Effects of Six Antibiotics on the Activity of the Photosynthetic Apparatus and
Ammonium Uptake of Thallus of Porphyra yezoensis
Min-Hyuk Oh, Yun Hee Kang1, Choon-Hwan Lee and Ik Kyo Chung1*
Department of Molecular Biology and 1Department of Marine Science, Pusan National University, Busan 609-735, Korea The modern integrated fish-seaweed mariculture has been tested to reduce the environmental impacts of an intensive fed culture. To obtain the best seaweed bioremediation performance, the effects of therapeutants used forfish disease control on the selected seaweed species should be considered. As a selected seaweed, Porphyra yezoensiswas tested with six commercial antibiotics including erythromycin thiocyanate_A, erythromycin thiocyanate_B,oxytetracycline, doxycycline, pefloxacin, and amoxicillin trihydrate under the batch incubation at a photon fluxdensity of 10 µmol · m-2 · s-1 at 15°C. Among the tested commercial antibiotics, erythromycin thiocyanate_A,erythromycin thiocyanate_B, oxytetracycline, and doxycycline showed decreases in Fv/Fm, the photochemicalefficiency of photosystem II, with a dose-dependant and time-dependant manner. From the quenching analysis ofchlorophyll fluorescence, three differential patterns were observed in the antibiotics-treated Porphyra: (1) high non-photochemical quenching (NPQ) and low photochemical quenching (qP) in the cases of Erythromycinthiocyanate_B and amoxicillin trihydrate, (2) high NPQ and high qP in the case of pefloxacin and (3) low NPQ andlow qP in the case of oxytetracycline. These results indicated that antibiotics affected in various ways on thephotosynthetic apparatus, reflecting differential lesion sites of antibiotics. In addition, the rates of ammoniumuptake also decreased with a decrease of Fv/Fm in P. yezoensis thalli treated with erythromycin thiocyanate_B andoxytetracycline. Therefore, the four antibiotics mentioned could affect the bioremediation capacity of the selectedseaweed species in the integrated fish-seaweed mariculture system due to the decrease of photosynthetic activityand the simultaneous decrease of ammonium uptake.
Key Words: ammonium uptake, antibiotics, chlorophyll fluorescence, Porphyra, seaweed
Although antibiotics are no longer used routinely, they INTRODUCTION
are still often used when a disease is diagnosed in thefish under cultivation. Nevertheless, antibiotics have The fed mariculture including the intensive finfish been widely used in some countries for the protection of aquaculture has caused many environmental problems diseases caused by bacteria such as Streptococcus, (Wu 1995). Modern intensive monoculture requires high Edwardsiella, Vivrio, and Aeromonas.
inputs of water, feeds, fertilizers and chemicals and There are many different types of drugs used in finfish inevitably produces considerable wastes. Some types of aquaculture. Erythromycin thiocyanate is most effective the fed aquaculture fish farm operations in the coastal against gram-positive bacteria, such as Streptococcus area can produce large amounts of waste. Therefore, species and is not very effective in a bath treatment and many aquaculture operations put enormous pressure on should only be administered by injection or mixed in to coastal habitats (Black 2001). To reduce the nutrient the feed. Tetracycline and related antibiotics are burden of the fish farm effluents, an integration of considered broad-spectrum antibiotics (effective against seaweed cultivation with fish aquaculture has been a wide variety of bacteria) and they work well when proposed (e.g., Chopin et al. 2001). However, in waste mixed with food. Terramycin is a brand of effluents, some chemicals and drugs, such as pesticides, oxytetracycline manufactured by Pfizer that is FDA disinfectants and antibiotics have been included and approved for use in the production of salmonids, which may negatively impact adjacent environment.
