Nutrient Bioassays of Growth Parameters for Algae in the North Bosque River of Central Texas 1

NUTRIENT BIOASSAYS OF GROWTH PARAMETERS FORALGAE IN THE NORTH BOSQUE RIVER OF CENTRAL TEXAS1

Jimmy S. Millican, Jeffrey A. Back, and Anne M.S. McFarland2

ABSTRACT: Nutrient dose-response bioassays were conducted using water from three sites along the NorthBosque River. These bioassays provided support data for refinement of the Soil and Water Assessment Tool(SWAT) model used in the development of two phosphorus TMDLs for the North Bosque River. Test organismswere native phytoplanktonic algae and stock cultured Pseudokirchneriella subcapitata (Korshikov) Hindak.Growth was measured daily by in vivo fluorescence. Algal growth parameters for maximum growth (lmax) andhalf-saturation constants for nitrogen (KN) or phosphorus (KP) were determined by fitting maximum growthrates associated with each dose level to a Monod growth rate function. Growth parameters of native algae werecompared between locations and to growth parameters of P. subcapitata and literature values. No significant dif-ferences in half-saturation constants were indicated within nutrient treatment for site or algal type. Geometricmean KN was 32 lg ⁄ l and for KP 7 lg ⁄ l. A significant difference was detected in maximum growth rates betweenalgae types but not between sites or nutrient treatments. Mean lmax was 1.5 ⁄ day for native algae and 1.2 ⁄ dayfor stock algae. These results indicate that watershed-specific maximum growth rates may need to be consideredwhen modeling algal growth dynamics with regard to nutrients.

(KEY TERMS: algae; bioassay; nutrients; rivers ⁄ streams; eutrophication; Pseudokirchneriella subcapitata;simulation.)

Millican, Jimmy S., Jeffrey A. Back, and Anne M.S. McFarland, 2008. Nutrient Bioassays of Growth Parametersfor Algae in the North Bosque River of Central Texas. Journal of the American Water Resources Association(JAWRA) 44(5):1219-1230. DOI: 10.1111 ⁄ j.1752-1688.2008.00219.x

INTRODUCTION

In 2001, two total maximum daily loads (TMDLs)for soluble reactive phosphorus were adopted forclassified segments along the North Bosque Riverin response to water quality concerns regardingexcessive nutrients and algae (TNRCC, 2001). TheseTMDLs were approved by the U. S. Environmental

Protection Agency (USEPA), but public concernshave lead to a reevaluation. The Soil and WaterAssessment Tool (SWAT) was used to support thedevelopment of these TMDLs (Santhi et al., 2001,2002), and as part of the TMDL reevaluation, newinformation regarding site-specific nutrient growthparameters for algae will be incorporated intoSWAT for simulating the North Bosque River(Hauck et al., 2003).

1Paper No. JAWRA-07-0066-P of the Journal of the American Water Resources Association (JAWRA). Received May 26, 2007; acceptedFebruary 6, 2008. ª 2008 American Water Resources Association. Discussions are open until April 1, 2009.

2Respectively (Millican and McFarland), Senior Research Associate, Research Scientist, Texas Institute for Applied EnvironmentalResearch, Tarleton State University, Stephenville, Texas 76402; and (Back) Graduate Research Assistant, Center for Reservoir and AquaticSystems Research, Department of Biology, Baylor University, One Bear Place #97388, Waco, Texas 76798-7388 (E-Mail ⁄ McFarland:mcfarla@tiaer.tarleton.edu).

JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION 1219 JAWRA

JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION

Vol. 44, No. 5 AMERICAN WATER RESOURCES ASSOCIATION October 2008

Parameter fitting is important in deterministicmodels, such as SWAT, because poorly fitted rate con-stants can lead to faulty results even when the gen-eral relationship is correctly defined (Bowie et al.,1985). Averages are often used in modeling, but site-specific values are preferred for better parameterestimates. Within SWAT, the biomass concentrationof phytoplanktonic algae is related to growth rateparameters lmax – the maximum specific algal growthrate (per day), KN – the Michaelis-Menton half-satu-ration constant for nitrogen (N), and KP – the Micha-elis-Menton half-saturation constant for phosphorus(P) as described by a Michaelis-Mention function(Neitsch et al., 2002). The Michaelis-Menton functionis often referred to as a Monod function when appliedto biological growth (see Tilman, 1982) and has thefollowing simple form:

l ¼ lmax�S

KS þ S; ð1Þ

where l equals the growth rate as a function of themaximum growth rate (lmax), the external nutrientconcentration (S), and the half-saturation constant ofthe nutrient of interest (KS), in this case either N orP. Other models, such as the Droop model that relieson intracellular concentrations of nutrients, may bebetter predictors of algal growth, but the Monodmodel is often preferred because it directly relatesalgal growth to an environmental parameter(Sommer, 1991).

The Monod model is widely accepted for describingalgal growth dynamics, but a wide range of parame-ter values for lmax and S have been produced thatvary greatly by algal species (e.g., Bowie et al., 1985;Sommer, 1991; Sterner and Grover, 1998). Dose-response bioassays are used to estimate lmax, KN,and KP by evaluating the growth rate of algae undervarying concentrations (both limiting and nonlimit-ing) of the nutrient of interest while maintaining allother nutrients as nonlimiting (Miller et al., 1978).Pseudokirchneriella subcapitata (formerly known asSelenastrum capricornutum) is often used for algalgrowth experiments for comparative purposesbetween water bodies. Within a given stream system,the aggregate growth response of the native algalcommunity may be more important than that of indi-vidual species. The use of native algae in dose-response bioassays provides a more realistic growthresponse that also allows for shifts in species abun-dance and dominance with manipulation of the nutri-ent environment (Lopez and Davalos-Lind, 1998).

While a number of studies on the North BosqueRiver have examined the in situ growth dynamicsfor periphyton in response to increasing nutrient

concentrations (Matlock et al., 1998, 1999; McFarlandet al., 2004), only limited efforts have been made tolook at phytoplankton growth. Growth responses ofphytoplankton have been evaluated indirectly throughrelating in-stream chlorophyll-a to nutrient concentra-tions, but this evaluation considers an aggregation ofdata over time and across stream locations, creatingconsiderable scatter (McFarland et al., 2000; Kieslinget al., 2001). This scatter likely represents theresponse of different algal types within chlorophyll-asamples taken at various locations along the river rep-resenting a range of nutrient conditions.

