Effects of chloramphenicol, erythromycin, and furazolidone on growth of Isochrysis galbana and Chaetoceros gracilis

Aquaculture 260 (2006) 145 150 www.elsevier.com/locate/aqua-online Effects of chloramphenicol, erythromycin, and furazolidone on growth of Isochrysis galbana and Chaetoceros gracilis A.I. Campa-Córdova a,, A. Luna-González b, F. Ascencio a, E. Cortés-Jacinto a, C.J. Cáceres-Martínez c a Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico b Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, Unidad Sinaloa, Km 1 Carretera a Las Glorias, Guasave, Sinaloa, Mexico c Universidad Autónoma de Baja California Sur (UABCS), Unidad Experimental de Maricultura, Apdo. Postal 19-B, La Paz, B.C.S. Mexico Received 24 October 2005; received in revised form 25 May 2006; accepted 1 June 2006 Abstract This study focused on determining the effects of antibiotics on microalgae used as food for scallop larvae. Six different dose levels of chloramphenicol, erythromycin, and furazolidone were added to cultures of Isochrysis galbana and Chaetoceros gracilis. An in vivo experiment was subsequently conducted to determine the effect of chloramphenicol and erythromycin on larval survival of the Pacific calico scallop Argopecten ventricosus in tanks and on the population of its associated bacteria. Results showed that growth of I. galbana was not significantly affected by chloramphenicol or erythromycin at the test doses of 0.5, 1.0, 3.0, 6.0, 9.0, and 12.0 mg/l. C. gracilis was significantly sensitive to erythromycin and chloramphenicol at doses higher than 0.5 and 3.0 mg/l, respectively. Furazolidone inhibited the growth of both I. galbana and C. gracilis at all test doses. Results showed that exposure of scallop larvae to a dose of 6 mg/l chloramphenicol or erythromycin did not significantly affect growth of I. galbana, significantly enhanced survival of the scallop larvae, and inhibited the growth of Vibrio spp. in tanks. This study demonstrated the adverse effect of chloramphenicol, erythromycin and furazolidone on I. galbana and C. gracilis microalgae but the positive effect on survival of the scallop larvae, decreasing associated bacterial population. 2006 Elsevier B.V. All rights reserved. Keywords: Antibiotics; Isochrysis galbana; Chaetoceros gracilis; Vibrio spp.; Mollusk larvae 1. Introduction Corresponding author. Tel.: +52 612 12 3 84 10; fax: +52 612 12 5 36 25. E-mail address: angcamp04@cibnor.mx (A.I. Campa-Córdova). Marine microalgae play a key role in aquaculture development (Riquelme and Avendaño-Herrera, 2003). Unicellular marine algae are commonly grown as food for commercially valuable marine organisms (Utting, 1985). The microalgal species Chaetoceros sp., Isochrysis sp., Skeletonema sp., and Tetraselmis sp. for instance are frequently used in the culture of marine organisms. Chaetoceros sp. and Isochrysis sp. have good nutritional qualities, particularly highly unsaturated fatty acids and small cell size, so they are widely used in aquaculture as food in the early larval stages of mollusks, fish, and crustaceans (Godínez et al., 2005). To enhance survival, rigorous cleaning and aseptic conditions are required. Careful control of the water temperature, salinity, ph, optimization of stocking densities, and balanced nutrition are also very important 0044-8486/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.06.014

146 A.I. Campa-Córdova et al. / Aquaculture 260 (2006) 145 150 (Beiras et al., 1994; Inglis, 1996). Normally, periodic changes of filtered, sterilized seawater, using filters from 10 to 1 μm and ultraviolet radiation, prevent bacterial infections in larval cultures. Antibiotics, used routinely, accompany these preventive measures. The requirement for use of antibiotics in a culture depends mainly on the quality of the water (Toranzo, 1990). Antibiotics commonly used to avoid the adverse effect of pathogens in aquaculture are furazolidone, chloramphenicol, streptomycin, erythromycin, kanamycin, oxytetracycline, neomycin, and oxolinic acid (Benbroock, 