Compounds with selective toxicity towards the off-flavor metabolite-producing cyanobacterium Oscillatoria cf. chalybea

Aquaculture 163 Ž1998. 85–99

Compounds with selective toxicity towards the off-flavor metabolite-producing cyanobacterium Oscillatoria cf. chalybea Kevin K. Schrader a,) , Marjan Q. de Regt a , Paula D. Tidwell a , Craig S. Tucker a , Stephen O. Duke b,1 a

Delta Research and Extension Center, Mississippi State UniÕersity, StoneÕille, MS 38776, USA b USDA, ARS, SWSRU, StoneÕille, MS 38776, USA Received 20 October 1997; accepted 20 January 1998

Abstract Oscillatoria cf. chalybea produces the musty, off-flavor compound 2-methylisoborneol, which can taint the flesh of channel catfish and render them unmarketable. Green algae are preferred over cyanobacteria in aquaculture ponds since they do not produce 2-methylisoborneol and because they are better in maintaining the primary productivity in pond ecosystems. The discovery of compounds exhibiting selective toxicity towards cyanobacteria is the first step in the development of a cyanobacterial algicide that would benefit the channel catfish industry. Herbicides and other synthetic compounds were screened using a microplate bioassay to determine their toxicity towards the cyanobacterium O. cf. chalybea and the green alga Selenastrum capricornutum. Diquat, paraquat, and diuron were most inhibitory to the growth of O. cf. chalybea Ž0.1, 0.1, and 1.0 m M, respectively. but only diquat and paraquat were selectively toxic towards O. cf. chalybea. Bromoxynil, cinmethylin, diclofop, isoxaben, and sodium carbonate peroxyhydrate were selective, with complete growth inhibition towards O. cf. chalybea. Of these compounds, sodium carbonate peroxyhydrate appears to be the most environmentally and toxicologically safe for use in aquaculture. Sodium carbonate peroxyhydrate may not be selectively toxic against all species of cyanobacteria, as evidenced by our additional screening of the compound using another cyanobacterium, Anabaena sp. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Aquaculture; Cyanobacteria; Green algae; Phytotoxins; Off-flavor; 2-Methylisoborneol; Selective toxicity

) Corresponding author. USDA, ARS, NPURU, P.O. Box 8048, University, MS 38677-8048, USA. Tel.: q1-601-232-1144; fax: q1-601-232-1035. 1 USDA, ARS, NPURU, School of Pharmacy, University of Mississippi, University, MS 38677-8048, USA.

0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 2 2 3 - 3

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1. Introduction Objectionable flavor, also referred to as off-flavor, is still one of the largest problems facing the catfish farming industry today. The most troublesome off-flavors in farm-raised channel catfish Ž Ictalurus punctatus . are earthy and musty flavors which are caused by the compounds geosmin Ž trans-1,10-dimethyl-trans-9-decalol. ŽFig. 1A. and 2-methylisoborneol Ž1,2,7,7-tetramethyl-exo-bicyclow2.2.1x-heptan-2-ol or MIB. ŽFig. 1B., respectively ŽLovell et al., 1986; Martin et al., 1987.. Such off-flavors result in unpalatable and unmarketable catfish. In aquaculture ponds in west-central Mississippi, MIB has been detected more frequently than geosmin Žvan der Ploeg et al., 1992.. Martin et al. Ž1988b. found that MIB was directly responsible for the musty-taint in the flesh of pond-raised channel catfish in west-central Mississippi. Channel catfish exposed to MIB can absorb the metabolite within hours ŽMartin et al., 1988a; Johnsen, 1989; Johnsen and Lloyd, 1992., while depuration of off-flavors by channel catfish may take days or weeks ŽLovell and Sackey, 1973; Johnsen and Lloyd, 1992.. Off-flavor problems in farm-raised channel catfish create delays in harvesting since off-flavor catfish must be held by the producer until they are deemed to be on-flavor. Such practices increase production costs, increase the risk of losses due to oxygen and disease problems, and can create inventory management dilemmas for catfish producers ŽEngle et al., 1995.. Off-flavor problems may increase the cost of producing channel catfish in the United States by 10–20% ŽPaerl and Tucker, 1995.. In 1996, off-flavor problems may have cost producers as much as US$50 million based on the calculations of off-flavor problem added-costs ŽKinnucan et al., 1988; Coats et al., 1989; Engle et al., 1995. and the revenues received by catfish producers ŽUS$417 million. ŽUnited States Department of Agriculture-National Agricultural Statistics Service, 1997.. Certain species of cyanobacteria Žblue-green algae. produce MIB ŽTabachek and Yurkowski, 1976; Izaguirre et al., 1982; Matsumoto and Tsuchiya, 1988; Negoro et al., 1988; Martin et al., 1991; Hosaka et al., 1995; Izaguirre and Taylor, 1995; Zimmerman et al., 1995., and the cyanobacterium Oscillatoria cf. chalybea has been found to be a contributor to MIB presence in Mississippi Delta aquaculture ponds Žvan der Ploeg et al., 1995.. Green algae have never been linked to musty off-flavor problems in Mississippi catfish production ponds, which is one reason they are the preferred type of phytoplankton to help maintain productive aquatic ecosystems for catfish production. In addition, green algae provide a more substantial base for aquatic food chains than cyanobacteria and are better oxygenators of the water than cyanobacteria due to faster