channel catfish and lobsters. The quinolones, includingnalidixic acid and oxolinic acid, like the tetracyclines, are *Corresponding author (ikchung@pusan.ac.kr) considered broad-spectrum antibiotics, and they work against a wide variety of bacteria. The sulfa drugs, Plant Efficiency Analyzer (PEA, Hansatech Instruments including Romet®, are also considered to be broad- Ltd., England) as described in Eu et al. (1996). The intensity of the saturating light beam was 1,200 µmol Antibiotics may affect critical metabolic processes such photons· m–2 · s–1. The Photochemical efficiency of PSII as mitochondrial and/or photosynthetic activities of (Fv/Fm) expressed as the ratio of variable fluorescence seaweeds growing in the biofiltration tank. If it is true, (Fv) to maximum yield of fluorescence (Fm) was then it is a critical problem for the seaweed integrated measured. In addition, chlorophyll a fluorescence for aquaculture system, because the metabolic activities of quenching analysis was analyzed using Diving-PAM seaweeds should be maintained in a proper level for the (Walz, Germany). Thallus samples were dark-acclimated biofiltration. Therefore, we investigated whether for 10 min at room temperature. Photochemical antibiotics frequently used in the fish aquaculture system quenching (qP) and non-photochemical quenching affect the activity of the photosynthetic apparatus and (NPQ) were calculated according to Schreiber et al.
the nutrient removal rates in seaweeds or not for the establishment of a sustainable seaweed integrated For recovery experiment, the antibiotics-treated thalli were rinsed with seawater to remove antibiotics,incubated to nutrient (NH + MATERIALS AND METHODS
and than measured Fv/Fm using a PEA.
Materials and Treatments of Antibiotics
Ammonium Uptake
Porphyra yezoensis samples used in this study were Ammonium contents and uptake of seawater were collected in Yeosu, Korea in February 2002. The thalli measured by detection at 640 nm using spectrophotometer were rinsed with seawater to remove sediment and (Shimadzu, Japan) using colorimetric methods (Parsons epiphytes. Before the experiment, the macroalgae were pre-equilibrated in the laboratory 4 days in filteredseawater at 10°C in darkness and used for experiments.
For treatments of antibiotics, we used six commercial antibiotics (Samyang Anipharm Co., Ltd) containing Effects of Antibioics Treatement on the Photochemical
different chemicals: erythromycin thiocyanate_A, Efficiency of PSII (Fv/Fm)
erythromycin thiocyanate_B, oxytetracycline hydrochloride, We investigated the change of the chlorophyll a doxyclclin hyclate, pefloxacin, amoxicillin trihydrate.
fluorescence to understand the effect of antibiotics on the Erythromycin thiocyanate_A and erythromycin photosynthetic activity of Porphyra yezoensis. When thiocyanate_B are two different commercial products antibiotics were treated with various concentration to containing erythromycin thiocyanate. The antibiotics filtered seawater, the photochemical efficiency of PSII were dissolved in filtered seawater and the macroalgae (Fv/Fm) showed the substantial differences in sensitivity were grown in the seawater under the cool-white to antibiotics as indicated by reduction of Fv/Fm (Fig. 1) fluorescence lamps with an intensity of 10 µmol and color change (data not shown). Thus, the effects of photons · m–2 · s–1 at 15°C. The lowest concentration of the the antibiotics for the photosynthetic activity of Porphyra antibiotics treated was similar to that recommended by sp. is divided into three groups (Fig. 1). (1) Type I: the manufacturers, and several higher concentrations pefloxacin and amoxicillin trihydrate; (2) Type II: were treated to test its effect when it was treated erythromycin thiocyanate_A and Erythromycin excessively compared with the recommended thiocyanate_B; (3) Type III: oxytetracycline concentration or to test its effect when the antibiotics was hydrochloride, and doxyclclin hyclate. The Type I treated for longer period. The period of the treatment antibiotics did not show the decrease of Fv/Fm value in was chosen from several hours up to 2 days depending all concentration points for 48 h treatment, but the Type on its effect. During experiments, the growth media were II and Type III show the dose- and time-dependant decreases of Fv/Fm. In case of erythromycinthiocyanate_A treatment, Fv/Fm was small-ranged Chlorophyll a Fluorescence and Quenching Analysis
between 0.5 and 0.6 at the concentration of 7.5 g/L for Chlorophyll a fluorescence was measured using a 18h, and erythromycin thiocyanate_A could be observed Oh et al.: Effects of Antibiotics on Porphyra yezoensis 123 Fig. 1. Changes in PSII photochemical efficiency (Fv/Fm) from thalli of Porphyra treated with various concentrations of several
antibiotics. (A) Type I antibiotics, (B) Type II antibiotics, (C) Type III antibiotics. The macroalgae were pre-equilibrated in the
laboratory 4 days in filtered seawater at 10°C in darkness, and then tested in filtered seawater under the light at 10 µmol
photons · m–2 · s–1 at 15°C during the subsequent experiment. Aeration was provided by bubbling with air pump. The subsequent
recovery experiments were carried out in 75 uM nutrient (NH +
4 )-containing seawater for 2h (gray shading). Chlorophyll a fluorescence was measured after 10 min dark adaptation at room temperature.
