Algal Toxicity of Antibacterial Agents Applied in Danish Fish Farming

H.-C. Holten Lutzhøft, B. Halling-Sørensen, S. E. Jørgensen

Section of Environmental Chemistry, Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy,Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark

Received: 24 February 1998/Accepted: 27 July 1998

Abstract. Algal toxicity of antibacterial agents applied in fishfarming was investigated. The growth-inhibiting effects ofamoxicillin (A), flumequine (F), oxolinic acid (OA), oxytetracy-cline hydrochloride (OT), sarafloxacin hydrochloride (SF),sulfadiazine (SD), and trimethoprim (T) were investigated by amodified test procedure based on the procedure described in theISO 8692 (1989) protocol on three algal species: the freshwatercyanobacteriaMicrocystis aeruginosa,the freshwater greenalga Selenastrum capricornutum,and the marine cryptophy-cean Rhodomonas salina.Algal growth was measured asincreased chlorophyll concentration by extraction with ethanolfollowed by measurement of fluorescence. Results were quanti-fied in terms of growth rates using the Weibull equation todescribe the concentration response relationship.M. aeruginosashowed higher sensitivity compared to bothR. salina andS. capricornutum,whereas the results for the latter two weremore or less identical. The toxicity (EC50 value, mg/L) indecreasing order were A (0.0037), SF (0.015), SD (0.135),F (0.159), OA (0.180), OT (0.207), and T (112) forM.aeruginosa;OT (1.6), OA (10), T (16), F (18), SF (24), SD(403), and A (3108) forR. salina;and OT (4.5), F (5.0), SD(7.8), OA (16), SF (16), T (130), and A (NOEC. 250) forS. capricornutum.Applying this test procedure the toxicity ofantibacterial agents, being mono- or polyprotic compounds,may be underestimated because of partitioning between ionizedand unionized forms.

More than 200 tons of antibacterial agents are used annually inDenmark for human as well as veterinary purposes (Halling-Sørensenet al.1998). Based on the consumption of amoxicillin,oxolinic acid and oxytetracycline in one Danish county in theyears 1994, 1995, and 1996 (Holm Sørensen and Landsfeldt1997) it is assumed that the application of antibacterial agentsin Danish fish farming is increasing. To treat bacterial infectionsin intensive fish farming, antibacterial agents are distributeddirectly to the water as feed additives. Biotic as well as abioticfactors affect absorption, disposition, metabolisation and elimi-nation of the antibacterial agents (Bjørklund and Bylund 1990;Bjørklundet al. 1992; Schneider 1994) in fish. The absorption

of antibacterial agents in fish varies from 10 to 80% for theindividual compounds, which, among other things, depend onthe environmental temperature (Cravediet al.1987; Hustvedtetal. 1991; Schneider 1994). The extent of metabolisation variesfrom 20 to 80% (Poppe 1990; Lunestadet al.1992; Miglioreetal. 1996) depending on the compound. Furthermore, sick fishdo often have a reduced consumption (Poppe 1990; Lunestadetal. 1992). Schneider (1994) states that about 70% of adminis-trated antibacterial agents applied in fish farming are releasedinto the environment. Antibacterial agents are often mono- orpolyprotic compounds. Schneider (1994) states that pKa valuesare important parameters to determine whether or not acompound will penetrate, persist, or be eliminated from theorganism. The bioavailability of the compounds in the environ-ment are therefore also pH-dependent. The application ofantibacterial agents in fish farming is consequently a directsource of exposure to the aquatic environment.

Jacobsen and Berglind (1988) and Miglioreet al. (1996)reported findings of flumequine and oxytetracycline, respec-tively, in water and sediment from the outflow of a breedingpond and below fish farms. From several investigations(Samuelsenet al. 1994; Hektoenet al. 1995; Marengoet al.1997) it is known that some antibacterial agents are relativelystable under environmental conditions, resulting in half-lives insediment over 100 days.

The toxic effect data of antibacterial agents on variousaquatic species found in the literature (Harraset al.1985; Macrı`et al.1988; Lanzky and Halling-Sørensen 1997; Miglioreet al.1997), show values in the mg/L range. Investigations haveprimarily been done on crustaceans and fish rather than algalspecies. The few tests done on algal species show on the otherhand that algae, especially cyanobacteria, may be sensitive toantibacterial agents (Harraset al. 1985; Lanzky and Halling-Sørensen 1997). Since algae take part in the photosynthesis, it isimportant to study whether certain compounds interact with thegrowth of these species or not.

