Kyle D. Hoagland

The occurrence of illicit and therapeutic pharmaceuticals in wastewater effluent and surface waters in Nebraska

The occurrence and estimated concentration of twenty illicit and therapeutic pharmaceuticals and metabolites in surface waters influenced by wastewater treatment plant (WWTP) discharge and in wastewater effluents in Nebraska were determined using Polar Organic Chemical Integrative Samplers (POCIS). Samplers were installed in rivers upstream and downstream of treated WWTP discharge at four sites and in a discharge canal at a fifth location. Based on differences in estimated concentrations determined from pharmaceuticals recovered from POCIS, WWTP effluent was found to be a significant source of pharmaceutical loading to the receiving waters. Effluents from WWTPs with trickling filters or trickling filters in parallel with activated sludge resulted in the highest observed in-stream pharmaceutical concentrations. Azithromycin, caffeine, 1,7-dimethylzanthine, carbamazepine, cotinine, DEET, diphenhydramine, and sulfamethazine were detected at all locations. Methamphetamine, an illicit pharmaceutical, was detected at all but one of the sampling locations, representing only the second report of methamphetamine detected in WWTP effluent and in streams impacted by WWTP effluent.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (I. A Model of Adhesives Based on Chemical Characterization and Localization of Polysaccharides from the Marine Diatom Achnanthes longipes and Other Diatoms)

Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cistula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHCO3-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64–70%) and fucosyl (32–42%) residues. In A. longipes polymers, 2,3-, t-, 3-, and 4-linked/substituted galactosyl, t-, 3-, 4-, and 2-linked fucosyl, and t- and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTA-soluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4f/5p-, and 3p-linked/substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized [alpha](1,2)-L-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Galactose residues were localized throughout the shafts of C. cistula and capsules of A. coffeaeformis. D-Mannose and/or D-glucose, D-galactose, and [alpha](t)-L-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorius, and Lotus tetragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.

Co-occurrence of the Cyanotoxins BMAA, DABA and Anatoxin-a in Nebraska Reservoirs, Fish, and Aquatic Plants

Several groups of microorganisms are capable of producing toxins in aquatic environments. Cyanobacteria are prevalent blue green algae in freshwater systems, and many species produce cyanotoxins which include a variety of chemical irritants, hepatotoxins and neurotoxins. Production and occurrence of potent neurotoxic cyanotoxins β-N-methylamino-l-alanine (BMAA), 2,4-diaminobutyric acid dihydrochloride (DABA), and anatoxin-a are especially critical with environmental implications to public and animal health. Biomagnification, though not well understood in aquatic systems, is potentially relevant to both human and animal health effects. Because little is known regarding their presence in fresh water, we investigated the occurrence and potential for bioaccumulation of cyanotoxins in several Nebraska reservoirs. Collection and analysis of 387 environmental and biological samples (water, fish, and aquatic plant) provided a snapshot of their occurrence. A sensitive detection method was developed using solid phase extraction (SPE) in combination with high pressure liquid chromatography-fluorescence detection (HPLC/FD) with confirmation by liquid chromatography-tandem mass spectrometry (LC/MS/MS). HPLC/FD detection limits ranged from 5 to 7 µg/L and LC/MS/MS detection limits were <0.5 µg/L, while detection limits for biological samples were in the range of 0.8–3.2 ng/g depending on the matrix. Based on these methods, measurable levels of these neurotoxic compounds were detected in approximately 25% of the samples, with detections of BMAA in about 18.1%, DABA in 17.1%, and anatoxin-a in 11.9%.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes).

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059–1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158–162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.

Effects of Solar Spectral Irradiance (Visible to UV) on a Prairie Stream Epilithic Community

The relative effects of light intensity, color, and UV radiation on the herbivory component of an epilithic food web were examined in a small spring-fed stream in the Sand Hills region of western Nebraska. Plexiglass, solar-radiation filters were suspended over artificial substrata at 4 sites with open riparian canopies. Periphyton and macroinvertebrate biomass and composition on unglazed, clay tiles were determined after 28 d of exposure to 6 treatments of spectral irradiance: green light (filters transmitted 470-600 nm), blue light (360-450 nm), red light (>600 nm), solar radiation with no UV below 380 nm (UV filter), solar radiation reduced equally for all wavelengths (neutral-density filter), and ambient solar radiation (no filter). Photon flux densities were reduced 80-93% by the colored and neutral density filters, and 10-17% by the UV filters. Unadjusted means for periphyton biomass were not significantly different among treatments, but periphyton biomass means adjusted for invertebrate herbivore densities (as a covariate) differed significantly. Consequently, direct effects of irradiance on periphyton were less apparent than indirect effects through herbivory. Both unadjusted and adjusted means for herbivore densities were significantly different among treatments, suggesting that herbivores were directly affected by irradiance, as well as indirectly through associated changes in periphyton biomass. Effects of light intensity on periphyton and herbivores were stronger (relatively greater) than effects of spectral quality of photosynthetically active radiation at a similar intensity. Periphyton and macroinvertebrate densities did not differ significantly between ambient and UV filter treatments.