Fig. 1. Chemical structures of geosmin ŽA. and 2-methylisoborneol ŽB..

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growth rates ŽPaerl and Tucker, 1995.. Furthermore, strains of several cyanobacterial genera are capable of producing toxins ŽCarmichael, 1992; Rinehart et al., 1994; Sivonen, 1996., and, in one case, the toxin-producing cyanobacterium Aphanizomenon flos-aquae has been attributed with causing the deaths of large numbers of pond-cultured channel catfish ŽEnglish et al., 1993.. Chemical treatment of aquaculture ponds with algicides is one method used to manage phytoplankton, and several synthetic compounds have been promoted as useful in selectively controlling cyanobacteria in aquaculture ponds. Simazine, although no longer approved by the United States Environmental Protection Agency ŽUSEPA. for use in food-fish production ponds, was reported to be effective and selective in the control of cyanobacteria without creating problems with low dissolved oxygen concentrations ŽNorton and Ellis, undated.. However, another study ŽTucker and Boyd, 1979. reported problems with low dissolved oxygen concentrations and lower rates of phytoplankton abundance in catfish ponds following simazine treatment. These results indicate that there may be little difference between the effective toxic concentration of simazine towards members of the division Cyanophyta Žcyanobacteria. when compared to the effective toxic concentration of simazine towards other divisions of phytoplankton. Potassium ricinoleate, derived from the saponification of castor oil with potassium hydroxide, has also been claimed to selectively inhibit the growth of cyanobacteria and eliminate off-flavor problems ŽVan Aller and Pessoney, 1982.. However, studies by Tucker and Lloyd Ž1987. and Scott et al. Ž1989. found that the compound did not selectively reduce the number of cyanobacteria or, for that matter, even total phytoplankton, and the incidence of off-flavor in channel catfish was not reduced. Sodium carbonate peroxyhydrate ŽSCP., a solid form of hydrogen peroxide with the molecular formula 2Na 2 CO 3 P 3H 2 O 2 , has been proposed for use as an algicide in sewage lagoons ŽOuzts et al., 1989. and was reported to be effective as an algicide in laboratory studies against two cyanobacterial species Ž Anabaena sp. and Raphidiopsis sp.. ŽQuimby et al., 1988.. Martin Ž1992. performed a field study in which SCP was applied to seven commercial catfish ponds in Mississippi to determine its effectiveness in eliminating or preventing off-flavor in catfish. Results of this study indicated a beneficial effect of SCP treatment of ponds producing off-flavor catfish although more successful treatment with SCP occurred when off-flavor episodes in ponds were less than 2 months in duration. However, Martin Ž1992. suggested that further studies were needed to identify target organisms of peroxide action and to determine the appropriate SCP dosage required to correlate with the amount of soluble organic matter present in a pond. Current methods for controlling cyanobacteria in aquaculture ponds include the use of copper-based compounds such as copper sulfate ŽCuSO4 P 5H 2 O.. Although these products are the only algicides approved by the USEPA for use in food-fish production ponds, they have broad spectrum toxicity towards phytoplankton, and their use can be poisonous and lethal to most or all members of the phytoplankton community. Large reductions in the number of phytoplankton and in the rates of metabolic processes of phytoplankton can result in the deterioration of water quality through oxygen depletion and large increases in ammonia and nitrite concentrations ŽTucker, 1996.. Ideally, methods of phytoplankton control or treatment in catfish production ponds should result in the complete removal of cyanobacteria while other divisions of algae are maintained