The antibacterial agents in question in this investigation are:amoxicillin, A, (2)-6-[2-amino-2-(p-hydroxyphenyl)acet-amido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid; flumequine, F, 9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid; oxolinicacid, OA, 5-ethyl-5,8-dihydro-8-oxo-1,3-dioxolol[4,5-g]quino-line-7-carboxylic acid; oxytetracycline hydrochloride, OT,[4S-(4a,4aa,5a,5aa,6b,12aa)]-4-(dimethylamino)-1,4,4a,5,5a,6,-Correspondence to:H.-C. Holten Lutzhøft

Arch. Environ. Contam. Toxicol. 36, 1–6 (1999) A R C H I V E S O F

EnvironmentalContaminationa n d Toxicologyr 1999 Springer-Verlag New York Inc.

11,12a- octahydro-3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide; sarafloxacin hydrochloride,SF, 6-fluoro-1-(4-fluorophenyl)-1,4-dihydro-4-oxo-7-(1-pipera-zinyl)-3-quinolinecarboxylic acid; sulfadiazine, SD, 4-amino-N-2-pyrimidinylbenzenesulfonamide; and trimethoprim, T,5-[(3,4,5-trimethoxyphenyl)methyl]-2,4-pyrimidinediamine.Chemical structures are shown in Figure 1. Registered applica-tion does not reveal application of F and SF in Danish fishfarming. The compounds have meanwhile been or consideredto be (Hektoenet al.1995) introduced in other countries and assuch are tested in the present investigation. The compoundsrepresent different groups of antibacterial agents;b-lactams, A;4-quinolones, OA, F, and SF; tetracyclines, OT; sulphonamides,SD; and dihydrofolate reductase inhibitors, T. It is shown thatOT is actively taken up by the bacterial cell (Hugo and Russell1992). Table 1 represents some physicochemical properties ofthe antibacterial agents, which implies that exposure to theenvironment of the compounds will distribute to the aquaticenvironment.

The objective of this investigation was to establish data foralgal toxicity of antibacterial agents, applied in,e.g., Danishfish farming, on three different algal species:Microcystisaeruginosaas model organism for cyanobacteria in freshwater,Rhodomonas salinaas model organism for algae in saltwater,and Selenastrum capricornutum,commonly used in standardalgal toxicity tests (ISO 1989), as model organism for algae infreshwater.

Materials and Methods

Algal Species

Nonaxenic unicultures of the test organisms were obtained fromScandinavian Culture Centre for Algae & Protozoa, University ofCopenhagen, Denmark,M. aeruginosa;Laboratory of Marine Biology,Helsingør, Denmark,R. salina; and Norwegian Institute of WaterResearch culture collection, Oslo, Norway,S. capricornutum.All algalspecies are usually found in Danish water streams and oceans. Toprevent contamination, the two freshwater algae were only culturedone at a time. Occurrence of contamination with other algal species,was controlled monthly using an Olympus BHM microscope.

Test Media

The growth medium for the two freshwater algal species was preparedin accordance to ISO (1989). Testing the cyanobacteria 750.0 µg/L ofthioamine hydrochlorid, 10.0 µg/L of cyanocobalamine, and 7.5 µg/Lof biotin were added to the ISO medium. The growth medium for themarine alga was prepared in accordance to Hansen (1989) with smallmodifications: KNO3 was applied instead of NaNO3, Na2HPO4 insteadof NaH2PO4, and CuCl2 · 2H2O instead of CuSO4 · 5H2O. The concen-trations of thioamine hydrochlorid, cyanocobalamine, and biotin werethe same as mentioned above. The water used for the growth media waspurified with a Millipore system.

Chemical Substances

Test compounds (purity, %) were purchased from the followingcompanies: K2Cr2O7 (.99.8), Riedel-de Hae¨n, Seelze, Germany;amoxicillin (plant-cell-culture-tested), Duchefa, Haarlem, The Nether-lands; flumequine (.99.9), Sigma Chemical Co., St. Louis, MO;

oxolinic acid (.98), oxytetracycline hydrochloride (100.7), sulfadia-zine (99.8), and trimethoprim (100), Unikem A/S, Denmark. Sarafloxa-cin hydrochloride (88.5) was obtained from Abbott Laboratories, NorthChicago, IL. Chemicals used for growth medium were all of analyticalgrade and purchased from Merck, Denmark. K2Cr2O7 was used asreference chemical.