Although P was identified as the nutrient mostlimiting algal growth for the TMDLs, N or N and Plimitation occurs in phytoplankton (Kiesling et al.,2001) and periphyton (McFarland et al., 2004) at loca-tions along the North Bosque River. In freshwaters, Pgenerally limits algal growth, but N-dependence,although less studied, is an important secondary limi-ting nutrient (Elser et al., 1990). Because theseTMDLs focus specifically on the North Bosque River,it was important to determine growth parameters forthe community of native algae within the river ratherthan single species. The objective of this study was todetermine growth response parameters for nativealgae to N and P at three locations along the NorthBosque River. These parameters were compared todetermine if they differed by location on the riverand to parameters measured for a stock culture of P.subcapitata. To mimic river conditions more closely,ambient river water was used in this study.

SITE DESCRIPTIONS

Three sites were selected representing a variety ofconditions along the North Bosque River (Figure 1).All three sites are also designated monitoring sites bythe Texas Commission on Environmental Quality(TCEQ). The upper most site, BO070 (TCEQ site11961), was located on the North Bosque River in thecity park of Hico, Texas. The drainage area aboveBO070 covers about 93,100 ha. The second site,BO080 (TCEQ site 11960), represented mid-river con-ditions and was located on the North Bosque River atthe crossing of Farm to Market (FM) 216 in Iredell,Texas. Site BO080 is about 25 river kilometers down-stream of site BO070 and has a drainage area ofabout 146,000 ha. The most downstream site wasBO090 (TCEQ site 11956) located on the North Bos-que River in Clifton, Texas at the crossing of FM 219.Site BO090 is about 56 km downstream of siteBO080 and has a drainage area of about 253,000 ha.Phosphorus concentrations generally decrease from

MILLICAN, BACK, AND MCFARLAND

JAWRA 1220 JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION

upstream to downstream with the highest P concen-trations occurring at BO070 of the three sitesselected (Adams et al., 2005).

METHODS

Seasonal nutrient dose-response bioassays wereconducted using water from the three stream sites oneight occasions between February 2004 and July2005 largely following SM8111B for the growth ofalgae using chlorophyll-a fluorescence (APHA, 1999).The method was adapted to obtain growth responsedata for native algae as outlined in Lopez and Dava-los-Lind (1998) with a stock culture of the alga P.subcapitata included with each bioassay experiment.The algal assay method was also modified to use testtubes rather than flasks for culturing algae as out-lined in Davalos et al. (1989). A further modificationinvolved the use of dilution water at sites with highbackground nutrient concentrations. High back-

ground nutrient concentrations, if not diluted, hadthe potential of providing nutrient saturated condi-tions, which would negate the ability to determineexpected low KN and KP concentrations. Using dilu-tion water when high ambient nutrient concentra-tions were present was also analogous to determiningthe mitigating effects of lowering the nutrient concen-tration (N or P) on algal growth rates of native algaeat these high nutrient sites. Dilution water was com-prised of ambient water from sites with lower nutri-ent concentrations following United StatesEnvironmental Protection Agency (USEPA) proce-dures (USEPA, 2002). Ambient water was used asthe dilution water to reduce changes in parameters,such as pH and specific conductance, which couldaffect algal growth rates. Historical instream mea-surements indicated fairly similar pH values for allthree stream sites (median values 8.1-8.4), althougha decrease in specific conductance was noted fromupstream (median 602 lmhos ⁄ cm) to downstreamsites (median 434 lmhos ⁄ cm) for grab samples col-lected between 2000 and 2004 (Adams et al., 2005).Sonde measurements of dissolved oxygen, pH, specific

FIGURE 1. Location of Algal Bioassay Sampling Sites Along the North Bosque River.

NUTRIENT BIOASSAYS OF GROWTH PARAMETERS FOR ALGAE IN THE NORTH BOSQUE RIVER OF CENTRAL TEXAS

conductance and water temperature were taken in-stream near the time when ambient samples werecollected to evaluate differences in these parametersprior to using site’s stream water as dilution water.

The overall algal bioassay procedure had four gen-eral steps: (1) collection of ambient water and nativealgae, (2) filtration of ambient water and concentra-tion of native algae, (3) preparation of dose-responsetreatment dilutions and concentrations includingnutrient analysis of ambient water, and (4) incuba-tion and measurement of algal growth.

Collection of Ambient Water and Native Algae

Samples were collected at each site on eight dates:February 2, May 11, June 21, August 4, and Novem-ber 9, 2004 and February 8, April 26, and July 12,2005. Enough river water was collected to fill a mini-mum of 12 one-liter plastic bottles. The river waterwas field-filtered through a 63-lm sieve to removezooplankton. An additional sample of river water wascollected and returned to the lab for analysis ofnitrite plus nitrate N (NO2-N + NO3-N) and solublereactive P measured as orthophosphate-P (PO4-P) todetermine background concentrations of soluble Nand P at each site. NO2-N + NO3-N was analyzedusing USEPA method 353.2 via colorimetric cadmiumreduction using an autoanalyzer (USEPA, 1983).PO4-P was analyzed colorimetrically using a spectro-photometer following EPA method 365.2 (USEPA,1983). Although ammonia (NH3-N) is also a form ofsoluble N available for algal growth, previous waterquality analyses at these sites indicated that NO2-N + NO3-N on average comprises over 80 percent ofthe soluble N present and that NH3-N concentrationsgenerally occur below the laboratory method detec-tion limit (see Adams et al., 2005). It is noted thatpreferential uptake of ammonium over nitrite andnitrate by algae occurs (e.g., Dortch, 1990) andemphasis on NH3-N would be important if it were thedominant N species as found for example in the PortAdelaide River estuary (Ault et al., 2000). BecauseNO2-N + NO3-N was the dominant form of soluble Nat these sampling sites, the analysis of NH3-N wasnot considered in the treatment calculations to allowa more timely turn around from the laboratory of thesoluble nutrient analysis.