2002). There is widespread concern that antibacterial agents in aquaculture have led to the emergence and selection of resistant bacteria. An improved understanding of how resistance emerges and is selected for among bacteria is essential in evaluating their impact in aquaculture, identifying high risk procedures, and designing ways to reduce the potentially dangerous effects. Bacteria acquire resistance by acquisition of foreign DNA or modification of chromosomal DNA (Inglis, 1996). Despite the risk of selecting for antibiotic-resistant strains and toxicity to the marine organisms, antibiotics are increasingly used. Chloramphenicol is the antibiotic most frequently used in hatcheries (Uriarte et al., 2001). This study evaluated the effect of chloramphenicol, erythromycin and furazolidone on Isochrysis galbana and Chaetoceros gracilis microalgae, Argopecten ventricosus larvae and associated bacterial population. 2. Materials and methods 2.1. Microalgal cultures I. galbana (strain ISX2) and C. gracilis (strain CHX1) were donated by Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE) and cultivated in 125 ml glass flasks containing 100 ml liquid medium (F/2 Guillard medium, 20 psu). Seawater was UVirradiated and 0.2 μm-filtered. Cultures were grown at 24±0.5 C under constant illumination. I. galbana and C. gracilis cells were collected in 5 ml glass tubes, fixed with 1% Lugol's Solution, and counted in a hemacytometer using a phase-contrast microscope (Nikon, Optiphot-2). A calibration curve was set up to determine the growth rate of I. galbana and C. gracilis during cultivation for 15 days based on sample readings at 750 nm in a Beckman DU 600 spectrometer. 2.2. Antibiotics Furazolidone (Roberts Laboratories, Inc.), chloramphenicol (Mediatech, Inc.), and erythromycin (Abbott Laboratories) were used in microalgal cultures. A stock solution of each antibiotic was prepared by dissolving 100 mg of the selected antibiotic with 100 ml 0.2 μmfiltered seawater. The concentration of antibiotics in microalgal cultures was adjusted from stock solution. 2.3. Growth of microalgae exposed to antibiotics Cultures of I. galbana and C. gracilis were exposed to 0.5, 1.0, 3.0, 6.0, 9.0, or 12.0 mg/l final concentrations of the test antibiotics following the method of Uriarte et al. (2001). An untreated control group was cultured under the same conditions as the treated groups. The microalgae were cultured in triplicate and samples were tested every 48 h for 15 days. 2.4. Broodstock Healthy adult Pacific calico scallop A. ventricosus (shell length, 58.9±3.4 mm) were collected from a culture facility in Bahía de La Paz, near La Paz, Baja California Sur, Mexico and transported to the Universidad Autónoma de Baja California Sur hatchery. They were conditioned for spawning for at least 20 days in a 1500 l fiberglass tank with 0.2 μm-filtered seawater containing 10 mg/l EDTA, under constant aeration at 24±1.0 C and salinity of 36 ppt (parts per thousand). Filtered seawater was maintained at ph 7.8 8.2. Broodstock were fed 1.5 10 5 cells/ml of a mixture of I. galbana, Chaetoceros calcitrans, and C. gracilis (1:1:2) algae. Tank water was changed at the rate of 50% daily. 2.5. Larvae Individual scallops were placed in 5 l plastic containers with 1 μm-filtered and aerated seawater, and were induced to spawn by thermal shock, a change from 18 to 28 C for 20 30 min at each temperature. After spawning, the trochophora stage larvae were cultured in 5000 l fiberglass conical tanks (5 larvae/ml), with constant aeration, filtered seawater at 24 ± 1.0 C and salinity of 36 ppt. At the veliger stage, a complete water exchange was made every 48 h. Larvae were fed 3.0 10 5 cells/ml I. galbana and C. calcitrans (1:1). 2.6. Survival of larvae exposed to antibiotics Based on the foregoing results, the effects of chloramphenicol and erythromycin on survival of the scallop larvae, A. ventricosus were evaluated in a bioassay experiment. Groups of larvae (15 larvae/ml) were cultured in 60 l fiberglass tanks under constant aeration.

