. Electrical Energy Dose (EED) varied between 1 and 0.27 KWh/m3 _{and UV transmission (UVT) ranged between 50% and 85% The EED} causing the greatest decrease in abundance was 0.6 KWh/m3_{, whereas it was 0.43} KWh/m3 _{for the highest decrease of phytoplankton activity (photosystem II efficacy).} Best efficacy was observed for UVT transmission ≥85%. The Equivalent Dose (power of UVC-only treatment to achieve the same mortality as a combined treatment such as AOPs) necessary to prevent phytoplankton regrowth was around 400-600 mJ/cm2_{. Additional treatment variables found to significantly increase} plankton mortality were increased water temperature and addition of coffee as a quenching agent.

Keywords: Ballast Water Treatment, Tetraselmis contracta, Design factors, transmission UV, Advanced Oxidation Processes.

INTRODUCTION

Colombia is a high biodiversity country, where the marine environment is used for aquaculture, recreation, biodiversity protection and at the same time for maritime transport. Shipping activity can cause local and broader scale environmental and human health problems by affecting water quality and its species composition, (Gollasch et al., 2015). In part, the problems are due to introduction of non-native species and pathogens carried in the ships’ ballast water which is deballasted in ports. Establishment of non-native species can affect local ecosystem composition, structure and function, with potential losses and impacts on coastal economies, biodiversity and human health, such as observed during the cholera outbreak in Latin America in 1991-1992 (McCarthy and Khambaty, 1994).

Ballast water is necessary for the safe operation of ships and these volumes are increasing in step with the increase of maritime activity. The International Maritime Organization-IMO (OMI, 2004) and the U.S Coast Guard-USCG (USCG, 2012) have developed regulations for ballast water management and treatment. There are 3 broad kinds of ballast water management options:1) exchange at sea, 2) treatment on board and 3) Isolation (Figure 21). Ballast water exchange at sea is the most commonly used management practice, even though outdated, but is still applied in many countries like Colombia. The treatment on board is a better solution, from the perspective of reducing the likelihood of species introductions, but it is not suitable for all ships due the space required and a large investment. On the other hand, the isolation with treatment in port can be more cost-effective option when strategically located in a specific port with a high risk of species introductions and a susceptible biodiversity.

Figure 21. Options for the management of ballast water, in red the option explored in this research. (Modified from: Wijnolst and Wergeland 2009).

The International Maritime Organization-IMO formulated the International Convention for the Control and Management of ballast water and sediment from ships, making it mandatory to evaluate the efficacy of the treatment option and ensuring it complies with maximum permissible limits. This regulation took effect in Colombia via Resolution 0477 from 2012 (Min. Defensa Nacional, 2012), and also went into effect in other countries in September 2017 (IMO, 2016). On a global scale this convention is very important for the responsible management of the ballast water. Seebens et al. (2013) modelled the risk of marine bioinvasions from ballast water, finding that there is a considerable decrease in global risk when the risk of species introductions is reduced is only a few selected high-risk ports. Additional, Pereira and Brinati (2012) studied the logistic viability for a treatment in ports in Latin America, concluding that it is technically feasible and effective to implement ballast water treatments in ports with high shipping volume.

In Colombia, López et al. (2015) identified the three ports with the highest ballast water volume and risk of non-native marine species introductions to be: Puerto

Bolívar, Santa Marta and Coveñas. Among these three ports, all located in the Caribbean Sea. Santa Marta has the highest discharges of ballast water (DIMAR- CIOH, 2009) and also the most numerous reports of new species (17) from the Colombian Caribbean (Ahrens et al, 2010). Garcia-Garay (2016) estimated volumes of 31.6 X 106 _{and 11.8 X 10}6 _m3_{/year being de-ballasted in Santa Marta and Puerto} Bolivar, respectively. Both ports are located in the north of Colombia, near the Panama Canal and close to busy international shipping routes, making them very relevant locations to explore the feasibility of the treatment in port.

To be able to compare treatment efficiency it is essential to have similar environmental conditions. The environmental conditions for the two ports of interest (Santa Marta and Puerto Bolivar) include the highest level of solar radiation in Colombia, as is the case for Puerto Bolivar (Vanegas et al. 2015), making a ballast treatment approach that includes solar radiation attractive. Also, the high daytime air temperatures of approx. 35°C, indicate conditions that are potentially hostile to marine organisms, if ballast water could be heated to these temperatures.