Timing of Wave Disturbance and the Resistance and Recovery of a Freshwater Epilithic Microalgal Community

Microalgal communities on clay tiles in Lake McConaughy (Nebraska, USA) were subjected to simulated wave disturbance after 6, 12, 18, or 24 d of development to examine the effects of community age on resistance and recovery in these communities. Six-day communities were less resistant than older communities; diatom standing crops in 6-d communities were reduced 47.6% by disturbance. Resistance of older communities was apparently a function of increased mat stabilization by diatom mucilages and overlying Oscillatoria surface layers. Diatom density reductions in 6-d communities were due to differential removal of large, chain-forming species of Fragilaria, and solitary, motile Navicula and Nitzschia. Sediment-trap data showed that Fragilaria and Navicula resettled quickly, but that once removed, many Nitzschia were not readily replaced through immigration. Differential removal of diatom taxa by disturbance at 6 d, as well as differential settling of taxa following disturbance affected community recovery patterns. Simulated wave disturbance also caused short-term increases in growth rates of four common taxa in 6-d communities. After 24 d, phaeophytin content of control communities exceeded chlorophyll a concentrations, indicating senescence of algal cells. Communities disturbed after 18 or 24 d had chlorophyll a concentrations similar to controls, but significantly lower phaeophytin content, suggesting that wave disturbance delayed senescence within algal mats. We conclude that wave disturbance can affect resistance and recovery of epilithic algal communities in lentic systems and that timing of disturbance influences community response.

SHORT‐TERM STANDING CROP AND DIVERSITY OF PERIPHYTIC DIATOMS IN A EUTROPHIC RESERVOIR1

Adverse weather and resultant turbulence caused sloughing of attached diatom communities during the first two weeks of colonization on artificial substrates. In contrast to Pacific macroalgal systems, older communities (8–12 d) were the most affected by wave action. Ash‐free dry weight of the periphyton and diatom cell density generally increased, except following periods of high turbulence when losses of up to 80% occurred. The maximum cell density (4 × 105 diatoms · cm−2) and ash‐free dry weight (2.9 mg · cm−2) are among the highest reported for similar conditions. Percent transmittance through the growth layer correlated significantly with cell density and ash‐free dry weight and is recommended as a rapid method for measuring the relative accumulation of periphyton. Diatom diversity generally increased throughout the study period and did not clearly reflect the effects of disturbance as did standing crop. Comparisons of diatom diversity and community composition indicated that the three overlapping growth series were not significantly different and samples collected on the same date were not more similar than those of identical age. Combined with previous evidence, the present study indicates that the first two weeks of substrate exposure in the summer represented a colonization phase followed by rapid microsuccession.

Genetic variation in the diatomFragilaria capucina (Fragilariaceae) along a latitudinal gradient across North America

Genetic variation in 126 clones of the planktonic diatom,Fragilaria capucina, collected from seven lakes from Manitoba to Texas, was determined by RAPD analysis. Five primers yielded 48 scorable RAPD fragments in 123 unique combinations. Patterns of genetic variation were analysed by cluster analysis, which showed that most clones grouped together according to the site from which they were collected. This pattern in genetic variation may be a result of the geographically disjunct nature of these populations and/or the different environmental conditions, especially temperature, in the different lakes.

Changes in benthic community composition in response to reservoir aging

The effects of reservoir aging on the benthic macroinvertebrate community in Pawnee Reservoir were documented by comparing species composition and biomass of samples collected from October 1991 through September 1992, to a similar survey conducted in 1968–70 by Hergenrader & Lessig (1980). Filling of the basin with sediment and associated material and the subsequent change in the benthic environment, has resulted in a relatively homogenous bottom substrate at each of the three sampling transects (dam, middle, and inflow). Sediment enrichment has limited the benthic fauna to species tolerant of brief periods of bottom anoxia and increased levels of organic matter, which has resulted in the disappearance of many taxa and a decrease in the abundance of remaining invertebrates. Significant differences in total biomass were found at each transect, as well as for the whole lake, between study periods. The dam, middle, inflow and total biomasses for the 1968–70 study period were 2.4, 1.5, 2.3 and 2.0 g m−2, respectively, compared to 0.2, 0.2, 0.3, and 0.2 g m−2 for the 1991–92 study period. The total disappearance of eight invertebrate taxa, in particular two sphaerid clam species, and significant declines in other dominant taxa such as Chaoborus punctipennis and Chironomus sp. accounted for these major differences in biomass between study periods. Reductions in the number of taxa present has resulted in an increase in benthic faunal similarity at each transect, with tubificid oligochaetes, Coelotanypus sp., C. punctipennis, and Chironomus sp., comprising 90% of both the total density and biomass of benthic invertebrates in Pawnee Reservoir.

EFFECTS OF CELL DENSITY, TEMPERATURE, AND LIGHT INTENSITY ON GROWTH AND STALK PRODUCTION IN THE BIOFOULING DIATOM ACHNANTHES LONGIPES (BACILLARIOPHYCEAE)

Achnanthes longipes Ag. is a marine stalk‐forming diatom that grows in dense biofilms. The effects of cell density, temperature, and light on growth and stalk production were examined in the laboratory to determine how they affected the ability of this diatom to form a biofilm. Stalk production abruptly increased when A. longipes was cultured at a density of 5.4 × 103 cells·mL− 1 , with a lag before stalk production occurring in cultures initiated at lower densities. Growth occurred at all temperatures from 8 to 32° C, with maximum growth at 26° C. Growth rate was light saturated at 60 μmol photons·m−2·s− 1 . Stalk production was determined as the proportion of cells producing stalks and stalk length in response to various temperatures and light intensities at high (5000 cells·mL− 1 ) and low (500 cells·mL− 1 ) densities. More cells formed stalks at high density, with no difference in stalk length. The proportion of cells producing stalks was maximal at 20° C, with little change at 17–32° C. Stalk length was at a maximum between 14 and 26° C. Stalk production showed little change in response to varying light intensity. The results of an earlier investigation on the effects of bromide concentration on stalk formation were expressed as the proportion of cells forming stalks and the lengths of the stalks. Both measures of stalk production varied with bromide concentration, with maximum values at 30 mM bromide. The increased stalk production at higher densities may be a means of elevating cells above the substrate to avoid competition in the dense biofilm.