A.I. Campa-Córdova et al. / Aquaculture 260 (2006) 145 150 147 Each group of larvae were collected on nylon screen (45 μm opening), and acclimated in culture tanks with 1 μm-filtered seawater for 30 min before treatment. Duplicate groups of larvae were treated with chloramphenicol or erythromycin at 6.0 mg/l for 10 days, from Fig. 2. Growth of Chaetoceros gracilis exposed to different concentrations of chloramphenicol (a), erythromycin (b), and furazolidone (c). *Significantly different than control. Fig. 1. Growth of Isochrysis galbana exposed to different concentrations of chloramphenicol (a), erythromycin (b), and furazolidone (c). *Significantly different than control. the early trochophora larvae to the pediveliger stage. A control group was cultured in filtered seawater without antibiotics. Temperature and salinity were recorded daily. Seawater in tanks was completely changed every 48 h and the treatment concentration of the antibiotic

148 A.I. Campa-Córdova et al. / Aquaculture 260 (2006) 145 150 Fig. 3. Survival of A. ventricosus larvae treated with 6.0 mg/l erythromycin and chloramphenicol. Whiskers=S.E. Data with different letters are significantly different. restored. For counting larvae, larvae were screened from each tank and placed in 10 l filtered seawater, from which 1 ml aliquots were obtained for counting and then measured in a Sedgwick-Rafter chamber, using an optical microscope (Nikon, Optiphot-2). Survival of larvae and density of bacterial mass were recorded on days 1, 3, 5, 7, 9, and 10. Larvae were fed daily during the experiment. 2.7. Bacterial count Density of bacteria in A. ventricosus culture tanks was estimated by the plate count method, using thiosulfate citrate bile sucrose (TCBS) medium to determine Vibrio spp. Samples were collected every 48 h before water change and incubated at 30 C for 24 h. The results were expressed as colony-forming units per ml (CFU/ml). 2.8. Statistical analysis Microalgal growth was estimated by multiple regression analysis, and larval survival and bacterial density were evaluated by ANOVA, with the Tukey test to measure differences (Statistica, StatSoft, Inc.). Values were considered significantly different at P < 0.05. 3. Results 3.1. Effect of antibiotics on microalgal growth Growth of I. galbana was not significantly affected by chloramphenicol and erythromycin (Fig. 1a and b). Furazolidone significantly inhibited growth of I. galbana at all treatment concentrations (Fig. 1c). Exposure of C. gracilis to chloramphenicol at 0.5 to 3.0 mg/l did not significantly affect growth (Fig. 2a). Doses higher than 0.5 mg/l erythromycin significantly affected growth of C. gracilis (Fig. 2b). Furazolidone inhibited growth of C. gracilis at all treatment concentrations (Fig. 2c). I. galbana showed higher tolerance to antibiotics than C. gracilis. Furazolidone inhibited the growth of both species. 3.2. Survival of larval scallops Results obtained from the bioassay of antibioticsmicroalgae at 6 mg/l chloramphenicol and erythromycin were selected to evaluate survival of larval A. ventricosus. Larvae showed a significantly higher survival than controls by day 9 of exposure (Fig. 3). Larvae treated with antibiotics displayed better larval health (active swimming in the water column, larvae sinking to the bottom with active movement of cilia, full gut, and nonphysical deformities or stunted growth) than untreated Table 1 Growth of Vibrio spp. (CFU/ml, mean±s.e.) during larval culture of Argopecten ventricosus treated with 6.0 mg/l erythromycin or chloramphenicol Treatment Days 1 3 5 7 9 10 Control 14± 2.0± 53± 42.5± 31.5± 5.5± 1.7 a 0.3 a 3.8 a 2.1 a 1.7 a 0.7 a Erythromycin 14± 0 b 0.5± 0.5± 0 b 0.5± 1.7 a 0.1 b 0.1 b 0.1 b Chloramphenicol 14± 1.5± 0.5± 0 b 0 b 0 b 1.7 a 0.2 a 0.1 b Means in the same column with different superscripts are significantly different.

A.I. Campa-Córdova et al. / Aquaculture 260 (2006) 145 150 149 controls. No significant differences between chloramphenicol and erythromycin treated larvae were observed (Fig. 3). Temperature in tanks during exposure to antibiotics was 27.5 28.5 C and salinity was 36 ppt. 3.3. Bacterial growth Table 1 displays the in vitro growth of Vibrio spp. treated with 6 mg/l of erythromycin and chloramphenicol. Bacterial numbers decreased significantly over control groups after exposure to the test antibiotics. Bacterial population also decreased in the controls on days 3 and 10 but increased from days 5 to 9. 