Chlorophyll Fluorometer for Intelligent Water Sampling by a Small Uncrewed Aircraft System (sUAS).

We describe a waterproof, lightweight (1.3 kg), low-power (∼1.1 W average power) fluorometer operating on 5 V direct current deployed on a small uncrewed aircraft system (sUAS) to measure chlorophyll and used for triggering environmental water sampling by the sUAS. The fluorometer uses a 450 nm laser modulated at 10 Hz for excitation and a standard photodiode and transimpedance amplifier for the detection of fluorescence. Additional detectors are available for measuring laser intensity and light scattering. Control of the fluorometer and communication between the fluorometer and the Raspberry Pi 4B computer controlling the sampler were provided by an Arduino microcontroller using the robot operating system (ROS). Calibrations were based on standards of dissolved chlorophyll extracted from Chlorella powder (a widely available dietary supplement). The detection limit for chlorophyll from these calibrations was found to be 0.2 µg per liter of water for a single 0.1 s differential measurement. The detection limit decreases with the square root of the integration time as expected. Detection limits increase by a factor of two to three when mounted in the sUAS due to electrical noise; sUAS acoustic noise and vibration do not appear to contribute significantly.

The influence of salinity in the domoic acid effect on estuarine phytoplankton communities.

Toxic species of the diatom genus Pseudo-nitzschia, observed worldwide from coastal waters to the open ocean, produce the neurotoxin domoic acid (DA). DA is an important environmental and economic hazard due to shellfish contamination with subsequent effects on higher trophic levels. Previous research has demonstrated that, among other environmental factors, salinity influences the abundance and toxicity of Pseudo-nitzschia. In this study, the environmental factors driving the growth of Pseudo-nitzschia and the production of dissolved DA (dDA) in North Inlet estuary were examined. The effect of salinity on the growth inhibition of phytoplankton induced by the initial presence as well as by an addition of dDA was also assessed. Initially, the diatom abundance was negatively correlated with the abundance of Pseudo-nitzschia and with the concentration of dDA. With the addition of a concentrated solution of dDA, the percent inhibition of cryptophytes and diatoms was significantly correlated with salinity and suggested a higher sensitivity to dDA at extreme salinities. These results emphasize the importance of salinity in assessing the properties of DA and potentially of other phycotoxins on phytoplankton.

Triclosan alterations of estuarine phytoplankton community structure.

Antimicrobial additives in pharmaceutical and personal care products are a major environmental concern due to their potential ecological impacts on aquatic ecosystems. Triclosan (TCS) has been used as an antiseptic, disinfectant, and preservative in various media. The sublethal and lethal effects of TCS on estuarine phytoplankton community composition were investigated using bioassays of natural phytoplankton communities to measure phytoplankton responses to different concentrations of TCS ranging from 1 to 200µgl-1. The EC50 (the concentration of an inhibitor where the growth is reduced by half) for phytoplankton groups (diatoms, chlorophytes, cryptophytes) examined in this ranged from 10.7 to 113.8µg TCS l-1. Exposures resulted in major shifts in phytoplankton community composition at concentrations as low as 1.0µg TCS l-1. This study demonstrates estuarine ecosystem sensitivity to TCS exposure and highlights potential alterations in phytoplankton community composition at what are typically environmental concentrations of TCS in urbanized estuaries.

Size-selective toxicity effects of the antimicrobial tylosin on estuarine phytoplankton communities.

The purpose of this study was to determine the lethal and sublethal effects of the antimicrobial tylosin on natural estuarine phytoplankton communities. Bioassays were used in experimental treatments with final concentrations of 5 to 1000 µg tylosin l(-1). Maximum percent inhibition ranged from 57 to 85% at concentrations of 200-400 µg tylosin l(-1). Half maximum inhibition concentrations of tylosin were ca. 5x lower for small phytoplankton (<20 µm) relative to larger phytoplankton (>20 µm) and suggests that small phytoplankton are more sensitive to tylosin exposure. Sublethal effects occurred at concentrations as low as 5 µg tylosin l(-1). Environmental concentrations of tylosin (e.g., 0.2-3 µg l(-1)) may have a significant sublethal effect that alters the size structure and composition of phytoplankton communities. The results of this study highlight the potential importance of cell size on toxicity responses of estuarine phytoplankton.