Irradiation by UV radiation (UVC) is a common approach to sterilize microorganisms in drinking water, but additional modifications are necessary to improve ballast water treatment, due to the wide range and kinds of organisms and sizes present. For example, combining UV-irradiation treatment with exposure to an oxidative catalyst such as H2O2 (UVC/H2O2 treatment) increases mortality and prevents re- growth of bacteria (Moreno-Andrés et al. 2016). The combination of oxidation processes (e.g. UVC/TiO2/H2O2, UVC/O3/H2O2) is likely to increase OH radicals, leading to better sterilization results than individual treatments (Meghana et al., 2007). The phytoplankton concentration is inversely correlated to OH concentration, which “causes direct cell death in phytoplankton from its strong oxidant capacity” (Llabrés, 2008).

One of the first steps for a full-scale ballast water treatment solution, is to perform simulations or smaller-scale prototypes. They provide cost-effective information about treatment designs and the efficacy and necessary improvements before constructing a full-scale equipment, where the corrections would be very expensive. The objective of this research is to define some operational parameters for the treatment in port of ballast water in tropical regions, such as Colombia. One of the most important parameters to study in the design of a ballast water treatment solution is the intensity range of transmitted UV-radiation (UVT) and the exposure dose (radiation received per unit time e.g. electrical energy dose, EED), which is related to flow rate. Garcia-Garay (2016) found that a realistic treatment volume for a prototype in a high-risk Colombian port is on the order of 360 L/h (0.36 m3_{/h). The} prototype design involves the use of pumps to achieve different flow rates. By changing flow rate, the UV exposure dose changes: for a high UV dose the flow rate is slow and for a lower dose the flow rate is faster. To minimize operational costs, it is important to define a maximum flow rate than can accomplish treatment efficiencies that comply with the IMO regulation.

One way to include the flow rate in the performance evaluation of AOP treatment solution with OH° radicals (Advanced Oxidation Processes) is to evaluate Electrical Energy Dose-EED (Equation 1) or the Electrical Energy Order Reduction-EEO (Equation 2) of the UV radiation received. These were taken into account in this research together with the Guidelines for Approval of Ballast Water Management Systems-G8 (IMO, 2013). For instance, the intake concentrations for the organisms of 10-50 µm diameter should be no less than 103 _{cel/mL, and preferably 10}4 _cel/mL, to match typical cell concentrations in natural waters. For the treated discharge they have to be less than 10 viable cel/mL. This means that the treatment efficacy has to be >2 orders (3).

With: P= UV power (kW), T= Irradiation time, 60 min/ hour, V = Total system volume.

With: C0 = initial concentration (cel/mL) and Ce = final concentration (cel/mL).

The efficacy of the treatment conditions can be calculated using a mass balance and percentage relation of the IMO guideline with the D2 and G8 (IMO, 2008) values for the intake water and treated water (3).

MATERIALS AND METHODS

Common aspects for the experiments Biological parameters

The present research included techniques to evaluate the vitality of exposed microphytoplankton by quantifying its abundance as well its viability by measuring the photochemical efficiency of photosystem II of the phytoplankton community. It used fluorescein diacetate (FDA) staining for direct microscope counting of live organism (cells/mL), Water-PAM fluorescence for measuring photosystem II efficiency (Fv/Fm) and the BallastCheck2 as an alternative technique for estimating phytoplankton abundance (cells/mL) and activity (Fv/Fm) (Figure 23).

Figure 23. Equipment and elements for the measurement of: Photosystem II photochemical efficiency with the PAM-Walz photometer (A), the count of vital microzooplankton in the Sedgewick-Rafter counting chamber with FDA fluorescence (B) and the abundance and activity of the phytoplankton with the BallastCheck2 (C). The techniques to evaluate the treatments and the dose included quantification of the abundance of live organisms (vitality) and the abundance of organisms that can multiply (viability). For determining vitality, the Fluorescein Diacetate-FDA count is a common method to stain living plankton by esterase activity (Peperzak and Brussaard, 2011). The FDA dye penetrates the cell of animals and plants and is transformed by cellular esterases into fluorescent fluorescein, resulting in a fluorescent colour inside the cells (Figure 24) The ETV protocol (Environmental Technology Verification) (NSF, 2010) uses FDA staining together with the CMFDA staining (5-Chloromethylfluorescein diacetate, green fluorescent dye), but the CMFDA is more expensive and can increase the false positives. For this reason, the present study only used FDA dye.