Filtration of Ambient Water and Concentration ofNative Algae

Eight liters of river water from each site wasvacuum filtered through a 47-mm diameter nylonmembrane filter (0.45 lm pore size). The vacuum

pressure was not directly monitored, but care wastaken to filter the algae under gentle pressure tominimize cell damage. About 10-15 hours wereneeded to filter the 24 l of water and nearly the samepressure was used for each filtration. Algae were har-vested from the filter by gently scraping algae with asterile stainless steel spatula into a beaker. To ensurethat a sufficient amount of native algae had beenharvested, 2 ml of the inoculum was pipetted into atest tube containing 28 ml of reverse osmosis water.If a minimum fluorescence of 0.200 fluorescence units(fu) was not attained, additional river water was fil-tered and native algae collected until the desired fluo-rescence response was achieved. This procedure fornative algae was implemented after the first bioassayexperiment in February 2004, when it was found thatlow algae concentrations were most likely responsiblefor very low fluorescence readings and erratic growthrate patterns observed for site BO090 treatments.Fluorescence readings for N and P treatments ofnative algae for the February 2004 trial for BO090were well below 0.2 fu for several days and showed agrowth pattern that undulated from day to dayrather than following the expected exponentialgrowth pattern.

Preparation of Dose-Response Treatment Dilutionsand Concentrations

Ambient nutrient concentrations for each site forPO4-P and NO2-N+NO3-N were used to determinedilutions and concentrations needed to reach targetdose concentrations for N and P treatments. A seriesof six doses for the N treatment were prepared thattypically ranged from 50 to 2000-lg ⁄ l N (Table 1).Phosphorus treatments consisted of seven separatedoses that typically ranged from 10 to 250-lg ⁄ l P.The highest target concentrations for N and P wereconsidered in excess to meet maximum algal growthassuming no other limiting factors. Specific targetconcentrations were adjusted between experiments tohelp obtain the best growth fit response given thespace limitations within the incubator. Ambientwater from each site spiked with a concentration ofP, N, or both P and N equal to the highest target con-centration was used to either concentrate or dilutenutrients to achieve target dose concentrations.Sodium nitrate for N and sodium phosphate dibasicheptahydrate for P were used to produce the nutrientspiked ambient water.

All treatments were implemented in 30-ml Pyrex�test tubes. Each treatment test tube contained 28 ml ofambient water, which had the limited nutrient (N or P)at a specific dose and the other nutrient (non-limited)provided at the highest target concentration, and 2 ml

of non-limited nutrient spiked algal inoculum. If theambient concentration of a nutrient was lower thanthe target dose concentration, the N and P spikedambient water was added in the appropriate amountto reach the target dose concentration. If the ambientconcentration was higher than the target dose concen-tration, then dilutions were accomplished by addingambient water spiked with the non-limited nutrientfrom the site with the lowest concentration of thenutrient being varied or by the addition of reverseosmosis water spiked with the non-limited nutrient.Reverse osmosis water spiked with the non-limitednutrient was used for dilution of ambient water onlywhen no other site had an ambient nutrient concentra-tion low enough to effectively dilute the treatment todesired dose concentrations. Ambient water was pri-marily used as dilution water to minimize changes inbackground parameters, such as pH or specific conduc-tance, which may alter algal growth based on instreammeasurements when ambient water was collected.Treatments were not evaluated after dilution forchanges in these parameters. Regardless of whether atreatment required dilution or concentration of nutri-ents, the non-limited nutrient was always provided atthe highest target concentration, which was consideredto be in excess for maximum algal growth assuming noother limiting factors.

Native algae treatments for each site were repli-cated five times. A separate set of treatments wereinoculated with the cultured algae P. subcapitata andwere prepared in the same manner except that eachtreatment was not replicated because of limited spacewithin the incubator. Controls containing 28 ml of

reverse osmosis water and 2 ml of native algae orP. subcapitata inoculum spiked with the non-limitednutrient (N or P) were prepared for each site. Foamplugs, which allowed for air diffusion yet preventedspillage, were used as caps for each test tube.

Incubation and Measurement of Algal Growth

For incubation, the test tubes were placed on clearPlexiglass� slant racks. The incubator was set to thecurrent light ⁄ dark cycle as indicated on the U.S.Naval Observatory website (http://aa.usno.navy.mil/data/docs/RS_OneDay.php) for Stephenville, Texasand the mean of the ambient water temperaturesrecorded on the day of sampling for the three sites(Table 2). Light intensity levels were above light sat-uration for photosynthesis.

Raw fluorescence readings were taken daily for eachtreatment using a model 10-AU-005-CE (TurnerDesigns) fluorometer equipped with a blue mercury

TABLE 1. Target Dose Concentrations by Assay Date for N and P.

Nutrient

Target Dose Concentrations by Assay Date (lg ⁄ l)

February2004

May2004

June2004

August2004

November2004

February2005

April2005

July2005

Nitrogen 0 0 0 0 0 0 0 050 50 - - - 50 50 50

100 100 100 100 100 100 100 100200 200 200 200 200 200 200 200300 300 300 300 300 - - -500 500 500 500 500 500 500 500

1000 1000 1000 1000 1000 1000 1000 1000- - 2000 2000 - 2000 2000 2000- - - - 2500 - - -

Phosphorus 0 0 0 0 0 0 0 010 10 10 10 10 10 10 1015 15 15 15 15 15 15 1530 30 30 30 30 30 30 3050 50 50 50 50 50 50 50

- - 75 75 75 75 75 75100 100 100 100 100 100 100 100250 250 250 250 - 250 250 250500 500 - - 500 - - -

TABLE 2. Incubator Settings for Algae Bioassay Experiments.

Sampling DateLight

(h)Dark

(h)Temperature

(�C)

February 2, 2004 10.95 13.05 7.3May 11, 2004 13.83 10.17 23.6June 21, 2004 14.16 9.84 29.3August 4, 2004 13.77 10.23 30.9November 9, 2004 10.79 13.21 16.6February 8, 2005 10.92 13.08 10.8April 26, 2005 13.59 10.41 19.3July 12, 2005 14.08 9.92 29.3

vapor lamp, a 436-nm excitation filter, a 680-nm emis-sion filter, and one neutral density square reference fil-ter. Prior to placing test tubes in the fluorometer,algae were evenly distributed within the test tube byusing a mini-vortexer. Daily readings were taken untilalgal growth either stabilized or started to decline asindicated by changes in the fluorescence readings.

