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    No exami-nation of impacts to integrated multispecies/mul-titrophic-level assemblages, however, has beenundertaken. The objective of this study was todetermine the effects of tebuthiuron on phyto-plankton productivity and standing crops ofinvertebrates and fish in multispecies mesocosmssuch that potential impacts of this chemical brushcontrol agent on aquatic food chains may beevaluated.Materials and methodsMesocosm description and experimental designTen multispecies mesocosms were establishedoutdoors on the Texas Tech University campus.Mesocosms used in this study were constructedto stimulate, as much as was possible, isolatedpools in west Texas streams. Pools are importantin these streams since flow is usually intermittent,restricting the aquatic biota to these habitatsduring periods of no flow. Tebuthiuron concen-trations may be appreciably higher in isolatedpools as opposed to habitats with inflow andoutflow (e.g., shallow runs and riffles), enhancingthe possibility of herbicide effects on aquatic orga-nisms. Round galvanized steel tanks (2846 1 ca-pacity) were partially filled with six centimeters oflocal topsoil (90.3% sand, 3.0% silt, 6.7% clay,7.8 pH, 1.5% organic matter, and 6.1 meq/100 gsoil cation-exchange capacity). Four 0.25 m2Hester-Dendy plate invertebrate samplers (Hes-ter & Dendy, 1962) were placed on the sedimentsurface and four sediment sampling devices(1 1 plastic bags attached to 185 cm2 metalframes) were buried in the sediment of each tank.Tanks were filled with water from a local playalake, and dechlorinated municipal water was usedto maintain the initial water level at a depth of0.55 m (2083 1). Thus a relatively high surface-to-volume ratio was created in each mesocosm,similar to that found for stream pools.The outdoor location allowed exposure to areaweather conditions and colonization by indi-genous macroinvertebrates and algae. Addition-ally, each system was innoculated with approxi-mately 45 1 of water from the littoral zone of alocal eutrophic pond. Phytoplankton and inver-tebrate communities were allowed to develop na- turally through colonization and succession for 30days before the mesocosms were dosed withtebuthiuron.
    A 30-day colonization period wasdeemed adequate since other researchers, investi-gating insect recolonization of previously stressedlotic systems, found that attainment of maximumdensity on artificial substrate samplers usuallyoccurred from 21 to 30 days (Sheldon, 1977;Meier et al., 1979).One day before treatment (22 July), sixty-five1.6 cm fathead minnow (Pimephales promelas)larvae were stocked in each mesocosm to allowthe assessment of potential tebuthiuron impactson a typical west Texas stream fish species. Theaddition of this level of organization was alsoimportant because macro-secondary consumersfrequently influence the rest of the system (Pilson& Nixon, 1980; Carpenter etal., 1985).The following nominal doses of tebuthiuron(97.5 technical material) were used in thisstudy: 0 (control), 10, 70, 200, 500, and1000 #g/l. Since 200 g/l approximates thehighest transient concentration of tebuthiuron yetrecorded in surface waters (180 ug/l, ElancoProducts Company, 1982), the control and the200 pg/l treatment level were replicated (threetanks each) to allow for additional statistical com-parisons of treatment effects. The remaining fournominal treatment levels were not replicated.Data generated from all mesocosms were used incorrelation and tebuthiuron fate analyses.Water samples (1 1) were collected eight times(- 1, 1, 3, 7, 14, 34, 64, and 108 days after treat-ment [DAT]) from each mesocosm for analysis oftebuthiuron concentrations. Each sample was acomposite of 250 ml aliquots collected from thesurface of each mesocosm quadrant. Sedimentsamples were collected four times ( - 3, 7, 64, 108DAT) from each tank for tebuthiuron analysis.Tebuthiuron concentration in water was deter-mined by gas chromatography using a flame pho-tometric detector. Spiked water samples of10#,g/1 and 1000 ig/1 indicated recoveries of93% (coefficient of variation (CV) = 7.44 ) and98 % (CV = 1.96 %), respectively. Sediment tebu-thiuron residues were determined using gas chro-matography/flame photometric methods for the- 3-DAT and 7-DAT samples and gas chromato-graphy/mass spectrometric methods for the 64-DAT and 108 DAT samples. These two methodswere found to be linearly correlated (r2 = 0.96,P < 0.001) and hence were considered to supplyessentially identical analyses. Spiked sedimentsamples of 100 4g kg - ' and 500 g kg - resultedin recoveries of 58% (CV = 12.00%) and 54%(CV= 18.00%), respectively. All tebuthiuronmeasurements in water and sediment in this studywere adjusted according to these standardi-zations. Analyses were conducted by the Analyti-cal Chemistry Group of Lilly Research Labora-tories at Greenfield, Indiana, according to in-house analytical procedures (Loh et al., 1978).The adsorption of herbicide by the sediments asa function of herbicide concentration in the waterwas described by the Freundlich equation (Pecketal., 1980): x m -= kc1 /n where x/m is theamount of tebuthiuron adsorbed per unit mass ofsediment (g kg '), c is the