APRIL MAE T. TABONDA MS Marine Science Critical Evaluation of Published Reports Journal Title: Photosynthetic response of ‘Neosiphonia sp. Epiphyte-infected’ and healthy Kappaphycus alvarezii (Rhodophyta) to irradiance, salinity and pH variations by Borlongan et al. 2016 I. Statement of the problem Borlongan et al. (2016) stated in the introduction of their study that Kappaphycus alvarezii farming is very crucial in the Philippine economy as it is an important source of kappa-carrageenan, however, seaweed industry in the country has been experiencing decline in the production due to poor quality of seaweed as a result of diseases such as “ice-ice” and epiphytic filamentous algae. Because of these observations, they have associated the quality deterioration of cultivars to ecological stresses (e.g extreme seawater temperature, salinity and pH changes, UV radiation, and high irradiance) triggering the infestation of epiphytic filamentous algae (EFA) Neosiphonia sp. that degrades the health and growth of the seaweed. The problem statement of this study can catch the interest of seaweed enthusiasts, especially those who are venturing in seaweed farming since epiphytic algae infestation has been one of the main problems of seaweed farmers. Additional key factor that might capture the interest of readers are the possible developments in agricultural technologies and knowledge on the physiological aspects of seaweed against epiphytic algae infestation that will help increase the production. II. Clear indication of the research hypothesis This study does not directly indicate the null (H0) and alternative (Ha) hypotheses. The dependent variable of this study was the photosynthetic performance through oxygen production per fresh weight biomass per unit time (mg O2 g-1 FW h-1), and the independent variables were irradiance, pH, and salinity. They have based their observations from the results of existing studies on K. alvarezii. For example, the study of Pang et al. (2011) reported that infection of the EFA N. savatieri on K. alvarezii influences the host by direct competition for light and nutrients. Ganzon-Fortes et al. (1993) also reported in their study that diseased seaweed had lower photosynthetic rates compared to the healthy seaweed. Due to rampant occurrence of diseases on farmed seaweed especially on K. alvarezii, they have observed that there was a decline on the production, and they have associated this decline to the effects of ecological stressors that triggers the infestation of diseases like EFA. Results from previous studies has become their basis in doing this experiment. As cited from previous study, it was clearly obvious that diseased and epiphyte infected K. alvarezii were susceptible to lower photosynthetic performance, thus, with the addition of stress treatment, it might have negative effects. They just need to compare and validate their results from existing studies. III. Identification of the experimental unit and/or sampling unit The experimental units used in this study were 1-3 g of epiphyte-infected and healthy K. alvarezii. Sampling units were portions of K. alvarezii (apical, mid or basal combined) measuring 6-8cm. The experiment material used was biological oxygen demand (BOD) bottle. The selection of the sampling unit was reasonable because they have used both young and adult portion of the seaweed which is crucial in determining the photosynthetic rate of the seaweed. Young portion may have lesser or underdeveloped photosynthetic apparatuses like chloroplast, responsible for photosynthesis, than adult portion. Using both young and adult portions may eliminate the occurrence of bias in the photosynthetic performance of the seaweed. IV. Presentation of treatment designed and experimental or observational study design including factors and levels of the treatment design The design used was comparative/experimental study performed in a closed system. The factors used in this experiment were different levels of irradiance (0, 70, 130, 270, 450, and 850 µmol photons m-2s-1), pH (6, 7, 8, 9) and salinity (20, 25, 30, 35, 40 psu) that may affect the photosynthetic rate of seaweed via dissolved oxygen concentration, as proxy for photosynthesis, in terms of amount of oxygen production per fresh weight biomass per unit time (mg O2 g-1 FW h-1). Exposure was carried out using light-dark bottle technique for 1 hour and 30 minutes (8:30 am to 10:00 am). The exposure time was reasonable because they have considered the effects of diurnal cycle on the photosynthetic rate result. In the natural set-up considering the irradiance, the irradiance that can be measured early in the morning (around 8:30 am) may reach 70 to 100 µmol photons m-2s-1 which is enough to achieve the photosynthetic saturation of the seaweed, and in the mid-day, the irradiance can reach up to 800 to 2000 µmol photons m-2s-1 which can already cause photoinhibition, reduction in photosynthetic rate when photon flux is more than the required level. However, they did not mention and indicate in their methods what light meter instrument they used prior to the experiment. One problem with this experimental design is the pH used as a factor that may affect photosynthetic rate of K. alvarezii. The pH value used was very unrealistic because in the natural set-up