Test Solutions

All solutions were prepared from water purified with a Milliporesystem. A, OT, and T were dissolved in H2O. F, OA, SF, and SD weredissolved in 0.05 M NaOH, pH was adjusted with 0.1 M HCl anddiluted to the desired concentrations. The initial pH was 7.96 0.2,7.36 0.2, and 7.96 0.4 in the tests withM. aeruginosa, R. salina,andS. capricornutum,respectively, except for A and K2Cr2O7 in the testwith R. salina,which were 6.4–7.2 and 6.5–6.6, respectively. The pHincrease during the tests did not exceed 1.5 pH unit. The lowestincrease was observed for the cyanobacteria. The pH was measuredwith a PHM95 pH/ION METER, Radiometer Denmark A/S. Thecompounds were tested at three concentration levels without replicates,whereas the controls were grown in triplicates. Number of tests were$2. No analytical determination was done concerning the concentra-tions of antibacterial agents, why their nominal concentration was usedas exposure concentration in the calculations of EC50 values, see Tables2–4; Tested concentration levels.

Test Procedure

The applied test procedures were modified versions of the testprocedure described in the ISO (1989) protocol. The duration of thetests withM. aeruginosawas set to 7 days in order to obtain at least a16 doubling in the cell numbers, which is prescribed in the ISOprotocol. Inoculations were made with algal precultures set up 1–3days before the experiment and propagated under same test conditionsas the subsequent test. The algal concentrations in the preculture weredetermined on a Coulter Countert Model TaII Multichannel particlecounter, Coulter Electronics LTD (Coldharbour Lane, Harpenden,

Table 1. Physicochemical properties of the antibacterial agents tested

Solubility(mg/L)f pKa Log Kow

Amoxicillin 4 · 103,g — —Flumequine 71h 6.4a 1.72a

Oxolinic acid 4.1h 6.9j 0.68b

Oxytetracycline 106,g 3.3, 7.3, 9.1k 21.12c

Sarafloxacin Slightly solublei — 0.84d

Sulfadiazine 2 · 103 (37°C)g 2.0, 6.5l 0.12c

Trimethoprim 400g 7.3m or 6.6g 0.91e

— not founda Takacs-Novak and Avdeef (1996)b Takacs-Novak et al.(1992)c Herbert and Dorsey (1995)d CLog P for Windows (1995), solubility set to 10 mg/Le Rekkeret al.(1993)f Solubility in waterg Budavari (1996)h Elema (1995)i Abbott Laboratories, North Chicago, IL, USAj Timmers and Sternglanz (1978)k Stephenset al.(1956)l Koizumi et al.(1964)m Watson and Stewart (1986)

2 H.-C. Holten Lutzhøftet al.

Herts, England) and were in the order of 1–23 106 cells/ml. Bothcontrol and test flasks were inoculated with exponential growingalgae so an initial concentration ofM. aeruginosa, R. salina,andS. capricornutumwere 2 3 104, 1 3 104, and 1 3 104 cells/ml,respectively. All glassware used in the tests was rinsed in 1 M HCl forat least 1 h prior to use. Algal toxicity tests were performed in 250-mlErlenmeyer flasks containing 100 ml algal culture. The flasks werecovered with perforated laboratory film to avoid contamination andevaporation but to allow gas exchange. Algal preculture as well as testsolutions were grown on a shaking table, with a shaking rate of 93 rpm,under Philips TLM 40W/33rs and TLD 36W/84o white fluorescentlight. The light intensity in the tests withM. aeruginosa, R. salina,andS. capricornutumwas 3.16 0.2, 3.3 6 0.3, and 6.86 0.4 Klux,respectively. The tests were performed at 216 1, 21 6 1, and 2361°C, respectively. Samples were taken from control flasks at the start aswell as at the end of the tests, whereas samples from test flasks weretaken only at the end.