DATA ANALYSIS

To determine the maximum specific growth ratefor each N or P dose (ldmax) of native algae or P. sub-capitata, the largest specific growth rate (ld) for eachreplicate within a treatment was determined andaveraged across replicates to obtain ldmax. Beforeevaluating the data for ld, the raw fluorescence val-ues of replicates for each dose were graphed on a nat-ural log scale and plotted over time to view the datafor potential outliers.

To determine if results from a replicate were an‘‘outlier’’ with regard to their impact on estimatingthe maximum growth rate, data were visuallyassessed by overlaying results from all five replicatesfor a given treatment. If the trajectory or slope of flo-rescence readings for a given replicate was obviouslydifferent than the other replicates, the replicate wasflagged as a potential outlier. Data results and labnotes were then evaluated to see if there were anyunusual observations noted with the replicate, andbasic statistics for the maximum slope were calcu-lated with and without the outlier observationbetween replicates. If there was not a clear reasonbased on lab notes to remove a replicate, best profes-sional judgment was used to determine if the flaggedreplicate inordinately influenced the calculated slopefor removal as an outlier. About 3% of project repli-cates were removed as outliers prior to calculatingthe largest specific growth rate for each treatment.

After removing outliers, if necessary, dates whenlargest ld occurred for each replicate were deter-mined by visually evaluating each plot for when thesteepest slope occurred. On a natural log scale, thesteepest slope closely approximates exponentialgrowth of the algae and generally occurred betweenday 1 and day 5. The two or three points defined in astraight line representing the steepest slope wereused to calculate the largest ld for a dose-replicate asfollows:

ld ¼ LnðF2=F1Þt2 � t1

; ð2Þ

where ld is the specific growth rate (per day) for agiven replicate, t1 is the first day selected, t2 is the

second day selected, F1 is the fluorescence on t1, and F2

is the fluorescence on t2. These slopes representing thelargest ld for each replicate were averaged across rep-licates to represent the maximum growth (ldmax) foreach dose.

The ldmax associated with each dose for a treat-ment was plotted (e.g., Figure 2), and the SAS PROCNLIN procedure was used to fit initial parameters forthe Monod growth model (SAS, 2000). For the exam-ple shown in Figure 2, ldmax represents l, lmax(unadj)

represents lmax, and the N dose concentrations repre-sent S from Equation (1). Of note, lmax is representedas an unadjusted value (lmax(unadj)), because for usein SWAT, l is adjusted for a standard photoperiodand temperature.

Estimated lmax values were adjusted for photope-riod and water temperature prior to comparison withliterature values using equations obtained from theSWAT theoretical documentation (Neitsch et al.,2002).

Photoperiod adjustments were performed as fol-lows:

lmaxðday adjÞ ¼ lmaxðunadjÞ�ð24=TDLÞ; ð3Þ

where lmax(day_adj) is adjusted for daily photoperiodand TDL is the total photoperiod or day length.

Temperature adjustments were made on lmax(day_adj)

using the following equation to obtain the adjustedlmax assuming a base temperature of 20�C:

lmax ¼lmaxðday adjÞ

1:047ðT�20Þ

� �; ð4Þ

FIGURE 2. Example Plot Showing Use of the Monod Function toEstimate Dose-Response Growth Rate Parameters. The maximumspecific growth rate (ldmax) was measured for individual dosetreatments. The horizontal dashed line represents the estimate oflmax(unadj) (1.43 ⁄ day) unadjusted for temperature and photoperiodand the vertical dashed line represents the estimate of half-satura-tion constant, KN (71 lg ⁄ l).

where T is incubator temperature used for each bio-assay experiment based on the mean ambient watertemperature.

Estimated growth rate parameters lmax, KN, andKP were compared between sites and algae types(native or stock culture) using analysis of varianceprocedures. The SAS UNIVARIATE procedure wasused to confirm that the dose-response data were nor-mally distributed based on site and nutrient type(SAS, 2000). The Hartley’s test was also applied toevaluate for equal variances (Ott, 1984). To be nor-mally distributed the half-saturation constants weretransformed to their natural log value. The analysisof variance test was implemented using the SASGLM procedure. A two-way ANOVA of site and algaltype was conducted by nutrient that included evalua-tion of an interaction factor between site and algaltype. Significance was indicated at a = 0.05. Theinteraction factor between site and algal was not sig-nificant, so significance by site and algal type wereconsidered separately, assuming the full model wassignificant. For lmax, a further evaluation was doneafter the initial two-way ANOVA to evaluate if differ-ences occurred between N and P treatments. If a sig-nificant difference (p < 0.05) existed for lmax and Kvalues between sites, then the Tukey’s honestly sig-nificant difference test was implemented to determinewhich sites were different from one another.

RESULTS AND DISCUSSION

As noted in the methods section, the fluorescencereadings for February 2004 for native algae at siteBO090 were very low and showed erratic patternsindicating a lack of exponential growth, which didnot occur with the P. subcapitata treatments.Because exponential growth did not occur, the Febru-ary 2004 BO090 native algal treatments could not beincluded in the growth parameter analyses. Correc-tive action to avoid this problem was taken by imple-menting procedures establishing minimum nativealgae fluorescence values for all other experimentdates (see Methods).

Ambient River Water Concentrations

Ambient nutrient concentrations for PO4-P wereconsistently highest at site BO070 and ranged from64 to 340 lg ⁄ l (Table 3). At BO080, PO4-Pconcentrations ranged from 1 to 54 lg ⁄ l, while atBO090, PO4-P concentrations ranged from 3 to11 lg ⁄ l. The NO2-N + NO3-N concentrations showed

a less consistent pattern across sites. The highestand lowest concentrations occurred at BO080 withvalues ranging from 9 to 2080 lg ⁄ l. Concentrations ofNO2-N + NO3-N at BO070 ranged from 20 to683 lg ⁄ l, while concentrations at BO090 ranged from30 to 502 lg ⁄ l (Table 3).