4. Discussion Live microalgae play an essential role in larval and juvenile mollusk development (Bougaran et al., 2003). However, there is a growing concern that the use and abuse of anti-microbial drugs in human medicine, agriculture, and aquaculture for disease control and promoting growth increases selective pressure on the microbial world, encouraging emergence of bacterial resistance (Inglis, 1996). Antibiotics used in aquaculture not only affect non-specific bacteria in culture tanks, but also microalgae. Uncontrolled use of antibiotics may destroy microalgal cells, preventing adequate feeding of cultured organisms and the quality of the microalgae may also decline (Nicolas et al., 1996). This study confirms that chloramphenicol and erythromycin did not affect the growth of the microalgae I. galbana. However,erythromycin and chloramphenicol adversely affected growth of C. gracilis at doses greater than 0.5 and 1.0 mg/l, respectively. By and large, C. gracilis was more sensitive to antibiotics than I. galbana. The results for furazolidone were different. Furazolidone was fatal to both microalgal strains. These findings correlates with those Campa- Córdova et al. (2005) who found lower survival rates of larvae, abnormal morphology, and empty gut in A. ventricosus when exposed to 6 mg/l furazolidone. In Europe, United States, Mexico, and other countries, furazolidone, chloramphenicol, and erythromycin are not permitted in food animals because of known toxicity to humans (Benbroock, 2002). Chloramphenicol has a wide anti-microbial spectrum, but is considered toxic to humans at doses greater than 25 mg/l in blood (Bevan, 1976). Erythromycin has a medium anti-microbial spectrum, less toxic than chloramphenicol, yet more effective against Gram positive bacteria, such as Streptococcus (Sahul Hammeed et al., 2003). The vast majority of bacteria that cause disease in fish are Gram negative, so erythromycin should only be used after culture and sensitivity testing confirms its usefulness. Furazolidone is derived from nitrofurane and it is used as an anti-microbial and anti-protozoan agent (Bevan, 1976). Oxytetracycline and sulfonamide are antibiotics approved by the US Food anddrugadministration(fda)fortreatingdisease,but in some aquatic animals (channel catfish, salmonids, and lobster, with oxytetracycline) it cannot be used prophylactically (Benbroock, 2002). Despite many years of research, there are many problems in hatchery production, i.e. high larval mortality attributed to pathogenic bacteria (Torkildsen and Magnesen, 2004). Vibrio species cause serious problems in hatcheries of marine and freshwater organisms (Sahul Hammeed et al., 2003). Currently, antibiotics, hypochlorite solutions, and formaldehyde have been effective in controlling bacterial infections among mollusk, crustaceans, and fish species (Sahul Hammeed and Balasubramanian, 2000; Mac Millan, 2001). In this study, enhanced survival occurred when larvae were treated with chloramphenicol and erythromycin at 6 mg/l, which did not alter larval development at the concentrations tested. Similarly, Braley (1986) reported higher survival in larval giant clam Tridacna gigas treated with 5 7 mg/ l erythromycin, oxytetracycline, streptomycin, or chloramphenicol during the first 2 days of development. Robert et al. (1995) also used chloramphenicol and erythromycin at 8 mg/l to enhance larval survival of the great scallop Pecten maximus. Uriarte et al. (2001) found that larvae of the Chilean scallop Argopecten purpuratus were successfully treated for bacterial contamination at concentrations of 2 and 8 mg/l chloramphenicol. Torkildsen and Magnesen (2004) reported significantly better survival rates in larval cultures of P. maximus when treated with chloramphenicol. The results of this study showed the importance of antibiotics in significantly decreasing the Vibrio spp. populations in culture tanks. The fewer Vibrio spp. populations in controls on days 3 and 10 could be related to the ingestion of bacteria by larvae, and the increase of bacteria on days 5 9 could be related to larval die-off recorded in control tanks as similar to the findings of Nicolas et al. (1996). Although antibiotics may be beneficial to reduce pathogens in larval cultures, its use must be studied with more care (Araya et al., 1999). Currently, antibiotics are an important tool for reducing disease in the aquaculture organisms, but there is increasing concern in the industry to control or eliminate anti-microbials (Gomez-Gil et al., 2000). An important concern is the evidence of secondary effects from antibiotics, such as breeding antibiotic resistant strains, eventually leading to increased pathogenicity (Ervik et al., 1994; Inglis, 1996). In hatcheries, oxytetracycline

150 A.I. Campa-Córdova et al. / Aquaculture 260 (2006) 145 150 and furazolidone has been replaced by ciprofloxin and nitrofurantoin because resistant bacteria have developed (Sahul Hammeed and Balasubramanian, 2000). Applications of antibiotics are advised only when there is positive evidence of Vibrio in the culture tanks (Uriarte et al., 2001). Sahul Hammeed et al. (2003) recommended prophylactic use of formalin at 50 mg/l to inhibit bacteria in culture tanks. 5. Conclusion Viability and growth of I. galbana was not affected by exposure to 0.5 12 mg/l chloramphenicol and erythromycin. Growth of C. gracilis was adversely affected by erythromycin at doses higher than 0.5 mg/ l and by chloramphenicol at doses higher than 3.0 mg/l. Furazolidone was toxic to both microalgal species at all treatment levels. Chloramphenicol and erythromycin at the test dose of 6 mg/l enhanced survival of scallop larvae and decreased Vibrio spp. populations. 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