Chlorophyll Determination

Algal chlorophyll were quantified using a modified version of thewhole water extract fluorescence method described by Mayeret al.(1997). The algal chlorophyll was extracted using 67% ethanol. Startcontrol extraction samples were thoroughly shaken for 20 s before theywere stored in the dark at 4°C until the end of the tests. End controlextraction samples and test solution samples were placed on a shakingtable in the dark for at least 2 h at 216 1°C together with the storedcontrol samples before chlorophyll determination. In the tests withR. salinathe ethanolic extracts were filtered through a sterile 0.22-µmMillipore filter to remove flocculates before measuring the chlorophyllcontent. The chlorophyll was fluorometrically determined; excitationwavelength: 430 nm and emission wavelength: 671 nm, on a PerkinElmer Luminescence Spectrometer LS 50B at ambient temperature.The slit width was set to 10 nm and a flow-through cell of 750 µl wasused. The flow-through cell was rinsed with approximately 3 ml samplebefore measurement.

Statistic Treatment of Data

The results of the algal toxicity tests were quantified in terms of growthrates calculated from pooled measurements of chlorophyll content inthree subsequent tests. Growth inhibitions,I, were calculated from

relative growth rates asI i 5 1-m i 0m c, whereIi is the growth inhibitionfor test concentration i,µi is the growth rate for test concentration i, andmc is the growth rate for the control. EC50 values were determined byweighted nonlinear regression analysis directly on the data using theWeibull equation to describe the concentration response relationship(Nyholm et al.1992). A regression program that calculates confidenceintervals by proper inverse estimation and also takes into account thecovariance with the control response was used (Andersen 1994).

Results

Tables 2–4 exhibit the algal toxicity results of this investigationquantified as EC50 values. It is observed thatM. aeruginosaisapproximately two to three orders of magnitude more sensitivetoward the antibacterial agents than bothR. salina andS. capricornutum.The two latter algal species have almost thesame level of sensitivity.

As regardsM. aeruginosa,the antibacterial agents could bedivided into three groups corresponding to EC50 values over 1mg/L, between 0.1 mg/L and 1 mg/L, and below 0.1 mg/L. Tothe first group belongs only T with an EC50 of 112 mg/L. SD, F,OA, and OT belongs all to the second group with EC50’s of0.135, 0.159, 0.180, and 0.207 mg/L, respectively. Both A andSF belongs to the third group with EC50’s of 0.0037 and 0.015mg/L, respectively. On the other hand, A was not toxic towardeitherR. salinaandS. capricornutum.An estimated EC50 valueof 3,108 mg/L and a NOEC of 250 mg/L were obtained,respectively, both in total unrealistic environmental concentra-tions. EC50 values in mg/L of the remaining antibacterial agentsin increasing order forR. salinawere: SD, SF, F, T, OA, and OT,403, 24, 18, 16, 10, and 1.6, respectively, and forS. capricornu-tum: T, SF, OA, SD, F, and OT, 130, 16, 16, 7.8, 5.0, and 4.5,respectively. These observations are exhibited in Figure 2,which shows the decreasing growth inhibiting effects of theantibacterial agents ranged with respect to algal specie.

Discussion

The statistical treatments of the data were performed on resultsfrom three subsequent tests. This is not in conformity with the

Fig. 1. Chemical structures of the antibacterial agents tested

3Algal Toxicity of Antibacterial Agents

ISO8692 (1989) protocol, but was done in order to obtainestimates of EC50 values from the Weibull equation. The resultsexhibited in Tables 2–4 are therefore obtained from pooledtests.M. aeruginosagenerally seems to be two to three ordersof magnitude more sensitive than bothR. salina and S.capricornutum,which have almost equal sensitivity. Theseresults do not surprise, since Harraset al. (1985) showed thatM. aeruginosawas approximately a factor of 10 more sensitive