Half-Saturation Constant Values

The N half-saturation constants (KN) were similarbetween native algae and P. subcapitata cultures,ranging from 200 lg ⁄ l to <10 lg ⁄ l (Figure 3). By sam-pling date, the KN for native algae showed less varia-tion than for P. subcapitata algae between sites,although temporal patterns in KN showed some simi-larities for the two algal types. Slightly higher KN

values were observed in May 2004 at all three sitesand again in April 2005 at BO070 (Figure 3a). For P.subcapitata, slightly elevated KN values were indi-cated in June 2004 and April 2005 for sites BO070and BO090 and for BO080 in August 2004 (Fig-ure 3b).

Phosphorus half-saturation constants (KP) rangedfrom 20 to 1 lg ⁄ l and were about an order of magni-tude less than KN values (Figure 4). Generally, higherKP concentrations occurred in the spring and summerthan in the winter. The highest KP for native algae

TABLE 3. Ambient River Water Concentrations forSoluble N and P Used to Set Treatment Concentrations.

Sampling Date SiteNO2-N +

NO3-N (lg ⁄ l)PO4-P(lg ⁄ l)

February 2, 2004 BO070 20 340BO080 9 3BO090 112 3

May 11, 2004 BO070 151 143BO080 14 54BO090 30 6

June 21, 2004 BO070 389 285BO080 12 95BO090 58 11

August 4, 2004 BO070 205 199BO080 2080 1BO090 122 5

November 9, 2004 BO070 145 252BO080 228 21BO090 343 9

February 8, 2005 BO070 112 74BO080 919 8BO090 502 4

April 26, 2005 BO070 683 85BO080 300 8BO090 328 4

July 12, 2005 BO070 85 64BO080 14 4BO090 299 4

occurred in June 2004 and July 2005 for site BO070,August 2004 and April 2005 for site BO080, and June2004 and April 2005 for site BO090 (Figure 4a). Thehighest KP concentrations for P. subcapitata occurredin June 2004 and July 2005 at site BO070, June 2004and April 2005 for site BO080, and August 2004 andApril 2005 at site BO090 (Figure 4b).

Seasonal variation in K values was anticipated,because dominant algal species will vary with temper-ature. A more intensive study over multiple seasonswould be needed to clearly evaluate potential seasonalfluctuations. Because the SWAT model currently usesa constant value for KN and KP and does not considerseasonal differences, KN and KP concentrations werecompared across sampling dates to determine if therewere significant differences between sites and betweenthe native and P. subcapitata algal species (Table 4).

Geometric mean KN values from both the nativealgae and P. subcapitata inoculated N treatmentswere well within the range suggested by Neitsch

et al. (2002) of 10-300 lg ⁄ l as potential parametervalues for SWAT (Table 4). The mean of the com-bined KN for all three sites for native algae wasapproximately 50% less than KN for P. subcapitata.By site, geometric mean KN values from native algaeN treatments were about 50% less for BO070 andBO080, and 45% less for BO090 when compared toP. subcapitata N treatments. It is possible thatbecause the cultured algae were maintained underoptimal nutrient conditions prior to the bioassay thatluxury uptake of nutrients occurred. If so, this luxuryuptake would have given the cultured algae anadvantage over the native algae under nutrient limit-ing conditions. Minimum KN values obtained from Ntreatments for both algae types were below the rangegiven in the SWAT manual for all sites except for siteBO090 P. subcapitata inoculated treatments.

Geometric mean KP concentrations for P dose-response treatments were similar between both algaetypes (Table 4), and all were within the range given inthe SWAT guidance of 0.001-0.05 mg ⁄ l (Neitsch et al.,2002). The KP concentrations obtained from all sitesfor native algae P treatments ranged from 1 to 20 lg ⁄ lwith a mean of 7 lg ⁄ l. Phosphorus treatments inocu-lated with P. subcapitata had KP values that rangedfrom 1 to 26 lg ⁄ l with a mean of 6 lg ⁄ l. Geometricmean KP values for native algae at sites BO080 andBO090 were about 50% lower than KP values forP. subcapitata. Unlike sites BO080 and BO090, thegeometric mean KP value at BO070 was slightly largerfor native algae than P. subcapitata treatments.

Although a wide variability occurred in KN and KP

values, statistically no significant differences(p > 0.05) were indicated for KN or KP concentrationsbetween sites or algae types when compared acrosssampling dates. While it is generally accepted that

FIGURE 3. Estimated Half-Saturation Constants (KN)for N Dose-Response Algal Bioassay Experiments by

Sampling Date for (a) Native and (b) P. subcapitata Algae.

FIGURE 4. Estimated Half-Saturation Constants (KP)for P Dose-Response Algal Bioassay Experiments bySampling Date for (a) Native and (b) P. subcapitata.

TABLE 4. Geometric Mean, Minimum, and Maximum Half-Saturation Estimates for Native Algae and P. subcapitata for

Individual Sampling Sites and All Sites Combined.

Site Statistic

Native Algae P. subcapitata

(lg ⁄ l)KP

(lg ⁄ l)KN

(lg ⁄ l)KP

(lg ⁄ l)

All sites Geometric mean 22 7 44 6Minimum 3 1 4 1Maximum 180 20 197 26

BO070 Geometric mean 31 7 61 6Minimum 4 2 7 1Maximum 142 18 197 26

lmax will increase with increasing temperatures, therelationship of K values with temperature is lessclear, but this relationship is included in some physi-cal models (e.g., Aksnes and Egge, 1991). KN indi-cated no significant relationships with temperature,while KP indicated marginal positive correlations forsites BO070 (r = 0.66, p = 0.0767, n = 8) and BO080(r = 0.57, p = 0.1401, n = 8). Sterner and Grover(1998) found a significant positive relationship of KN

with temperature, but also found that inclusion of atemperature dependence for KN only marginallyimproved their ability to predict algal growth rates.

