Fate of Antimicrobial Agents During Treatment and Resistance Levels Quantification of antimicrobial agents during treatment showed levels inside the treated pond of approximately 10 µg/L. Inasmuch as different numbers of ponds were treated and the drugs posses different physic-chemical properties, the concentration detected in the effluent water varied but showed a steady, continuous concentration during and just after the treatment period. As an example, water concentrations of sulfadiazin during the first day of treatment (Figure 4a), during the treatment period (Figure 4b) and during the final day of treatment (Figure 4c) are shown. The pond concentrations were measured in one of the five treated ponds. Bacterial total-count resistance levels varied between samplings but no association to the use of antimicrobial agents or sampling sites was evident. No significant differences were found in resistance levels of bacteria from water in and around the fish farm compared to samples collected where the stream is unaffected by any fish farms and also in comparison to previous investigations (Schmidt et al. 2000). Apparently there is no cumulative effect on resistance levels in view of the fact that antimicrobial agents have been used in Danish aquaculture since the 1950's. Resistance levels of fish pathogenic bacteria (Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida) were very low in accordance with previous findings (Bruun et al. 2000). Measured Concentrations Compared to Le�islative Threshold Values Effluent peak concentrations with a few hours duration of formaldehyde, chloramines and hydrogen peroxide from pond treatments were less than 0.5 mg/L, though exceeding the proposed WOC. Antibiotic concentrations in effluent were in the range of or exceeded the WQC. There is still some debate on how to administer the WQC in practice and both short term and long term (more than 24 hours) WQCs have been proposed. For both types, it will probably be a matter of average discharge concentrations during the treatment period and not peak values. The findings must be assessed in view of the fact that 1) only a limited number of pon9s, one to six ponds out of a total of 24 ponds, were used in the experiments, 2) the design of the flow through a fish farm causes relatively high water turnover and a short retention time before discharge and, 3) field experiments were not replicated at different types of fish farms. As a worst-case scenario where all ponds are included, the discharge concentration would be proportionately higher and give raise to an even larger discrepancy between actual discharge concentration and WQC. Can Effluent Concentrations Be Lowered? Various measures can be taken if discharge values exceed 60 MARCH 2007 the WQC to comply with the legislation. A first approach to reduce discharge is to minimize the use of chemicals and antibiotics. This can be a possible solution, as long as it does not hamper disinfection/treatment efficacy. Lowering the dose concentration and prolonging the treatment period, by recirculation of treatment water, might result in similar response efficacy. But this must be tested. Removal of chemicals and antibiotics from a fish farm depends on the degradability of the agents and the duration of retention. As decomposition is time dependent, increased retention time caused by water recirculation or by insertion of large settling ponds or plant lagunas will lead to a larger removal within the fish farm. Preliminary experiments have shown that recirculation over a biofilter amplify formaldehyde and hydrogen peroxide removal and, hence, could be a post treat�ent solution of contaminated system water (Pedersen et al. in press). Increased knowledge of chemical kinetics and actual concentrations will provide a basis for optimized dosage (Rach et al. 1997), which will benefit fish health and concomitant discharge. Detected Concentrations of Antimicrobial Agents and the Potential for Resistance Selection The use of antimicrobial agents to treat bacterial diseases in aquaculture causes release of the drugs to the surrounding environment at low concentrations. Even if the concentrations are lower than minimum inhibition concentrations for most sensitive fish pathogenic and environmental bacteria, the discharges might conflict with national regulations. Occurrences of antimicrobial agents were detected on the fish farm at a maximum of 10 µg/ L in the pond medicated with sulfadiazin and 3.9 µg/L at the outlet after treatment of five ponds with sulfadiazin. At farm A the dilution of effluent in the stream water was approximately 1:4½ indicating that water further downstream in this case should contain 0.9 µg/L. Stream water was only sampled during florfenicol treatment, and showed a maximum concentration of 0.08 µg/L after treatment of one pond. No direct correlation between concentration of antimicrobial agents and resistance level was detected and apparently there is no cumulative effect on resistance levels in view of the fact that antimicrobial agents have been used in Danish aquaculture for 50 years. Perspectives Further work should focus on minimizing discharge, investigate p�ssible environmental effects and provide a stronger scientific basis for developing threshold values. This could be achieved by optimizing husbandry and hygiene, investigate alternative dose-response possibilities and determine effective effluent treatment measures. The indigenous as well as the specific fish pathogenic bacteria should be monitored for accumulation of antibiotic resistance and finally ecotoxicological effects of the chemicals and antibiotics should be studied further.
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