pH 6, 7, and 9 do not exist in seawater. Even if in ocean acidification scenario, the pH will remain greater than 7 or else, other organisms will be compromised. They should use existing pH values of the seawater from current literature as basis for the natural pH values because 1 magnitude difference in pH can already implicate a great impact the marine organisms, especially if it will be applied in the natural set-up. Instead of adding HCl and NaOH to adjust the pH of the seawater, they should have used air bubbling of CO2 since seaweed utilizes CO2 as their substrate for photosynthesis. With this, they might have more efficient and reasonable results. Ideally, they have managed to consider and eliminate the effect of temperature on the photosynthetic performance of the seaweed by maintaining a constant temperature on all the treatments at 29 degrees Celsius. Also, they have considered the correlation between the concentration of pigments (chlorophyll a and phycobiliproteins) to the photosynthetic efficiency of the seaweed, which is good because pigments are the main drivers of photosynthesis. V. The use of randomization or random sampling and replication Obviously, the experimental design of this study employs pseudo replication. By assessing how the different experimental treatment was conducted and how they measured it, it was an obvious indication of pseudo replication. It was stated in their methods on photosynthesis-irradiance experiment that they have not performed “true” replication. Salinity-pH treatment were conducted for one (1) hour and 30 minutes exposed to specific irradiance, and the dissolved oxygen (DO) in seawater was measured thrice, which served as their replicates. They have not mentioned in their paper that they have replicates among different treatments, which makes them guilty of pseudo replication. Different portions of K.alvarezii may have different photosynthetic response with different treatments, therefore, they should not assume that measuring the DO in seawater will have a general response to the seaweed as a whole. Randomization was not indicated in the methods of the study. In considering the portions of thalli as sampling unit, it was not randomly selected, instead, it might have been chosen in order to remove the bias in the photosynthetic efficiency of the sampling unit that may affect the result.
VI. Clear indication of the statistical analysis/method used This study was statistically analyzed using D’ Agostino-Pearson normality test, Bartlett’s method for homogeneity of variances. Two-way ANOVA followed by Bonferroni’s post hoc test (at p=0.05 significance level) was used to compared photosynthetic rates with pigment contents of infected and healthy K. alvarezii after treated with pH and salinity. The statistical methods were used in order to determine the normal distribution of the data gathered. VII. Conclusions and reliability of conclusions The main point of their conclusion was both Neosiphonia-infected and healthy K. alvarezii had broad photosynthetic tolerance to short-term changes in irradiance, seawater salinity, and pH. They have also presented in their conclusion that the results from this research had indicated the effect of Neosiphonia sp.-epiphytes on the K. alvarezii but the main objective of the study was not achieved because they have stated in their discussion that they have failed to show the significant effects of pH and salinity on the photosynthetic rate of both infected and healthy K. alvarezii. They have also indicated in their discussion that short-term exposure (1 hour 30 mins) cannot sufficiently provide reliable results, however, they should suggest and recommend in their conclusion that long term exposure should be done to validate and determine the effects of pH, irradiance, and salinity to the photosynthetic performance as well as to the physiology of K. alvarezii. Long term exposure is more reliable with “true” replicates because in the natural set-up changes in the water parameters occur and took place for a certain period, and this might provide knowledge on the consequences of changes in pH, irradiance, and seawater salinity to farmed K. alvarezii in the long run. Also, they should not present in their conclusion that they have verified the optimum farming conditions for maximum production and control of epiphytes because they have done pseudo-replication. There might have greater uncertainties that the effects of the treatment they employ are the same to another K. alvarezii individual since they do not have replicates. VIII. Evaluation of the study The study was a good start in assessing the effects of EFA together with interrelated factors such as irradiance, pH, and seawater salinity to farmed-seaweed K. alvarezii. However, after their paper was critically assessed, it was not a recommended paper as basis for seaweed farming, especially to those who wants to venture in this field. There were lots of discrepancies in the methods and experimental design leading to unreliable results. After they have stated in their paper that they have failed to show the significant effects of their treatments on the photosynthesis of infected and healthy K. alvarezii, there should have further validation and experiments to conduct in order to have reliable data and results.