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at sustainable and productive levels without becoming a nuisance, detriment, or hindrance to catfish production ŽTucker, 1996.. Due to the limitations of copper-based products in controlling cyanobacteria in aquaculture ponds, the discovery of other compounds that exhibit greater selective toxicity towards cyanobacteria would be beneficial. The objective of this study was to discover herbicides and other synthetic compounds previously unidentified as exhibiting selective toxicity towards O. cf. chalybea that might be used to help prevent musty off-flavors problems in catfish production ponds. A rapid bioassay utilizing 96-well cell culture plates ŽSchrader et al., 1997. was used to screen herbicides and other synthetic compounds against O. cf. chalybea and the green alga Selenastrum capricornutum. Several compounds previously identified as possessing selective toxicity towards cyanobacteria, i.e., ricinoleate, SCP, and a copper-based product ŽCopper Control w ; Argent Chemical Laboratories, Redmond, WA., were also screened. Several herbicides andror synthetic compounds that exhibited a high degree of selective toxicity towards O. cf. chalybea were analyzed further to determine 96-h IC50 Ž50% inhibition concentration. values. Also, SCP was screened against another cyanobacterium Ž Anabaena sp.. and another green alga Ž Pediastrum simplex . to determine if the difference in toxicity exhibited by SCP towards O. cf. chalybea and S. capricornutum was similar between other genera from the phytoplankton divisions Cyanophyta and Chlorophyta Žgreen algae..

2. Materials and methods 2.1. Continuous culture conditions Algal cultures of O. cf. chalybea and S. capricornutum were grown and maintained separately in continuous, steady-states at 298C under continuous light by the method of van der Ploeg et al. Ž1995., except that medium pH was maintained in the range of 7.6–9.0, photon flux density ranged from 18–29 m mol my2 sy1 , and air flow rate was 16–33 l hy1 . O. cf. chalybea was obtained through isolation work performed by van der Ploeg et al. Ž1995. on water samples from a Mississippi catfish pond. O. cf. chalybea had also been isolated previously by Martin et al. Ž1991. from the water of several catfish research ponds in Mississippi. S. capricornutum was provided by Dr. J.C. Greene, USEPA, Corvallis, OR, and was used as a representative species for green algae in the screening process due to its frequent use in toxicity studies of compounds by the USEPA and because Selenastrum spp. are commonly found in freshwater ponds in the southeastern United States ŽBoyd, 1990.. Continuous cultures of O. cf. chalybea and S. capricornutum provided a source of cells growing at a fairly constant rate for use in the screening process. Cell density in the continuous cultures was maintained at 0.18–0.27 A Žabsorbance. for O. cf. chalybea and at 0.19–0.26 A for S. capricornutum when measured at 750 nm with a spectrophotometer ŽGilford model Response UV–VIS, Gilford Instrument Laboratories, Oberlin, OH..

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2.2. Determination of continuous culture cell r natural unit numbers Standard curves relating spectrophotometric absorbance reading Ž750 nm. with cell or natural unit Žfilaments were counted for O. cf. chalybea. numbers were developed for continuous cultures of O. cf. chalybea and S. capricornutum. These curves were used to determine cell or natural unit numbers in continuous culture samples used as inoculum for the screening bioassay. Data used to produce the standard curves was obtained from absorbance measurements and phytoplankton enumeration performed on continuous culture samples taken during a 2-week period after the establishment of steady-state conditions. Phytoplankton were enumerated in a Sedgwick-Rafter counting chamber by field counting under 150 = and 300 = magnification ŽAmerican Public Health Association, American Water Works Association, and Water Pollution Control Federation, 1989.. 2.3. Screening of herbicides and other synthetic compounds A rapid bioassay developed previously ŽSchrader et al., 1997. for screening compounds for selectivity as cyanobacterial algicides was used in this study. Technical grade herbicides and other synthetic compounds were used in this study, and compensations for purity were applied when preparing stock solutions. Stock solutions of herbicides and other synthetic compounds insoluble in water were prepared in either methanol or ethanol, added by micropipet to the empty wells Ž10 m l per well. of a 96-well cell culture plate ŽCostar, Cambridge, MA., and allowed to completely evaporate before aseptically adding continuous culture samples Ž200 m l per well. of either O. cf. chalybea or S. capricornutum. Controls for the water-insoluble compounds screened contained only 200 m l of culture per well. Stock solutions of water-soluble herbicides and other compounds to be screened were prepared in double-deionized water, filtersterilized Žsterile Acrodisc with 0.2 m m pore size; Gelman Sciences, Ann Arbor, MI., and then added to wells Ž50 m l per well. already containing continuous culture samples Ž150 m l per well. of either O. cf. chalybea or S. capricornutum. Filter-sterilized double-deionized water was added to the controls for water-soluble compounds screened. Haloxyfop, isoxaben, and ricinoleate were initially dissolved in dimethyl sulfoxide ŽDMSO. and then diluted further in double-deionized water to make the final stock solutions. Final DMSO concentrations in the wells did not exceed 0.01% Žvrv.. Filter-sterilized, double-deionized water containing DMSO wfinal DMSO concentration in well of 0.01% Žvrv.x were added to the controls for haloxyfop, isoxaben, and ricinoleate. For the preliminary screening, stock solutions were prepared in 10-fold concentration increments, and four replications were used for each concentration and control. Table 1 lists the herbicides and other synthetic compounds screened, purity of the screened compounds, the solvent used to make stock solutions of each particular compound, and the source Žmanufacturer. of each compound screened. Plates were held in a growth chamber at 25–278C and were illuminated by three overhead fluorescent lamps Ž40 W, cool white. at a photon flux density of 20–28 m mol my2 sy1 . Optical densities of each well were measured daily for 5 days at 650 nm using a Bio-Tek model EL311 microplate reader ŽBio-Tek Instruments, Winooski, VT.. Mean