Maximum Growth Rates

Variation in maximum growth rates was anticipatedbecause of variations in dark light cycles in the incuba-tor associated with day length and ambient water tem-perature. Higher maximum growth rates wereexpected with longer photoperiods and higher temper-atures and were adjusted for using Equations (3 and 4)prior to conducting statistical comparisons. Maximumgrowth rates prior to adjustment [lmax(unadj)] generallyshowed the highest values in August 2004 and the low-est values in February 2004 and 2005 because ofextremes in temperature and photoperiod (Figure 5).Adjusted lmax values showed relatively little variationbetween sites or algal types (Figure 6), althoughbetween dates, there appeared to be a slight drop inthe adjusted lmax values in June 2004.

Maximum growth rates (lmax) for native algae ran-ged from 0.84 to 2.49 ⁄ day for both N and P treat-ments (Table 5). Native algae produced similar meanlmax results of 1.48 ⁄ day for N treatments and1.51 ⁄ day for P treatments. Overall lmax results forP. subcapitata inoculated nutrient treatments weregenerally lower than those obtained for native algae.Across sites and nutrient treatment, lmax results forP. subcapitata ranged from 0.62 to 1.81 ⁄ day. Similarto native algae, overall mean lmax values for P. sub-capitata showed little variation between nutrienttreatments with values of 1.20 ⁄ day for N and1.26 day for P treatments. Mean lmax results for bothalgae and nutrient types decreased slightly fromupstream (BO070) to downstream (BO090) sites.

A relatively wide range of lmax values from litera-ture and model documentation are referenced forboth total phytoplankton and green algae in theUSEPA manual, Rates, Constants, and KineticsSurface Water Quality Modeling (Bowie et al., 1985).The lmax values for native algae obtained from thisstudy were within the range of literature sourcevalues given by Jorgensen (1979) that proposed atypical range for total phytoplankton of 0.58-3.0 ⁄ dayat 20�C. The native algae lmax values for this study

were also within the range of model documentationvalues of 0.20-8.0 ⁄ day at 20�C from Grenney andKraszewski (1981) and Baca and Arnett (1976) ascited in Bowie et al. (1985).

The range of lmax values cited in the USEPAmodeling manual (Bowie et al., 1985) for green algaewere generally higher than those obtained for theP. subcapitata inoculated treatments from this study.Typical lmax values given by Collins and Wlosinski(1983) of 0.7-2.1 ⁄ day at 20�C would encompass therange of lmax values obtained for P. subcapitata fromthis study with the exception of the minimum valuesfor site BO080 of 0.62 and 0.66 day for N and P treat-ments, respectively. The mean lmax values obtainedfor P. subcapitata treatments for all sites were withinthe above-referenced values.

In comparing lmax values, the analysis of varianceprocedure indicated no significant interactionbetween site and algae or differences between sites ornutrient treatments within an algal type. Significantdifferences were indicated for lmax between thenative and P. subcapitata algae. The native algaehad a significantly higher lmax than the P. subcapita-

FIGURE 5. Maximum Growth Rates Unadjusted for Water Tem-perature and Photoperiod [lmax(unadj)] for (a) Native Algae N Treat-ments, (b) P. subcapitata Algae N Treatments, (c) Native Algae PTreatments and (d) P. subcapitata Algae P Treatments by Sam-pling Date.

ta algae. The structure of the native algae communityvs. the single alga in the P. subcapitata treatmentmay have allowed for greater maximum growth rates

for the native algae through more efficient assimila-tion of nutrients (Piehler et al., 2002). Because nativealgae represent a community rather than a singlealga, different algal taxa may dominate at differentnutrient concentrations. These results indicate thatwatershed-specific maximum growth rate parametersmay need to be considered when modeling algalgrowth dynamics with regard to nutrients.

Estimates of lmax were more reliable than esti-mates of KN or KP. This reliability is due in part tothe inherent difficulties in preparing dose treatmentsrepresentative of the low concentrations generallyassociated with KN and KP (Grover, 1989). For KN,even though NH3-N concentrations in the ambientwater were considered a minor contribution comparedto NO2-N + NO3-N, further work should consider con-centrations of both for low concentration bioassays indefining KN. In addition, internal storage of nutri-ents, particularly for the cultured algae, may compli-cate low dose bioassays allowing growth whenexternal nutrients alone would be limiting (Droop,1973). The Monod model relates growth only to exter-nal concentrations. Although accounting for internalconcentrations would allow a more realistic represen-tation of growth dynamics, the difficulty in reliablydoing this limits the use of internal algal concentra-tions as a parameter in most widely used water qual-ity models.

While the parameter estimates for lmax, KN, andKP derived from this study could be yet furtherrefined, the derived values provide a better estimateof parameter values to be used in the SWAT modelfor the native algae in the North Bosque River thanthe broad range of literature values. This study alsoclearly indicates for lmax that native algae ratherthan ‘‘test’’ specimens, such as P. subcapitata, shouldbe used for evaluating growth parameters.

SUMMARY

Half-saturation constant mean values from nativealgae N treatments were considerably lower thanthose obtained from P. subcapitata treatments; how-ever, mean values for both algae types fell within therange of typical values given in the SWAT user’smanual. Mean K values for P treatments were typi-cally higher for native algae treatments with theexception of site BO090 when compared to P. subcapi-tata treatments. For both types of algae, mean KP

values were lower than mean KN values because ofthe lower amounts of P required for algae growth.Analysis of variance results indicated no significantdifference in K values within each nutrient treatment

FIGURE 6. Maximum Growth Rates (lmax) Adjusted for WaterTemperature and Photoperiod for (a) Native Algae N Treatments,(b) P. subcapitata Algae N Treatments, (c) Native Algae P Treat-ments and (d) P. subcapitata Algae P Treatments by SamplingDate.

TABLE 5. Mean, Minimum, and MaximumGrowth Rate (lmax) Estimates for Native Algae forIndividual Sampling Sites and All Sites Combined.

Site Statistic

Native Algae(lmax ⁄ day)

P. subcapitata(lmax ⁄ day)

PTreatment

NTreatment

PTreatment

NTreatment

All sites Mean 1.51 1.48 1.26 1.20Minimum 1.00 0.84 0.66 0.62Maximum 2.49 2.34 1.81 1.75

BO070 Mean 1.64 1.62 1.29 1.24Minimum 1.32 1.18 0.80 0.91Maximum 2.49 2.34 1.71 1.75

between either sites or algae types. The high tempo-ral variability in the data may have reduced the abil-ity to detect differences between sites or algae types,but evaluation of temporal variability was beyond thescope of this study.