Ecotoxicology of bromoacetic acid on estuarine phytoplankton.

Bromoacetic acid is formed when effluent containing chlorine residuals react with humics in natural waters containing bromide. The objective of this research was to quantify the effects of bromoacetic acid on estuarine phytoplankton as a proxy for ecosystem productivity. Bioassays were used to measure the EC50 for growth in cultured species and natural marine communities. Growth inhibition was estimated by changes in chlorophyll a concentrations measured by fluorometry and HPLC. The EC50s for cultured Thalassiosira pseudonana were 194 mg L(-1), 240 mg L(-1) for Dunaliella tertiolecta and 209 mg L(-1) for Rhodomonas salina. Natural phytoplankton communities were more sensitive to contamination with an EC50 of 80 mg L(-1). Discriminant analysis suggested that bromoacetic acid additions cause an alteration of phytoplankton community structure with implications for higher trophic levels. A two-fold EC50 decrease in mixed natural phytoplankton populations affirms the importance of field confirmation for establishing water quality criteria.

Towards an Understanding of the Interactions between Freshwater Inflows and Phytoplankton Communities in a Subtropical Estuary in the Gulf of Mexico.

Subtropical estuaries worldwide face increased pressure on their ecosystem health and services due to increasing human population growth and associated land use/land cover changes, expansion of ports, and climate change. We investigated freshwater inflows (river discharge) and the physico-chemical characteristics of Galveston Bay (Texas, USA) as mechanisms driving variability in phytoplankton biomass and community composition between February 2008 and December 2009. Results of multivariate analyses (hierarchical cluster analysis, PERMANOVA, Mantel test, and nMDS ordination coupled to environmental vector fitting) revealed that temporal and spatial differences in phytoplankton community structure correlate to differences in hydrographic and water quality parameters. Spatially, phytoplankton biomass and community composition responded to nutrient loading from the San Jacinto River in the northwest region of the bay (consistent with nutrient limitation) while hydraulic displacement (and perhaps other processes) resulted in overall lower biomass in the Trinity River delta (northeast region). The influence of inflows on phytoplankton diminished along a north to south gradient in the bay. Temporally, temperature and variables associated with freshwater inflow (discharge volume, salinity, inorganic nitrogen and phosphorus concentrations) were major influences on phytoplankton dynamics. Dissolved inorganic nitrogen: phosphorus (DIN:DIP) ratios suggest that phytoplankton communities will be predominately nitrogen limited. Diatoms dominated during periods of moderate to high freshwater inflows in winter/spring and were more abundant in the upper bay while cyanobacteria dominated during summer/fall when inflow was low. Given the differential influences of freshwater inflow on the phytoplankton communities of Galveston Bay, alterations upstream (magnitude, timing, frequency) will likely have a profound effect on downstream ecological processes and corresponding ecosystem services.

Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae, Stramenopiles).

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans-neoxanthin, cis-neoxanthin, α and ß carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan-thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well-described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell-specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.

Sublethal effects of the antibiotic tylosin on estuarine benthic microalgal communities.

Pharmaceuticals are common chemical contaminants in estuaries receiving effluent from wastewater and sewage treatment facilities. The purpose of this research was to examine benthic microalgal (BMA) community responses to sublethal exposures to tylosin, a common and environmentally persistent antibiotic. Bioassays, using concentrations of 0.011-218 µmol tylosin l(-1), were performed on intertidal muddy sediments from North Inlet Estuary, SC. Exposure to tylosin resulted in a reduction in total BMA biomass and primary productivity. Furthermore, exposure seemed to retard diatom growth while having a minimal effect on cyanobacteria biomass. Estuarine systems receiving chronic inputs of trace concentrations of tylosin, as well as other antibiotics, may experience significant reductions in BMA biomass and primary productivity. Given the well-documented role of BMA in the trophodynamics of estuaries, these impacts will likely be manifested in higher trophic levels with possible impairments of the structure and function of these sensitive systems.

Antagonistic interactions between heterotrophic bacteria as a potential regulator of community structure of hypersaline microbial mats.