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Table 1 Herbicides and other synthetic compounds screened Herbicider synthetic compound

Purity Ž%.

Solvent used

Manufacturer of herbicider synthetic compound

Diquat Paraquat Acifluorfen Atrazine Diuron Fluometuron Norflurazon Imazaquin Diclofop Haloxyfop Isoxaben Clethodim Sethoxydim Bromoxynil Diclobenil Sulfometuron Cinmethylin Difenzoquat Endothall Fluridone Glyphosate Copper Control w Ricinoleate SCP c

98.0 94.0 97.5 99.7 80.0 99.2 98.0 96.0 99.0 Unknowna 92.2 99.0 99.0 99.0 97.0 99.0 Unknowna 99.0 95.0 95.0 99.0 100.0 99.0 77.0

H 2O H 2O H 2O H 2O H 2O H 2O H 2O Methanol Ethanol DMSOrH 2 O b DMSOrH 2 O b Ethanol H 2O Ethanol Ethanol H 2O H 2O H 2O H 2O Methanol H 2O H 2O DMSOrH 2 O b H 2O

Chem Service Sigma Chem Service Ciba-Geigy Sigma Ciba-Geigy Sandoz-Wander Amer. Cyanamid Chem Service BASF DowElanco Valent USA BASF Chem Service Chem Service Chem Service Shell Development Chem Service Chem Service Eli Lilly Rhone-Poulenc Ag ˆ Argent Chemical Lab. Sigma Aldrich Chemical

a

For unknown purities, no correction for purity was applied when making stock solutions. Dimethyl sulfoxide ŽDMSO. was used to initially dissolve the herbicide before further dilution using H 2 O; highest DMSO concentration used in screening was no greater than 0.01%. c SCPssodium carbonate peroxyhydrate. b

values of the optical density measurements for each concentration and controls were calculated and graphed. Graphs were used to determine herbicidersynthetic compound LCIC ŽLowest-Complete-Inhibition Concentration. and LOEC ŽLowest-Observed-Effect Concentration. which were used to calculate differential sensitivity values ŽLCIC of S. capricornutumrLCIC of O. cf. chalybea and LOEC of S. capricornutumrLOEC of O. cf. chalybea.. From the graphs, LCIC was determined to be the lowest concentration which completely inhibited the growth Žalgistatic. of the test organism while LOEC was determined to be the lowest concentration which had any inhibitory effect on the growth of the test organism but was not completely inhibitory Žalgisensitive.. 2.4. 96-h IC50 determinations of selected herbicidesr synthetic compounds The 96-h IC50 were determined for cinmethylin, diclofop, diuron, diquat, paraquat, and SCP on O. cf. chalybea and S. capricornutum. The same bioassay set-up and conditions outlined previously for herbicide and other synthetic compound screening

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was used. Estimation of the IC50 was determined using graphs obtained by plotting 96-h absorbance readings against logarithmic dilution values of the phytotoxic compounds. 2.5. SCP screening against other genera of phytoplankton Following the initial screening of herbicides and other synthetic compounds against O. cf. chalybea and S. capricornutum, SCP was screened further against two other genera of phytoplankton. SCP is more environmentally acceptable wi.e., SCP forms hydrogen peroxide ŽH 2 O 2 . and sodium carbonate ŽNa 2 CO 3 . upon contact with waterx than the herbicides and other synthetic compounds screened. Anabaena sp. LP 691 Žobtained from George Izaguirre, The Metropolitan Water District of Southern California, La Verne, CA., and P. simplex UTEX aLB 1601 Žobtained from the Culture

Table 2 Lowest-complete-inhibition concentrations ŽLCIC. of phytotoxic compounds screened for toxic selectivity towards O. cf. chalybea Herbicidersynthetic compound

Diquat Paraquat Aciflurofen Atrazine Diuron Fluometuron Norflurazon Imazaquin Diclofop Haloxyfop Isoxaben Clethodim Sethoxydim Bromoxynil Diclobenil Sulfometuron Cinmethylin Difenzoquat Endothall Fluridone Glyphosate Copper Control c Ricinoleate SCP d a