These growth parameters provide a more realisticparameterization of the SWAT model for studies onthe TMDL within the North Bosque River. Similari-ties appeared to exist in lmax values obtainedbetween sites within algal type (native or cultured).When comparing different algal types, a significantdifference was detected. Larger mean lmax valueswere indicated for the native algae than the P. sub-capitata. Although not statistically different, meanlmax values for native algae appeared to decreasefrom upstream to downstream for both nutrient treat-ments. Differences in ambient nutrient concentra-tions along the North Bosque River may account forthe slight site differences noted in lmax for the nativealgae, because algae that exist in areas withhigh-nutrient concentrations tend to have higher lmax

values (Dodds, 2002). Because statistically significantdifferences were not indicated between sites orbetween nutrient treatments, a single lmax value maybe appropriate for SWAT simulations of native algalgrowth.

ACKNOWLEDGMENTS

Support for this research was provided through the Texas Com-mission on Environmental Quality, Work Order 582-6-70859-04 aspart of the project, Monitoring to Support North Bosque RiverModel Refinement. We take full responsibility for the informationpresented herein, but would like to recognize the initial guidanceand advice given us by Dr. Owen Lind and Dr. Laura Davalos-Lindof the Department of Biology at Baylor University in Waco, Texason procedures for conducting algal bioassay experiments.

LITERATURE CITED

Adams, T., N. Easterling, and A. McFarland, 2005. SemiannualWater Quality Report for the North Bosque River Watershedand Lake Waco: Monitoring Period January 1, 2000 – December31, 2004. Texas Institute for Applied Environmental Research,Technical Report 0508. Tarleton State University, Stephenville,Texas. http://tiaerweb.tarleton.edu/pdf/TR0508.pdf, accessedMay 7, 2007.

Aksnes, D.L. and J.K. Egge, 1991. A Theoretical Model for NutrientUptake in Phytoplankton. Marine Ecology Progress Series70:65-72.

APHA (American Public Health Association). 1999. Standard Meth-ods for the Examination of Water and Wastewater (TwentiethEdition). APHA, Washington, D.C.

Ault, T., R. Velzeboer, and R. Zammit, 2000. Influence of NutrientAvailability on Phytoplankton Growth and CommunityStructure in the Port Adelaide River, Australia: BioassayAssessment of Potential Nutrient Limitation. Hydrobiologia429:89-103.

Baca, R.G. and R.C. Arnett, 1976. A Limnological Model for Eutro-phic Lakes and Impoundments. Battelle, Inc., Pacific NorthwestLaboratories, Richland, Washington, D.C.

Bowie, G.L., W.B. Mills, D.B. Porcella, C.L. Campbell,J.R. Pagenkopf, G.L. Rupp, K.M. Johnson, P.W.H. Chan,S.A. Gherini, and C.E. Chamberlin, 1985. Rates, Constants, andKinetics Formulations in Surface Water Quality Modeling (Sec-ond Edition). U.S. Environmental Protection Agency,EPA ⁄ 600 ⁄ 3-85 ⁄ 040, Environmental Research Laboratory, Officeof Research and Development, Athens, Georgia.

Collins, C.D. and J.H. Wlosinski, 1983. Coefficients for Use in theU.S. Army Corps of Engineers Reservoir Model, CE-QUAL-R1.U.S. Army Corps of Engineers, Waterways Experiment Station,Vicksburg, Mississippi.

Davalos, L., O. Lind, and R.D. Doyle, 1989. Evaluation of Phyto-plankton-Limiting Factors in Lake Chapala, Mexico: Turbidityand Spatial and Temporal Variations in Algal Assay Response.Lake and Reservoir Management 5(2):99-104.

Dodds, W. K., 2002. Freshwater Ecology Concepts and Environ-mental Applications. Academic Press, London.

Dortch, Q., 1990. The Interaction Between Ammonium and NitrateUptake in Phytoplankton. Marine Ecology Progress Series61:243-264.

Droop, M.R., 1973. Some Thoughts on Nutrient Limitation inAlgae. Journal of Phycology 9:264-272.

Elser, J.J., E.R. Marzolf, and C.R. Goldman, 1990. Phosphorus andNitrogen Limitation of Phytoplankton Growth in Freshwaters ofNorth America: A Review and Critique of Experimental Enrich-ments. Canadian Journal of Fisheries and Aquatic Sciences47:1468-1477.

Grenney, W.J. and A.K. Kraszewski, 1981. Description and Appli-cation of the Stream Simulation and Assessment Model: VersionIV (SSAM IV). U.S. Fish and Wildlife Service, Instream FlowInformation Paper, Cooperative Instream Flow Service Group,Fort Collins, Colorado.

Grover, J.P., 1989. Phosphorus-Dependent Growth Kinetics of 11Species of Freshwater Algae. Limnology and Oceanography34:341-348.

Hauck, L.M., A. McFarland, A. Saleh, and G.H. Ward, Jr, 2003.Work Plan and Schedule for North Bosque River TMDL Model-ing System. Texas Institute for Applied EnvironmentalResearch, Project Report 0303, Tarleton State University,Stephenville, Texas.

Jorgensen, S.E. (Editor), 1979. Handbook of Environmental Dataand Ecological Parameters. International Society for EcologicalModeling, Pergamon Press, New York, New York.

Kiesling, R.L., A.M.S. McFarland, and L.M. Hauck, 2001. NutrientTargets for Lake Waco and North Bosque River: DevelopingEcosystem Restoration Criteria. Texas Institute for AppliedEnvironmental Research, Technical Report 0107, Tarleton StateUniversity, Stephenville, Texas. http://tiaerweb.tarleton.edu/pdf/TR0107.pdf. accessed May 7, 2007.

Lopez, E.L. and L. Davalos-Lind, 1998. Algal Growth Potential andNutrient Limitation in a Tropical River-Reservoir System onthe Central Plateau, Mexico. Aquatic Ecosystem Health andManagement 1:345-351.