Figure 24. FDA stained cells with as seen under a fluorescent microscope (blue light excitation).

The BallastCheck2 is an equipment to estimate the abundance and activity of phytoplankton by quantifying chlorophyll fluorescence. For assessing abundance, shorter wavelength light emitted from the fluorometer is absorbed by algae and the phytoplankton and emitted as a fluorescence that is related to a numerical estimate of abundance.

C B

For quantifying photosynthetic activity, the BallastCheck2 and the Pulse Amplitude Modulation Water-PAM-(PAM) both use two LEDs with variable intensity. The first light with low intensity measures the minimum fluorescent value of the sample (Fo). The second LED (saturating) blasts the photosystem II to a maximum fluorescence state (Fm). The difference between (Fm) and (Fo) is called variable fluorescence (Fv) and is related with the Fm to obtain the Activity (4). Both instruments follow the same principle, but the PAM approach is more robust. The response parameter is called yield or Photosystem II Photochemical Efficiency-PSII (Turner 2016, Heinz- Walz 2013).

(4) The ratio (Fv/Fm) is a good measure of algal activity, which tends to range between 0.01 and 0.75 (Turner, 2016). Moreover, Fv/Fm can be related to the viability of phytoplankton using the following criteria (Stehouewer et al., 2010):

• Fv/Fm ≥ 0.5 healthy population.

• 0.3< Fv/Fm < 0.5 population is not in optimal condition.

• < Fv/Fm < 0.3 population is dying.

• <0.1 population is considered dead.

Phytoplankton abundance and activity were also measured with a BallastCheck2 instrument (Turner Designs). The samples were stored in dark at least 15 minutes before measurement. The calibration was verified with a solid standard. The measures were done by initially measuring an unfiltered sample and then a 10 µm filtered sample to obtain the measures of the phytoplankton >10 µm. Each sample was measured 3 times.

For the photosystem II (PSII) photochemical efficiency, a Water-PAM fluorometer was used (Walz Heinz GmbH, Germany). A volume of 10 ml of 0.2 µm filtered sample was used as the blank. A solid fluorescent standard and ultra pure (UP) water were used for the calibration and a negative control. The gains (amplitude fluorescence signal parameter) for the solid fluorescent standard were 2-5, and were 5,10,15, 20 and 25 for the ultra pure water. The samples were stored in dark for a least 15 min before measurement. The current fluorescence (ft) for the samples ranged between 100-1000 mV. Each sample was measured 5 times.

The FDA reagent (Invitrogen F1303), was dissolved in acetone 99+% (Alfa Aesar) at stock concentrations of 500 µM FDA and aliquoted in separate vials. The samples had a final concentration of 5 µM FDA by adding 15 µL of stock to 1.5 mL of the sample. These stained samples of 1.5 mL were incubated in the dark and then counted in a Sedgewick-Rafter chamber of 1 mL volume under an epifluorescence microscope (Zeiss Axioplan II) at 495 nm. Glass counting

chambers (#3220) with 1000 squares (each one with a volume of 1µL) were used. The number of microzooplankton counted was divided by the number of squares surveyed, to obtain a density of individuals per mL. The higher densities were counted until reaching 200 cells/volume (7% error) whereas for the low densities the entire chamber was counted. The samples were counted in duplicate, subtracting the background counts of naturally fluorescent particle fluorescence, determined in negative controls of samples heated to 50°C for 30 min.

Physicochemical parameters and UVC intensity

The parameters measured in intake and treated water were Dissolved Oxygen (DO), pH, Temperature, Dissolved Organic Carbon (DOC), Salinity, Turbidity (NTU), Nitrogen, UV transmission (UVT), Total Suspended Solids (TSS) and Particulate Organic Carbon (POC) (Table 9) The measurements were done according to the standard operating procedure (SOP) in its last version (June 2016) of the Ballast Water Lab from the Royal Netherlands Institute for Sea Research (NIOZ) (confidential). The equipment used are listed in the Table 9.

Table 9. Water quality parameters and equipments.