Differential sensitivity a

LCIC Ž m M. O. cf. chalybea

S. capricornutum

0.1 0.1 )100.0 10.0 1.0 100.0 )1000.0 )100.0 100.0 )100.0 10.0 1000.0 )1000.0 100.0 )100.0 100.0 10.0 10.0 1000.0 100.0 1000.0 10.0 )10.0 100.0

10.0 10.0 100.0 10.0 1.0 100.0 100.0 )100.0 )10,000.0 )100.0 )10.0 1000.0 )1000.0 )1000.0 )100.0 10.0 1000.0 10.0 1000.0 1.0 1000.0 10.0 )10.0 1000.0

2 2 0 0 0 0 0 0 3b 0 1b 0 0 2b 0 0 2 0 0 0 0 0 0 1

Sensitivity expressed in terms of order of magnitude; derived by LCIC of S. capricornutumr LCIC of O. cf. chalybea. b Differential sensitivity value could be higher than listed since LCIC of S. capricornutum is greater than the highest concentration of compound screened. c Product of Argent Chemical Laboratories, Redmond, WA. d SCPssodium carbonate peroxyhydrate.

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Collection of Algae, University of Texas, Austin, TX. were used as additional representatives of Cyanophyta and Chlorophyta, respectively. Anabaena sp. LP 691 was isolated from a water reservoir in California and produces the earthy off-flavor compound geosmin. For this additional screening, the same continuous culture conditions, bioassay procedures, and data analyses were used as those outlined previously, except that continuous culture cell density was maintained at 0.28–0.33 A for Anabaena sp. LP 691 and 0.20–0.24 A for P. simplex when measured spectrophotometrically at 750 nm. 3. Results Cultures were considered to be in a continuous, steady-state after biomass had been constant through five generation Ždoubling. times. Natural unit numbers of O. cf.

Table 3 Lowest-observed-effect concentrations ŽLOEC. of phytotoxic compounds screened for toxic selectivity towards O. cf. chalybea Herbicidersynthetic compound

Diquat Paraquat Aciflurofen Atrazine Diuron Fluometuron Norflurazon Imazaquin Diclofop Haloxyfop Isoxaben Clethodim Sethoxydim Bromoxynil Diclobenil Sulfometuron Cinmethylin Difenzoquat Endothall Fluridone Glyphosate Copper Control c Ricinoleate SCP d a

Differential sensitivity a

LOEC Ž m M. O. cf. chalybea

S. capricornutum

0.1 0.1 )100.0 1.0 1.0 10.0 1000.0 )100.0 10.0 )100.0 0.1 1000.0 )1000.0 10.0 )100.0 100.0 10.0 10.0 1000.0 10.0 1000.0 10.0 1.0 1.0

1.0 1.0 10.0 1.0 1.0 10.0 100.0 )100.0 )10,000.0 )100.0 )10.0 1000.0 )1000.0 1000.0 )100.0 1.0 1000.0 1.0 1000.0 1.0 1000.0 10.0 )10.0 1000.0

1 1 0 0 0 0 0 0 4b 0 3b 0 0 2 0 0 2 0 0 0 0 0 2b 3

Sensitivity expressed in terms of order of magnitude; derived by LOEC of S. capricornutumr LOEC of O. cf. chalybea. b Differential sensitivity value could be higher than listed since LOEC of S. capricornutum is greater than the highest concentration of compound screened. c Product of Argent Chemical Laboratories, Redmond, WA. d SCPssodium carbonate peroxyhydrate.