Matlock, M.D., M.E. Matlock, D.E. Storm, M.D. Smolen, andW. J. Henley, 1998. A Quantitative Passive Diffusion Periphy-tometer for Lotic Ecosystems. Journal of the American WaterResources Association 34:1141-1147.

Matlock, M.D., D.E. Storm, M.D. Smolen, M.E. Matlock,A.M.S. McFarland, and L.M. Hauck, 1999. Development andApplication of a Lotic Ecosystem Trophic Status Index. Transac-tions of the American Society of Agricultural Engineers 42:651-656.

McFarland, A., R. Kiesling, and J. Back, 2004. Using Periphytome-ters to Evaluate Impairment Due to Nutrient Enrichment. Texas

Institute for Applied Environmental Research, Technical Report0404, Tarleton State University, Stephenville, Texas. http://tiaer-web.tarleton.edu/pdf/TR0404.pdf, accessed May 7, 2007.

McFarland, A., R. Kiesling, L. Hauck, and M. Matlock, 2000. Link-ing Chemical and Biological Monitoring Components in theTMDL Process. In: Proceedings of the National Water QualityMonitoring Council National Monitoring Conference 2000, Moni-toring for the Millennium. National Water Quality MonitoringCouncil, Washington, D.C., pp. 425-434

Miller, W.E., J.C. Greene, and T. Shiroyama, 1978. The Selena-strum Capricornutum Printz Algal Assay Bottle Test: Experi-mental Design, Application, and Data Interpretation Protocol.United States Environmental Protection Agency, EPA-600 ⁄ 9-78-018, Office of Research and Development, Corvallis Environ-mental Research Laboratory, Corvallis, Oregon.

Neitsch, S.L., J.G. Arnold, J.R. Kiniry, and J.R. Williams, 2002.(SWAT) Soil and Water Assessment Tool User’s Manual Version2000. Grassland, Soil and Water Research Laboratory, UnitedStates Department of Agriculture, Agricultural ResearchService, Temple, Texas.

Ott, L., 1984. An Introduction to Statistical Methods and DataAnalysis (Second Edition). Duxbury Press, Boston, Massachu-setts.

Piehler, M.F., J. Dyble, P.H. Moisander, J.L. Pinckney, and H.W.Paerl, 2002. Effects of Modified Nutrient Concentrations andRatios on the Structure and Function of the Native Phytoplank-ton Community of the Neuse River Estuary, North Carolina,USA. Aquatic Ecology 36:371-385.

Santhi, C., J.G. Arnold, J.R. Williams, W.A. Dugas, R. Srinivasan,and L.M. Hauck, 2001. Validation of the SWAT Model on aLarge River Basin With Point and Nonpoint Sources. Journal ofthe American Water Resources Association 37:1169-1188.

Santhi, C., J.R. Williams, W.A. Dugus, J.G. Arnold, R. Srinivasan,and L.M. Hauck, 2002. Water Quality Modeling of Bosque RiverWatershed to Support TMDL Analysis. In: Total MaximumDaily Load (TMDL) Environmental Regulations: Proceedings ofthe March 11-13, 2002 Conference, Fort Worth, Texas Spon-sored by the American Society of Agricultural and BiologicalEngineers, St. Joseph, Michigan, pp. 33-43.

SAS. 2000. The SAS System for Windows. Ver. 8. SAS Institute,Cary, North Carolina.

Sommer, U., 1991. A Comparison of the Droop and the Monod Mod-els of Nutrient Limited Growth Applied to Natural Populationsof Phytoplankton. Functional Ecology 5:535-544.

Sterner, R.W. and J.P. Grover, 1998. Algal Growth in Warm Tem-perature Reservoirs: Kinetic Examination of Nitrogen, Tempera-ture, Light, and Other Nutrients. Water Research 32:3539-3548.

Tilman, D., 1982. Resource Competition and Community Structure.Princeton University Press, Princeton, New Jersey.

TNRCC (Texas Natural Resource Conservation Commission). 2001.Two Total Maximum Daily Loads for Phosphorus in the NorthBosque River for Segments 1226 and 1255. TNRCC, StrategicAssessment Division, TMDL Team, Austin, Texas. http://www.tceq.state.tx.us/implementation/water/tmdl/06-bosque.html,accessed May 7, 2007.

USEPA (United States Environmental Protection Agency). 1983.Methods for Chemical Analysis of Water and Wastes. USEPA,EPA-600 ⁄ 4-79-020, revised March 1983) Environmental Moni-toring Support Laboratory, Office of Research and Development,Cincinnati, Ohio.

USEPA (United States Environmental Protection Agency). 2002.Short-Term Methods for Estimating the Chronic Toxicity ofEffluents and Receiving Waters to Freshwater Organisms(Fourth Edition). USEPA, EPA-821-R-02-013, Office of Water,Washington, D.C.

Nutrient Bioassays of Growth Parameters for Algae in the North Bosque River of Central Texas 1

Documents

1. Protists A. Algae

Coralline algae of New Zealand

Algae - Spirogyra Chara Vaucheria

Algae Britannicae

Indirect effects of algae on coral: algae-mediated, microbe-induced coral mortality

Cytoskeleton and Morphogenesis in Brown Algae

BROWN ALGAE

Manual covid versión final - Las Cañadas - Bosque de niebla

FLORÍSTICA DEL BOSQUE MESÓFILO DE MONTAÑA DE

proyecto - Fondo de Investigación del Bosque Nativo

Funciones alometricas para bosque mediterraneo chileno

Potency Database: Results ofAnimal Bioassays Published in

PROYECTO DE INVESTIGACION DIGI Valoracion Economica Agua Bosque

Algae in paleolimnological studies in Argentina

A genotoxic assessment of environmental tobacco smoke using bacterial bioassays

Report on activities of WG-2 on Bioassays - THE NORMAN

ALGAE TO ALKANES - ScholarlyCommons

FRESHWATER ALGAE OF SIKKIM

Manual-Arhuaco-para-la-Conservación-de-Bosque-Seco

Ecosistemas de páramo y bosque alto andinos - BIVICA