than S. capricornutumtoward streptomycin, an aminoglyco-side. It is impossible with the data presented in the presentinvestigation to generalise toxic relations among the antibacte-rial agents for any of the three algal species. Lanzky andHalling-Sørensen (1997) investigated the toxicity of metronida-zole to organisms at different trophic levels. They showed thatboth Chlorella sp.and S. capricornutumhad a much highersensitivity compared withAcartia tonsa(marine crustacean)and Brachydanio rerio (zebrafish). Miglioreet al. (1997)investigated the toxicity of bacitracin and F, among others,towardArtemia,and showed that both compounds were mostactive against nauplii and cysts. Macrı` et al.(1988) investigatedthe acute toxicity of furazolidone to different species and foundLC50 values toArtemia salina, Daphnia magna,and Culexpipiens larvae to be 250 mg/L, 60 mg/L, and 40 mg/L,respectively. Dojmi Di Delupiset al. (1992) investigated thetoxicity of different antibacterial agents towardD. magnaandshowed that only bacitracin had an EC50 value (48 h) below 100mg/L; furthermore, they showed that both bacitracin andlincomycin lowered the phototactic behavior at concentrationsof 10 and 5 mg/L, respectively, whereas aminosidine, atconcentrations of 10 mg/L, increased it. An overview of thetoxicity data, on algae, crustaceans, and fish, found in theliterature, are shown in Table 5. From toxicity data establishedin this investigation as well as data found in the literature, it isrealized that algae have a higher sensitivity toward antibacterialagents compared to crustaceans and fish. Furthermore, amongalgae, cyanobacteria have shown to be the most sensitive algalspecies, due to their structure being more like bacteria. Theseobservations indicate that effects on higher trophic levelsprimarily would be indirect. In order to perform a properenvironmental risk assessment of antibacterial agents, it wouldbe necessary to include a cyanobacteria as test organism in thetest battery.

In algal batch cultures the biomass density may quickly reacha level where the carbon demand by the growing algae exceedsthe transfer rate of CO2 from the gas phase to the liquid phase.In this situation, dissolved CO2 for algal growth will also bederived from medium bicarbonate, which results in an increaseof medium pH during the 3 days of test performance. Due to thetechnical problems of maintaining constant pH during an algalbatch toxicity test, a pH increase as large as up to 1.5 units isaccepted (ISO 1989). The antibacterial agents tested in thisinvestigation are all protic, with pKa values around 6–7; due toincreasing pH during test performance, increasing ionization ofthe compounds will occur. An increase in pH is of specialimportance in batch tests with protic substances. The pHincrease observed in this investigation were within the limits forboth R. salina and S. capricornutum.For the tests withM.aeruginosaalmost no pH increase was observed; all tests werewithin 0.2 units of increase, except F and T, which were 0.8 and20.2, respectively. The low pH increase is due to lower growthrate ofM. aeruginosacompared withR. salinaandS. capricor-nutum.Increasing pH will reduce the bioavailable concentra-tions of weak acids, which, for compounds with passivediffusion across the cell membranes, may lead to (fairly)underestimated results. The phenomenon of changed toxicitydue to different pH values is recognized for chlorinatedphenols. Koenemann and Musch (1981) found that a decreasein pH from 8 to 6 increased the toxicity of pentachlorophenolwith a factor of approximately 10. To avoid the influence of pHon the test results a test setup including a buffering capacity

Table 2. Results for the tests withM. aeruginosa

TestedConcentrationLevels(mg/L)

EC50

(mg/L)

95%ConfidenceInterval(mg/L) n

Amoxicillin 0.0009–0.0038 0.0037 — 2Flumequine 0.094–0.369 0.159 0.066–0.382 3Oxolinic acid 0.060–0.540 0.180 — 2Oxytetracycline 0.040–0.360 0.207 0.175–0.246 3Sarafloxacin 0.004–0.009 0.015OMR 0.009–0.023 3Sulfadiazine 0.020–0.180 0.135 0.082–0.223 3Trimethoprim 100–132 112 100–126 3K2Cr2O7 0.044–0.400 0.211 0.029–1.506 3µcontrol, d21 0.5–0.7

— not obtainableOMR Out of measured range

Table 3. Results for the tests withR. salina

TestedConcentrationLevels(mg/L)

EC50

(mg/L)

95%ConfidenceInterval(mg/L) n

Amoxicillin 5.0–500 3,108OMR 320–30,199 2Flumequine 10–160 18 10–31 3Oxolinic acid 5.0–20 10 5.5–19 3Oxytetracycline 0.8–3.0 1.6 0.4–6.1 3Sarafloxacin 10–40 24 11–52 3Sulfadiazine 5.0–45 403OMR 146–1,113 3Trimethoprim 5.0–20 16 9.3–27 3K2Cr2O7 1.0–8.0 3.9 3.6–4.4 3µcontrol, d21 1.3–1.5

OMR Out of measured range

Table 4. Results for the tests withS. capricornutum

TestedConcentrationLevels(mg/L)

EC50

(mg/L)