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chalybea continuous cultures used for the screening bioassay ranged from approximately 1.75 = 10 4 filaments mly1 to 2.65 = 10 4 filaments mly1 while cell numbers of S. capricornutum continuous cultures used for screening ranged from approximately 1.92 = 10 6 cells mly1 to 2.25 = 10 6 cells mly1 . The bipyridilium herbicides diquat and paraquat had the lowest LCIC Ž0.1 m M. towards O. cf. chalybea ŽTable 2.. Diuron also had a low effective toxic concentration ŽLCIC of 1.0 m M. towards O. cf. chalybea, but was not selectively toxic when compared to the LCIC for S. capricornutum. Based on the LCIC results ŽTable 2., diquat and paraquat were found to be two orders of magnitude more toxic towards O. cf. chalybea than S. capricornutum wi.e., differential sensitivity ŽDS. values of 2x. In addition, O. cf. chalybea was at least three orders of magnitude more sensitive to diclofop, at least two orders of magnitude more sensitive to bromoxynil, two orders of magnitude more sensitive to cinmethylin, and one order of magnitude more sensitive to isoxaben and SCP. Diquat, paraquat, and isoxaben had the lowest LOEC Ž0.1 m M. towards O. cf. chalybea and were also selectively toxic ŽTable 3.. Atrazine, diuron, ricinoleate, and SCP also had low LOECs Ž1.0 m M. for O. cf. chalybea, but only ricinoleate and SCP were selectively toxic towards O. cf. chalybea. Diclofop was at least four magnitudes more toxic towards O. cf. chalybea than S. capricornutum ŽDS ) 4.. DS values were also high for isoxaben and SCP Ž) 3 and 3, respectively.. Bromoxynil, cinmethylin, and ricinoleate were two orders of magnitude more toxic towards O. cf. chalybea than S. capricornutum, while diquat and paraquat had DS values of 1. Diquat and paraquat had the lowest 96-h IC50 for O. cf. chalybea Ž0.036 and 0.056 m M, respectively. ŽTable 4.. Diuron was not selective towards O. cf. chalybea based on 96-h IC50 results. Comparison of 96-h IC50 ŽTable 4. revealed diquat to have the highest differential selectivity value. Differential selectivity could not be determined for diclofop since no growth inhibition was observed even at the highest concentration of diclofop screened Ž1 = 10 4 m M.. Higher concentrations of diclofop Ž) 1 = 10 4 m M.

Table 4 96-h IC50 of selected herbicides and other synthetic compounds Herbicidercompound

Differential selectivity a

IC50 O. cf. chalybea

Diquat Paraquat Cinmethylin Diclofop Diuron SCP b

S. capricornutum

mM

m g ly1

mM

m g ly1

0.036 0.056 7.9 50.1 0.1 22.4

12.2 12.9 2180 1.7=10 4 28.0 8600

5.0 0.9 159 )1=10 4 0.13 177.8

1730 229 4.4=10 4 ) 3.4=10 6 36.4 6.8=10 4

138.9 17.8 20.0 Unknown 1.3 7.9

a IC50 of S. capricornutumrIC50 of O. cf. chalybea; higher differential selectivity values higher selective toxicity towards O. cf. chalybea. b SCPssodium carbonate peroxyhydrate.

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Fig. 2. Effects of different concentrations of SCP on the growth of O. cf. chalybea ŽA., S. capricornutum ŽB., Anabaena sp. LP 691 ŽC., and P. simplex UTEX aLB 1601 ŽD.. Symbols: v, Control Žno SCP.; B, 1 m M SCP; ', 10 m M SCP; %, 100 m M SCP; l, 1000 m M SCP. Vertical bars represent standard errors Ž ns 4..

could not be screened due to the limits of diclofop solubility in ethanol at such high concentrations. SCP completely inhibited the growth of O. cf. chalybea at 100 m M ŽFig. 2A., while 1000 m M SCP was required to completely inhibit the growth of S. capricornutum ŽFig. 2B., Anabaena sp. LP 691 ŽFig. 2C., and P. simplex UTEX aLB 1601 ŽFig. 2D.. SCP at 1.0 and 10.0 m M caused clumping of O. cf. chalybea filaments which resulted in higher absorbance readings than those obtained for the controls ŽFig. 2A..

4. Discussion Diuron is an effective herbicide for inhibiting the growth of O. cf. chalybea ŽIC50 s 0.1 m M.. However, diuron was not selectively toxic towards O. cf. chalybea as indicated by an IC50 s 0.13 m M for S. capricornutum. Maule and Wright Ž1984. also found diuron to be inhibitory to several species of cyanobacteria and green algae at low concentrations with EC50 values ranging from 0.1 to 2.3 m M, and they also found no indication of selective toxicity of diuron towards cyanobacteria. LCIC and LOEC results reveal several selectively toxic phytotoxic compounds which might be useful in control-