serially the decrease of Fv/Fm with the dose-dependant decrease of dry weight (data not shown). From these manner within 12h. In the oxytetracycline hydrochloride results, we could observed some antibiotics down- and doxyclclin hyclate as a Type III, rapid reduction of regulates the photosynthetic activity in Pophyra sp.
Fv/Fm could be observed within 2h although low Interestingly, it was notable that some antibiotics-treated concentrated antibiotics were treated, and also showed a Porphyra leaves showed the recovery pattern in terms of chlorosis, differ to Type II, resulting the significant increase of Fv/Fm when antibiotics-treated thalli were oxytetracycline hydrochloride is reduced both NPQ andqP shown that depressed electron transport and wholephotosynthetic capacity. From chlorophyll a fluorescencequenching analysis, we could differentiate antibioticsinto 3 groups with different action mechanism inphotosynthetic apparatus, indicating the different targetor action site of antibiotics in the photosyntheticapparatus.
The Effects of Antibiotics in Ammonium Uptake of

To understand interrelation between the degeneration of photosynthetic apparatus and the ammonium uptakein Porphyra, we tested the changes of the concentration of Fig. 2. Quencing analysis from thalli of Porphyra treated with
the soluble ammonium in seawater after transfer the several antibiotics. The experimental conditions are the antibiotic-treated samples to the 75 µM ammonium same as in Fig. 1. The concentration of antibiotics weretreated during 32 h as followed: 20 g/L Amoxicillin standard solution (Fig. 3). Ammonium uptake in the trihydrate, 7.5 g/L Erythromycin thiocyanate_B, 5 g/L Porphyra were showed biphasic pattern: (1) Surge Pefloxacin, and 1.0 g/L Oxytetracycline hydrochloride.
uptake, initially high uptake rate with time; (2) Constant Chlorophyll fluorescence was measured after 10 min dark uptake, comparatively constant uptake at a fixed rate; after surge uptake (Dy and Yap 2001). The decrease ofammonium uptake in the erythromycin thiocyanate_B and oxytetracycline hydrochloride-treated Porphyra also rinse with filter seawater and than were incubated for was similar with decrease in Fv/Fm. Generally, the ability of ammonium uptake in Porphyra wassignificantly decreased in antibiotics-treated leaves Quenching Analysis of Chlorophyll a Fluorescence of
compared with control showing the dose-dependant the Antibiotics-Treated Porphyra
manner. It has been demonstrated that concurrent As shown in Fig. 2, in order to find the difference in oscillations in chlorophyll a fluorescence and changes in the effects among the antibiotics, the chlorophyll a oxygen exchange after additions of N to N-stressed fluorescence quenching analysis were conducted at Dunaliella tertiolecta reflect tight coupling between concentration and time with near 0.5 value of Fv/Fm photosynthesis and N metabolism (Young and Beardall after treatment of the antibiotics. NPQ is induced by 2003). The effect of the antibiotics on the decrease in the changes in the transthylakoid pH-gradient, as well as by ammonium uptake causally linked to its effect on activation xanthophyll cycle; whereas qP changes when decrease of Fv/Fm, suggesting the quantity of the photochemical energy conversation by the charge antibiotics treated often limits of aquaculture production.