concentration inwater (gl-'), n is a constant, and k is theFreundlich adsorption coefficient.Primary production in each mesocosm wasassessed 15 times ( - 9, - 7, - 1, 1, 2, 3, 5, 7, 11,14, 35, 42, 57, 64, 106 DAT) using the lightbottle/dark bottle technique (Vollenweider, 1974).Initial dissolved oxygen levels were measured andduplicate light and dark bottles were placed atmid-depth (0.3 m) in each tank from 1000 h to1600 h. After removal, bottles were immediatelyplaced in ice, transported to the lab, and analyzedfor dissolved oxygen content with a YSI oxygenmeter (within two hours).Density and biomass of aquatic invertebratespecies were measured four times ( - 3, 7, 64, 106DAT). Hester-Dendy samplers were removedfrom each mesocosm and all attached materialscraped and rinsed from the plates. This materialwas preserved in 70% ethanol for later identifi-cation, separation by taxa, enumeration, and dryweight biomass determination (Lind, 1979).Chironomid larvae (Diptera: Chironomidae)were the only macroinvertebrates analyzed for thefollowing reasons: 1) chironomids are abundantin most natural aquatic and semiaquatic habitatsand usually account for at least 50% of the com- bined macroinvertebrate species composition(Coffman, 1978); 2)chironomids are very valu-able as fish food organisms (Lee etal., 1980);3) chironomids often have been used as indica-tors of lentic productivity (Saether, 1980); and4) chironomids composed 99 % of the density andbiomass of the macroinvertebrate 'assemblage'collected on the artificial substrate samplers ofthis study.Fathead minnows were sampled by electro-fishing six times (3, 7, 14, 41, 56, 116 DAT). Incontrast to the other biotic components, charac-terization of the initial state of the fish componentin each mesocosm was unnecessary. Inpiduallive weights from each sample date were summedto estimate total biomass in each system.Spearman Rank Correlation analysis was usedat each sample date to determine if linear relation-ships existed between either primary production,chironomid density, chironomid biomass, or fishbiomass and tebuthiuron concentration (n = 10).Post-treatment data for each biotic component(except fish) was adjusted by subtraction of pre-treatment values. To illustrate, for a particularmesocosm during a sample date, median pre-treatment primary production was subtractedfrom the post-treatment primary productionvalue. Adjusted primary production values andcorresponding median tebuthiuron concentra-tions were used in correlation analysis at thesample date. For correlation analysis betweentwo biotic components (over all time), the medianof all adjusted values for each component wasused.The analysis of variance (ANOVA) repeated-measures procedure was employed to test fortreatment effects in phytoplankton production,chironomid density and biomass, and fishbiomass at the 200/,g 1-' nominal treatmentlevel. As with correlation analysis, all post-treat-ment values were adjusted by subtraction of pre-treatment values.Results and discussionWater quality variables were similar among meso-cosms (Table 1). Continuous evaporation andmunicipal water addition caused consistent in-creases in alkalinity, hardness, pH, and total dis-solved solids during the experimental period. De-spite these increases, all water quality values arewithin those typically observed in some westernTexas streams (USGS 1979).Tebuthiuron fate:The partitioning of tebuthiuron between waterand sediments in the mesocosms (Table 2) canbe described by the Freundlich equationx/m = 3.24c °6 8 (Peck et al., 1980). The good fit ofthe experimental data to the Freundlich model(r2= 0.83) indicates that tebuthiuron partitions inresponse to normal chemical equilibria.Whereas tebuthiuron concentrations in waterdecreased in each mesocosm over the experimen-tal period, no consistent concentration pattern insediment was observed (Table 2). At the finalsample date (109 DAT), 14.9 to 32.0% of theactual initial inoculum was unaccounted for ineach system. Although not volatile (2 x 10 - 6 mmHg at 25 C), tebuthiuron has been found to besusceptible to breakdown through both metabo-lism and direct photodecomposition (Morton &Hoffman, 1967; Elanco Products Company,1983). Since residue analysis was performed forthe parent compound only, biodegradation andphotodecomposition of the parent compoundcould account for the loss of a significant portionof the herbicide.Tebuthiuron effects:Pretreatment measurements of primary produc-tion, chironomid density, and chironomidbiomass were tested for correlations with mediantebuthiuron concentration. Although chironomiddensity (r = 0.42, p < 0.223) and biomass(r = 0.19, p < 0.608) were not correlated withmedian concentration, there was a significant ne-gative relationship of pre-treatment primary pro-duction and median concentration (r = - 0.63,p < 0.051). To compensate for this pre-treatment pattern, post-treatment primary productionmeasurements in each mesocosm were adjustedby subtraction of mesocosm median pretreatmentprimary production values. We also felt that itwas appropriate to adjust post-treatment chiro-nomid density and biomass data in the samemanner.
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