95%ConfidenceInterval(mg/L) n

Amoxicillin 2.5–250 250a — 2Flumequine 3.0–27 5.0 1.6–16 3Oxolinic acid 9.3–37 16 9.1–29 3Oxytetracycline 3.0–12 4.5 2.3–8.6 3Sarafloxacin 10–40 16 9.8–25 3Sulfadiazine 3.0–27 7.8 4.5–14 3Trimethoprim 30–270 130 81–211 3K2Cr2O7 0.3–2.7 0.6 0.5–0.7 3µcontrol, d21 1.9

— not obtainablea NOEC

4 H.-C. Holten Lutzhøftet al.

should be applied. Several modifications (Halling-Sørensenetal. 1996) of the standard open flask test method have beensuggested to reduce the pH development in the test procedure ofthe ISO8692 (1989) protocol. Halling-Sørensenet al. (1996)calculated the quantity of [HCO32] in the medium needed tobuffer pH to different endpoint values. Initial algal densitycould be lowered to,e.g., 103 cells/ml. Test time could bereduced from the recommended 3 days in ISO (1989) standardtest withS. capricornutumto 1 or 2 days. If an initial pH of 7 isallowed a reduction of the test duration from 3 to 2 days wouldsignificantly reduce the problems of pH increase. Application of

a chemostat setup with buffered medium at pH 7 or even lowermight also increase the fraction of unionized compound.

Conclusion

In this investigationM. aeruginosais found to be about two tothree orders of magnitude more sensitive than eitherR. salinaand S. capricornutum.A and SF seem to be the most toxiccompounds towardM. aeruginosa,with EC50 values below 0.1mg/L; SD, F, OA, and OT seem to constitute a group with

Fig. 2. Decreasing EC50 values ranged withrespect to algal specie with corresponding95% confidence intervals

Table 5. Overview of different effect data

Compound Organism Effect Effect Value (mg/L)

Streptomycina M. aeruginosa Minimum inhibitory concentration 0.3S. capricornutum Minimum inhibitory concentration 2.1

Metronidazoleb Chlorella sp. Growth inhibition, EC50 38.8S. capricornutum Growth inhibition, EC50 39.1A. tonsa No effect level 100B. rerio No effect level 500

Bacitracinc Artemia Acute toxicity, EC50 (48 h) 21.8Artemianauplii 100% mortality 6.3Artemiacysts Hatching 25

Flumequinec Artemia Acute toxicity, EC50 (72 h) 96.4Artemianauplii 22% mortality 6.3Artemianauplii Transparency induced 1 ppm

Furazolidoned A. salina LC50 250D. magna LC50 60C. pipienslarvae LC50 40

Aminosidinee D. magna EC50 (48 h) 502.8Bacitracine D. magna EC50 (48 h) 30.5Erythromycine D. magna EC50 (48 h) 210.6Lincomycine D. magna EC50 (48 h) 379.4Aminosidinee D. magna Phototactic behavior, increased 10Bacitracine D. magna Phototactic behavior, decreased 10Erythromycine D. magna Phototactic behavior No effectLincomycine D. magna Phototactic behavior, decreased 5

a Harraset al.(1985)b Lanzky and Halling-Sørensen (1997)c Migliore et al.(1997)d Macri et al.(1988)e Dojmi Di Delupiset al.(1992)

5Algal Toxicity of Antibacterial Agents

EC50 value between 0.1 mg/L and 1 mg/L; and T seems to be theleast toxic compound with an EC50 value over 1 mg/L. Acyanobacteria has to be included in the test battery as testorganism, if an environmental risk assessment is to be per-formed for antibacterial agents. IfR. salinaandS. capricornu-tumare applied as test organisms, changes have to be made inthe standard batch test procedure, to avoid the difficultiesregarding pH increase.

Acknowledgments.The grammatical and technical help and assis-tance provided by Klavs Mulvad, Susanne Hermansen, Johan ChristianFriis, and Flemming Ingerslev is gratefully acknowledged. Thisinvestigation was partly funded by a grant from the Danish Centre forSustainable Land Use and Management of Contaminants, Carbon andNitrogen, under the Danish Strategic Environmental Research Pro-gramme, Part 2, 1997–2000. Sarafloxacin hydrochloride was gentlysupplied by Abbott Laboratories, North Chicago, Illinois, USA.

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