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ling cyanobacteria in fish ponds. Cost of compound, compound persistence in the environment, toxicity to fish and mammals, and toxicity to other microorganisms in the aquatic ecosystem also need to be considered in selecting a compoundŽs. for use in aquaculture. Although diquat and paraquat were found to be selectively toxic towards O. cf. chalybea and possess lower LCIC for O. cf. chalybea than any of the other compounds screened, both are highly toxic towards fish and mammals and, therefore, are not alternatives for reducing cyanobacterial numbers in food-fish ponds. These results do, however, suggest that some species of cyanobacteria are more sensitive to oxidative damage than green algae. Bromoxynil, cinmethylin, diclofop, and isoxaben are also not worth pursuing for USEPA approval for use in food-fish ponds due to the concentrations required for complete growth inhibition of O. cf. chalybea ŽTable 2. and their toxicity to fish at similar or lower levels ŽAhrens, 1994.. The concentration of bromoxynil and diclofop required for complete growth inhibition of O. cf. chalybea Ž100 m M. is about 100 times greater than the concentrations of both compounds reported for rainbow trout 96-h LC50 ŽAhrens, 1994.. However, SCP may provide an environmentally acceptable method for controlling noxious cyanobacteria and has been evaluated for use as an algicide previously ŽQuimby et al., 1988; Martin, 1992.. Martin Ž1992. found that SCP treatment of catfish ponds reduced numbers of O. cf. chalybea only in those ponds not dominated by high numbers of O. cf. chalybea, and other cyanobacterial species appeared to have been unaffected by the levels of SCP treatment used in his study. Microscopic observation Ž20 = . of cell cluster wells revealed that diquat, paraquat, and SCP were actually algicidal towards O. cf. chalybea as noted by the clumping, bleaching, and subsequent disappearance of filaments Žcells. from wells containing toxic levels of these compounds. The mechanism of diquat, paraquat, and SCP toxicity towards O. cf. chalybea may be similar. In plants, the mechanism of toxicity of diquat and paraquat is due to the reduction of the herbicide to a free radical by photosystem I, which, in turn, reduces molecular oxygen to a superoxide radical ŽHalliwell, 1982.. Superoxide can generate H 2 O 2 , which can directly cause toxic oxidations. Superoxide and H 2 O 2 can also react to form hydroxyl radicals which destroy membrane lipids and chlorophyll by hydrogen abstraction, forming lipid peroxides ŽHalliwell, 1982.. Van Rensen Ž1975. demonstrated that diquat has the same mode of toxic action in the green alga Scenedesmus obtusiusculus as is observed to occur in plants. SCP forms H 2 O 2 upon contact with water, and Kay et al. Ž1984. suggest that observed light potentiation of H 2 O 2 activity and its bleaching effect on algal chlorophyll indicate similar mechanisms of toxicity exist between H 2 O 2 and the bipyridylium herbicides. Kay et al. Ž1982. found that threshold toxicities of H 2 O 2 for Ankistrodesmus sp. ŽChlorophyta. was 6.8 to 10.2 mg ly1 Ž200 to 300 m M., less than 3.4 mg ly1 Ž100 m M. for Raphidiopsis sp. ŽCyanophyta: Nostocaceae., and 1.7 mg ly1 Ž50 m M. for Microcystis sp. ŽCyanophyta: Chroococcaceae., suggesting that cyanophytes may be more sensitive to H 2 O 2 than chlorophytes. In another study, Kay et al. Ž1984. found that chlorophyll loss Žbleaching. occurred in Ankistrodesmus sp. and Raphidiopsis sp. after 48 h with 200 m M H 2 O 2 at 90 m mol my2 sy1 while Anabaena sp. ŽNostocaceae. experienced no significant chlorophyll destruction with 200 m M H 2 O 2 at 90 m mol my2