separation in reaction centers of PSII lowers due tochanging linear electron transport rate to PSI through Conclusions
cytochrome b6/f complex (Rohacek 2002). The effect of In an attempt to avoid the creation of resistant bacteria the antibiotics in Porphyra is divided into three groups in a facility, some farms will rotate the antibiotics they for pattern of NPQ and qP compared with control (Fig.
use every few months or every year. However, the best 2). (1) High NPQ and low qP; These pattern may be solution is to positively identify the bacteria by running results of the activated delta pH or xanthophyll cycle culture and sensitivity tests, and thereby avoid data in case of amoxicillin trihydrate and erythromycin unnecessary, costly and potentially harmful treatments thiocyanate_B, whereas suppress electron transport rate in modern aquaculture. Our study revealed that misuse and the amount of actual fraction of PSII reaction centers of any antibiotic can lead to loss of the ammonium that are in the open state. (2) High NPQ, high qP; Both uptake through degradation of the photosynthetic reaction, NPQ and qP, due to re-oxidation of the QA in apparatus in Pophyra. Therefore, we assume that the the case of pefloxacin. (3) Low NPQ, low qP; proper management of the antibiotics is important to Oh et al.: Effects of Antibiotics on Porphyra yezoensis 125 was also given to M.-H. Oh. We thank the entire SSIASgroup for a fruitful collaboration and Drs. J.A. Shin andT.H. Seo for their assistance in the field at Yeosu andfinally to the Marine Research Institute (ContributionNo. 32) in Pusan National University.
Black K.D. 2001. Environmental Impacts of aquaculture. Scheffield Chopin T., Buschmann A.H., Halling C., Troell M, Kautsky N., Neffus A., Kraemer G.P., Zertuche-Gonzalez J.A., Yarish C.
and Neefus C. 2001. Integrated seaweeds into marine
aquaculture systems: a key toward sustainability. J. Phycol.
37: 975-986.
Dy D.T. and Yap H.T. 2001. Surge ammonium uptake of the culture seaweed, Kappaphycus alvarezii (Doty) Doty
(Rhodophyra: Gigartinalse). J. Exp. Mar. Biol. Ecol. 265: 89-
Eu Y.-J., Ha, S.-B. and Lee C.-H. 1996. Effects of chilling injury in the light on chlorophyll fluorescence and D1 proteinturnover in cucumber and pea leaves. J. Biochem. Mol. Biol.
Fig. 3. Ammonium uptake expertiment. Time course in
29: 398-404.
depletion of ammonium by Porphyra yezoensis in the initial Parsons T.M., Maita Y. and Lalli C.M. 1984. A manual for 75 uM concentration of NH4 . The antibiotics-treated thalli chemical and biological methods for seawater analysis.
were rinsed with seawater to remove antibiotics, incubated to nutrient (NH4 )-containing seawater for 2h, and than Rohacek K. 2002. Chlorophyll fluorescence parameters: the measured ammonium concentration by detection at 640 nm definitions, photosynthetic meaning, and mutual relationships. Photosynthetica 40: 13-29.
Schreiber U., Bilger W. and Neubauer C. 1994. Chlorophyll eliminate or reduce all contributing stresses for seaweed fluorescence as a nonintrusive indicator for rapid plant to remove fish-excreated inorganic nutrient, assessment of in vivo photosynthesis. In: Schulze E.D. andCaldwell M.M. (eds), Ecophysiology of Photosynthesis. Wu R.S.S. 1995. The environmental impacts of marine fish ACKNOWLEDGEMENTS
culture: towards a sustainable future. Mar. Pollut. Bull. 31:
This work has been financed by the Ministry of Science Young E.B. and Beardall J. 2003. Papid ammonium- and nitrate- and Technology through the International Science & induced pertubationsd to Chl a fluorescence in nitrogen- Technology Cooperation Program (KISTEP: M6-0203-00- streessed Dunaliella tertiolecta (Chlorophyta). J. Phycol. 39:
0041) to I.K. Chung and by the Ministry of MarineAffairs and Fisheries through Korean Sea Grant Program to C.-H. Lee. A financial support from the Korea Science and Engineering Foundation (M02-2003-000-20089-0)

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