Measurement Equipment and model Calibration Oxygen pHenomenal OX4100H portable O2

meter 0 and 100% saturation

pH Metrohm 826 pH mobile with unitrode

electrode with Pt sensor (Applikon) Buffer solutions 4, 7, 9 Salinity Digital conductivity Meter GMH 3430

with Pt sensor (B) (Gresinger, Germany) LRM 15 g/kg IAPSO: CRM Temperature Digital conductivity Meter GMH 3430

with Pt sensor (B) (Gresinger, Germany)

NIOZ Department of marine Technology Electronics Turbidity Oakton T-100 Turbidimeter Oakton Standards UVC VWR UV-1600 PC Spectrophotometer

UP-water; Hellma Analytics 667.301-UV Calibration Standard Set

Samples for DOC were filtered with GF/F filters (0.7 μm), acidified and sent to the CO2 Laboratory at NIOZ-Texel (Netherlands) for analysis. The samples for TSS and POC were filtered by triplicate onto pre-weighted filters (Figure 25) and heated in an oven at 60°C overnight, dried in a desiccator and weighted to obtain the TSS concentration. Finally, the filters were combusted in a muffle furnace at 550°C for 12 hours and weighted again to obtain POC content.

Figure 25. Filtration system for the Total Suspended Solids-TSS and Particulate Organic Carbon-POC measures (a) and GF/F filtration system for the DOC measures (b) thermometer and salinity meter (c).

UVC intensity

The UVC intensity was measured in a same central point for three distances from the lamp (0, 2.1 cm and 4.2 cm) ( Figure 26). The lamp was preheated for 45 min before each measurement, taking triplicate readings over three minutes. An air refrigeration system was used for the lamp.

Figure 26. UVC Sensor and radiometer for measuring UVC intensity at different distances from the UVC light source.

Adjustment of water temperature and initial abundance

b

c

a

Temperature

The natural marine water had conditions that varied according to the season, daytime, tide and other factors. To store the natural water, Nalgene bottles (15 L) and IBC tanks made of polyethylene (600 L) were used. The water volumes for the experiments were 10 L in the bottles and 200 L in the tanks. To simulate the temperature of the Colombian ports, the water was heated to 35°C, with aquarium heaters of 300 W (Resun). Nevertheless, in the first experiment the temperature did not increase sufficiently in the IBC, requiring a thermal isolation of the sides of the tanks (Figure 27).

Figure 27. Heaters for the Nalgene bottle of 15 L and the polyethylene IBC tanks and the isolation material for the IBC.

Population increase from the intake water

To increase in the number of phytoplankton organisms, natural marine plankton communities were amended with cultures of Tetraselmis contracta (recommended by the Environmental Technology Verification protocol, NSF 2010). The culture medium used for growing T. contracta was Mix 50%/50% (Mix of F/2 medium and ES medium) with nutrient and vitamin addition (provided by Plankton lab-NIOZ). The culture was stored in Erlenmeyer flasks of 2000 mL (day 1-4) and 5000 mL (day 5-7) and the maximun culture volume added was 2000 mL. The culture temperature was kept at 19°C at a salinity of 20-27 g/kg. Temperature and the light were controlled in an incubator before the experimental treatments.

77 García-Garay, J.

Figure 28. Nutrients and vitamins (A) added to the Tetraselmis contracta culture media and refrigerator with light (B) for UV transmitance-UVT decrease.

The intensity of UVT was decreased according with the NIOZ protocol by addition of instant coffee from the producer Douwe Egberts, which is an effective quencher of UVC radiation. To know the relation between coffee added and the decrease of the UV transmission (UVT) a preliminar test was done. This found that an amount of 7 and 30 mg/L of coffee was needed to obtain a 75% and 50% UVT respectively. Experimental set-up

In the experiments a peristaltic pump Easy-load II (ColeParmer) was included, a cartridge filter of 50 µm (Moises), three voltage converters 230-110 V (HQ), the oxidant hydrogen peroxide 30% (Anal R VWR) and a UV Reactor (FOTOX, diameter 3 inch and 30 Watts power) provided for Biohidroingenieria. A plastic hose was used to connect the pump, the filter and the reactor. The final hydrogen peroxide concentration in the water for the treatment was 5 ppm. The catalyst in the UV reactor was TiO2 from Evonik P-25, at saturated concentration and absorbrf onto cloth (Figure 29).

Figure 29. Diagram for the experiment: Continuous flow with Filter and UVC reactor with catalyst.

For the pilot scale experiments an industrial pump of 30 Hp was used, with a maximum flow rate of Qmax 450m3_{/h from the manufacturer Calpeda. The natural}