Microbial mats are laminae of self-sustaining microbial communities with a high level of competition for resources. We tested the hypothesis that chemically mediated antagonism is a potential mechanism for structuring the bacterial community. In the co-culturing assay, 57% of the isolates expressed antagonistic behavior toward one or more isolates and 5% of the isolates inhibited more than 80% of the isolates. We observed greater levels of antagonism between isolates from adjacent laminae than within. The bacterial isolate library derived from the mat was predominately Gram-positive, and inhibition within this group was greater than against the few Gram-negative isolates. Microdiversity of 16S rRNA gene was observed for Bacillus marisflavi isolates, which represented 23 of the 75 isolates in the library. Within this and other groups, the patterns of inhibition and sensitivity varied greatly, suggesting rapid gain and loss of the ability to produce antagonistic secondary metabolites and resistance toward such molecules. Our observations are consistent with the hypothesis that antagonistic interactions are a potential mechanism in addition to physiochemical properties that regulate the vertical distribution of aerobic heterotrophic bacteria in hypersaline microbial mats.

Toxic effect of the combined antibiotics ciprofloxacin, lincomycin, and tylosin on two species of marine diatoms.

The role that antibiotics and other "emerging contaminants" play in shaping environmental microbial communities is of growing interest. The use of the prokaryotic metabolic inhibitors tylosin (T), lincomycin (L), and ciprofloxacin (C) in livestock and humans is both global and extensive. Each of these antibiotic compounds exhibits an affinity for sediment particles, increasing the likelihood of their deposition in the benthos of aquatic systems and each are often present in environmental samples. The purpose of this study was to determine if T, L, and C and their mixtures exhibit significant toxicity to two species of marine diatoms, an algal class comprised of ubiquitous eukaryotic primary producers. Subpopulations from laboratory cultures of Cylindrotheca closterium and Navicula ramosissima were reared in 24-well microtiter plates in the presence of single or combined antibiotics in dilution series. Population growth rates were assessed via epifluorescent microscopic cell counts, from which the half-max inhibitory concentrations (IC(50)) were calculated and used as part of a toxic unit (TU) method for assessing mixture interactions. The single-compound IC(50)'s were, for C. closterium: T = 0.27 mg L(-1), L = 14.16 mg L(-1), C = 55.43 mg L(-1), and for N. ramosissima: T = 0.99 mg L(-1), L = 11.08 mg L(-1), C = 72.12 mg L(-1). These values were generally higher than similar metrics for freshwater species. Mixture IC(50)'s were generally synergistic against C. closterium and additive for N. ramosissima. Both single and combined treatments reduced or eliminated diatom motility. Monochemical responses were similar between species and were not useful for predicting mixture interactions. Mixtures had compound-specific and species-specific effects, favoring N. ramosissima. These results suggest that anthropogenic antibiotics may play a significant role in the ecology of environmental benthic microbial communities. They also suggest single-compound/species studies do not yield useful predictions of the ecological impact of anthropogenic pharmaceuticals.

Fluorometric estimation of surface associated microbial abundance.

Surface associated microbes have historically been difficult to accurately and effectively enumerate. In the current study, we propose a rapid and simple method for estimating abundance of surface associated microbial cells by fluorescence of SYBRGreen stained bacteria and in vivo chlorophyll a fluorescence of benthic diatoms in 24 and 48-well microtiter plates. The effectiveness of this high-throughput technique is demonstrated by assessing sensitivity of a clinical strain of Vibrio cholerae, a benthic bacterial isolate and the benthic microalgae Cylindrotheca closterium to three antibiotics--tylosin, lincomycin and ciproflaxacin. We report on the significant linear relationships between spectral chl a fluorescence and cell abundance and between microalgal growth rates derived from cell counts and fluorescence. Additionally, we provide a simplified and improved method for preparation of a silica gel matrix (SGM), which is an ideal plating media for fluorescence applications. These findings indicate that spectrofluorometry is an inexpensive tool for rapidly estimating abundance of surface associated microbiota and can be employed for assessing antibiotic sensitivity.

Spectral fluorometric characterization of phytoplankton community composition using the Algae Online Analyser.