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sy1 . Growth of O. cf. chalybea was inhibited with 100 m M SCP Žequivalent to 32.5 m M H 2 O 2 . at 20–28 m mol my2 sy1 while 1000 m M SCP Žequivalent to 325 m M H 2 O 2 . at 20–28 m mol my2 sy1 was required to completely inhibit the growth of Anabaena sp. LP 691. H 2 O 2 tolerance may be greater for species of Anabaena than for members of Oscillatoriaceae. We found that SCP’s algicidal activity occurs quickly, e.g., within 24 h ŽFig. 2A–D.. SCP may be more ideal for use in aquaculture ponds than other ‘fast-acting’ algicides since it is not inhibitory to the same degree to all cyanobacteria and, therefore, may actually be less disruptive to the aquatic ecosystem by not eliminating all of the cyanophytes so quickly as to create low dissolved oxygen levels which could stress and kill fish. Ricinoleate is structurally similar to allelopathic compounds that have been isolated from aquatic macrophytes such as Eleocharis microcarpa ŽVan Aller et al., 1985.. These allelopathic compounds may play a role in algal diversity, succession, and inhibition in natural aquatic ecosystems. Van Aller and Pessoney Ž1982. found that under laboratory conditions the growth of cyanobacteria was inhibited to a greater extent than the growth of green algae by approximately 2 mg ly1 of potassium ricinoleate Žequivalent to approximately 6 m M as ricinoleate.. At the same treatment level in field studies, numbers of Oscillatoria spp. were reduced ŽVan Aller and Pessoney, 1982.. LCIC results indicate that ricinoleate is not selectively toxic towards O. cf. chalybea but does have differential sensitivity based on LOEC results ŽTable 3.. Growth inhibition by ricinoleate at 1 and 10 m M occurred after 24 h, and growth inhibition was greater at 10 m M than 1 m M but was still not completely inhibitory at 10 m M as evidenced by an increase in absorbance readings at 3–4 days. Tucker and Lloyd Ž1987. treated five ponds with 0.8 mg ly1 of potassium ricinoleate Žequivalent to approximately 2.4 m M ricinoleate. three times per week from May through October and found that potassium ricinoleate did not reduce the percentage of cyanobacteria in phytoplankton communities in treated ponds. This treatment regimen was greater than recommended application rates. Precipitation of ricinoleate as relatively insoluble salts of divalent cations and the lack of persistence of ricinoleate in pond water were suggested as possible reasons for the failure of ricinoleate to inhibit the growth of cyanobacteria in fish ponds, and greater application rates or more frequent applications of ricinoleate were suggested as possibilities to selectively eliminate cyanobacteria although such applications might not be economically sound ŽTucker and Lloyd, 1987.. Higher levels of ricinoleate Ž10 m M. are still not completely inhibitory to the growth of O. cf. chalybea in the laboratory, and, therefore, higher application rates of potassium ricinoleate in fish ponds might not completely eliminate cyanobacteria. Among the other phytotoxins screened, acifluorfen, norflurazon, sulfometuron, and fluridone were found to be more selectively toxic towards S. capricornutum than O. cf. chalybea ŽTable 2.. Acifluorfen exerts its effects through peroxidative damage; however, unlike diquat or paraquat, its activity is dependent upon the porphyrin pathway ŽDayan and Duke, 1996.. Fluridone used as an aquatic herbicide to control aquatic plants in canals, rivers, and lakes may also selectively kill green algae and decrease competition in the phytoplankton community for nutrients, light, and oxygen thereby resulting in an increase in the numbers of cyanobacteria which are a less favorable base for aquatic food chains ŽPaerl and Tucker, 1995.. Conversely, diclofop appears to be almost

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completely nontoxic to S. capricornutum based on a 96-h IC50 greater than 3.4 g ly1 ŽTable 4. but, as stated previously, effective toxic concentrations of diclofop towards O. cf. chalybea are above the reported toxic concentrations towards fish. Copper Control w , a chelated copper compound which is claimed to selectively kill cyanobacteria, was found to have a DS of less than one order of magnitude ŽTables 2 and 3. which indicates that O. cf. chalybea and green algae may have little differential sensitivity to copper. Maloney and Palmer Ž1956. found that copper sulfate was more toxic towards cyanobacteria than green algae, and Gibson Ž1972. found that an Anabaena sp. was more sensitive to copper than a Chlorella sp. ŽChlorophyta.. However, Fitzgerald Ž1959. reported species of Oscillatoria and Phormidium ŽCyanophyta. to be more resistant to copper sulfate than Chlorella, and Stokes Ž1981. reported strains of Scenedesmus ŽChlorophyta. to have different sensitivities to copper. These results suggest that copper offers very little toxic selectivity between cyanobacteria and green algae in general but may be selectively toxic for particular strains and species of cyanobacteria and green algae. This species- and strain-specific sensitivity to copper may be due in part to the amount of copper that is transported into the cell as explained by Butler et al. Ž1980. who found less copper in cells of a copper-tolerant Chlorella Õulgaris than a nontolerant C. Õulgaris strain. The variation of sensitivities of green algae and cyanobacteria towards copper, as well as our results showing that Anabaena sp. LP 691 fails to have the same degree of sensitivity as O. cf. chalybea towards SCP, indicate the difficulty in discovering a compound that is selectively toxic towards all members of Cyanophyta. A ‘cocktail’ algicide consisting of several phytotoxic compounds may need to be used to help selectively control the growth of cyanobacteria in aquaculture ponds. 5. Conclusions Of the compounds determined to be selectively toxic towards O. cf. chalybea, SCP appears to be the most desirable compound to pursue for use as a cyanobacterial algicide in food-fish ponds due to its environmentally-safe nature Žie. no toxic residuals are formed from the breakdown of SCP.. However, SCP may be limited in its effectiveness as a ‘general’ cyanobacterial algicide used to selectively kill all species of cyanobacteria since SCP was found to not be selectively toxic towards the cyanobacterium Anabaena sp. LP 691. Another drawback of using SCP in aquaculture ponds to help prevent off-flavor problems is the high concentration of SCP required to kill O. cf. chalybea. Other oxidizing compounds, especially environmentally-safe natural compounds, need to be screened to determine their potential for use as selective cyanobacterial algicides. Acknowledgements Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the Mississippi Agricultural and Forestry Experiment Station or by the United States Department of Agriculture Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.

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