The utility of a multiple-fixed-wavelength spectral fluorometer, the Algae Online Analyser (AOA), as a means of quantifying phytoplankton biomass and community composition was tested using natural communities from two southeastern United States estuaries, North Inlet, South Carolina, and the Neuse River Estuary, North Carolina. Estimates of biomass (as chlorophyll a) were correlated with HPLC values and variations (usually over-estimates) were consistent with effects of light intensity and nutrient availability on fluorescence quenching. AOA estimates of taxonomic structure were consistent with those from HPLC-derived marker pigments by ChemTax, with both methods indicating domination by chromophytes and green algae in North Inlet and chromophytes and cyanobacteria in the Neuse. We recommend frequent calibration by discrete sample collection, and calibration with species representative of the region of interest. Overall, the AOA appears to be a useful tool for monitoring of phytoplankton community composition, especially as an early warning system for the detection of harmful algal blooms.

Microbial indicators of aquatic ecosystem change: current applications to eutrophication studies.

Human encroachment on aquatic ecosystems is increasing at an unprecedented rate. The impacts of human pollution and habitat alteration are most evident and of greatest concern at the microbial level, where a bulk of production and nutrient cycling takes place. Aquatic ecosystems are additionally affected by natural perturbations, including droughts, storms, and floods, the frequency and extent of which may be increasing. Distinguishing and integrating the impacts of natural and human stressors is essential for understanding environmentally driven change of microbial diversity and function. Microbial bioindicators play a major role in detecting and characterizing these changes. Complementary use of analytical and molecular indicator tools shows great promise in helping us clarify the processes underlying microbial population, community, and ecosystem change in response to environmental perturbations. This is illustrated in phytoplankton (microalgal and cyanobacterial) and bacterial community changes in a range of US estuarine and coastal ecosystems experiencing increasing development in their water- and airsheds as well as climatic changes (e.g., increasing hurricane frequency). Microbial indicators can be adapted to a range of monitoring programs, including ferries, moored instrumentation, and remote sensing, in order to evaluate environmental controls on microbial community structure and function over ecosystem to global scales.

Estuarine phytoplankton group-specific responses to sublethal concentrations of the agricultural herbicide, atrazine.

Atrazine is a common agricultural herbicide that is readily transported into estuaries through surface water runoff. In this study, we determined the short-term (24-48 h) sublethal effects of atrazine on estuarine phytoplankton biomass and community composition. Phytoplankton group-specific responses to atrazine exposure (25 microgh(-1)) were measured using natural water samples collected from two locations in Galveston Bay, Texas. Addition bioassays, coupled with HPLC pigment analysis, were used to quantify changes in the relative abundances of algal groups. For all algal groups except prasinophytes, the addition of atrazine in combination with nitrate was not significantly different from nitrate additions alone. These results suggest no significant negative effect of atrazine on phytoplankton under the specified environmental conditions for the bioassays. Although low concentrations of atrazine may have minimal impacts on phytoplankton, herbicide loadings need to be further characterized before generalizations can be applied to estuarine and coastal ecosystems.

Characterizing man-made and natural modifications of microbial diversity and activity in coastal ecosystems.

The impacts of growing coastal pollution and habitat alteration accompanying human encroachment are of great concern at the microbial level, where much of the ocean's primary production and biogeochemical cycling takes place. Coastal ecosystems are also under the influence of natural perturbations such as major storwns and flooding. Distinguishing the impacts of natural and human stressors is essential for understanding environmentally-induced change in microbial diversity and function. The objective of this paper is to discuss the applications and merits of recently developed molecular, ecophysiological and analytical indicators and their utility in examining anthropogenic and climatic impacts on the structure and function of coastal microbial communities. The nitrogen-limited Neuse River Estuary and Pamlico Sound, North Carolina are used as examples of ecosystems experiencing both anthropogenic (i.e., accelerating eutrophication) and climatic stress (increasing frequencies of tropical storms and hurricanes). Additional examples are derived from a coastal monitoring site (LEO) on the Atlantic coast of New Jersey and Galveston Bay, on the Gulf of Mexico. In order to assess structure, function, and trophic state of these and other coastal ecosystems, molecular (DNA and RNA-based) characterizations of the microbial taxa involved in carbon, nitrogen and other nutrient transformations can be combined with diagnostic pigment-based indicators of primary producer groups. Application of these methods can reveal process-level microbial community responses to environmental variability over a range of scales. Experimental approaches combined with strategic monitoring utilizing these methods will facilitate: (a) understanding organismal and community responses to environmental change, and (b) synthesizing these responses in the context of ecosystem models that integrate physical, chemical and biotic variability with environmental controls.