Jennifer L. Graham

Cyanotoxin mixtures and taste-and-odor compounds in cyanobacterial blooms from the midwestern united states

The mixtures of toxins and taste-and-odor compounds present during cyanobacterial blooms are not well characterized and of particular concern when evaluating potential human health risks. Cyanobacterial blooms were sampled in twenty-three Midwestern United States lakes and analyzed for community composition, thirteen cyanotoxins by liquid chromatography/mass spectrometry and immunoassay, and two taste-and-odor compounds by gas chromatography/mass spectrometry. Aphanizomenon, Cylindrospermopsis and/or Microcystis were dominant in most (96%) blooms, but community composition was not strongly correlated with toxin and taste-and-odor occurrence. Microcystins occurred in all blooms. Total microcystin concentrations measured by liquid chromatography/mass spectrometry and immunoassay were linearly related (r(s) = 0.76, p < 0.01) and LC/MS/MS concentrations were lower than or similar to ELISA in most (85%) samples. Geosmin (87%), 2-methylisoborneol (39%), anatoxin-a (30%), saxitoxins (17%), cylindrospermopsins (9%), and nodularin-R (9%) also were present in these blooms. Multiple classes of cyanotoxins occurred in 48% of blooms and 95% had multiple microcystin variants. Toxins and taste-and-odor compounds frequently co-occurred (91% of blooms), indicating odor may serve as a warning that cyanotoxins likely are present. However, toxins occurred more frequently than taste-and-odor compounds, so odor alone does not provide sufficient warning to ensure human-health protection.

Cyanotoxins in inland lakes of the United States: Occurrence and potential recreational health risks in the EPA National Lakes Assessment 2007

A large nation-wide survey of cyanotoxins (1161 lakes) in the United States (U.S.) was conducted during the EPA National Lakes Assessment 2007. Cyanotoxin data were compared with cyanobacteria abundance- and chlorophyll-based World Health Organization (WHO) thresholds and mouse toxicity data to evaluate potential recreational risks. Cylindrospermopsins, microcystins, and saxitoxins were detected (ELISA) in 4.0, 32, and 7.7% of samples with mean concentrations of 0.56, 3.0, and 0.061μg/L, respectively (detections only). Co-occurrence of the three cyanotoxin classes was rare (0.32%) when at least one toxin was detected. Cyanobacteria were present and dominant in 98 and 76% of samples, respectively. Potential anatoxin-, cylindrospermopsin-, microcystin-, and saxitoxin-producing cyanobacteria occurred in 81, 67, 95, and 79% of samples, respectively. Anatoxin-a and nodularin-R were detected (LC/MS/MS) in 15 and 3.7% samples (n=27). The WHO moderate and high risk thresholds for microcystins, cyanobacteria abundance, and total chlorophyll were exceeded in 1.1, 27, and 44% of samples, respectively. Complete agreement by all three WHO microcystin metrics occurred in 27% of samples. This suggests that WHO microcystin metrics based on total chlorophyll and cyanobacterial abundance can overestimate microcystin risk when compared to WHO microcystin thresholds. The lack of parity among the WHO thresholds was expected since chlorophyll is common amongst all phytoplankton and not all cyanobacteria produce microcystins.

Challenges for mapping cyanotoxin patterns from remote sensing of cyanobacteria

Using satellite imagery to quantify the spatial patterns of cyanobacterial toxins has several challenges. These challenges include the need for surrogate pigments - since cyanotoxins cannot be directly detected by remote sensing, the variability in the relationship between the pigments and cyanotoxins - especially microcystins (MC), and the lack of standardization of the various measurement methods. A dual-model strategy can provide an approach to address these challenges. One model uses either chlorophyll-a (Chl-a) or phycocyanin (PC) collected in situ as a surrogate to estimate the MC concentration. The other uses a remote sensing algorithm to estimate the concentration of the surrogate pigment. Where blooms are mixtures of cyanobacteria and eukaryotic algae, PC should be the preferred surrogate to Chl-a. Where cyanobacteria dominate, Chl-a is a better surrogate than PC for remote sensing. Phycocyanin is less sensitive to detection by optical remote sensing, it is less frequently measured, PC laboratory methods are still not standardized, and PC has greater intracellular variability. Either pigment should not be presumed to have a fixed relationship with MC for any water body. The MC-pigment relationship can be valid over weeks, but have considerable intra- and inter-annual variability due to changes in the amount of MC produced relative to cyanobacterial biomass. To detect pigments by satellite, three classes of algorithms (analytic, semi-analytic, and derivative) have been used. Analytical and semi-analytical algorithms are more sensitive but less robust than derivatives because they depend on accurate atmospheric correction; as a result derivatives are more commonly used. Derivatives can estimate Chl-a concentration, and research suggests they can detect and possibly quantify PC. Derivative algorithms, however, need to be standardized in order to evaluate the reproducibility of parameterizations between lakes. A strategy for producing useful estimates of microcystins from cyanobacterial biomass is described, provided cyanotoxin variability is addressed.

Experimental manipulation of TN:TP ratios suppress cyanobacterial biovolume and microcystin concentration in large-scale in situ mesocosms

Abstract A global dataset was compiled to examine relations between the total nitrogen to total phosphorus ratio (TN:TP) and microcystin concentration in lakes and reservoirs. Microcystin concentration decreased as TN:TP ratios increased, suggesting that manipulation of the TN:TP ratio may reduce microcystin concentrations. This relationship was experimentally tested by adding ammonium nitrate to increase the TN:TP ratio in large-scale (70 m3), in situ mesocosms located in a eutrophic reservoir that routinely experiences toxic blooms of cyanobacteria. At a TN:TP ratio >75:1, chlorophytes dominated the phytoplankton community in the mesocosms, while cyanobacterial biovolume was significantly reduced and microcystin was not detected. In contrast, the unmanipulated reservoir was dominated by cyanobacteria, and microcystin was detected. Secchi depths were 1.1 to 1.8 times greater in the mesocosms relative to the reservoir. Cladoceran zooplankton had a larger body size (0.14 mm on average) in the mesocosms compared to conspecifics in the reservoir, which was likely related to the higher quality food. Combined, these empirical and experimental data indicate that although nutrient addition is counterintuitive to current cyanobacteria management practices, increasing the TN:TP ratio by adding nitrogen may be a potential short-term management strategy to reduce cyanobacteria and cyanotoxins when other alternatives (e.g., phosphorus reduction) are not possible. Additional experimental studies with careful controls are needed to define best management practices and identify any potential unintended consequences before nitrogen addition is implemented as a lake and reservoir management practice.

Combined effects of nitrogen to phosphorus and nitrate to ammonia ratios on cyanobacterial metabolite concentrations in eutrophic Midwestern USA reservoirs

Abstract Recent studies have shown that the total nitrogen to total phosphorus (TN:TP) ratio and nitrogen oxidation state may have substantial effects on secondary metabolite (e.g., microcystins) production in cyanobacteria. We investigated the relationship between the water column TN:TP ratio and the cyanobacterial secondary metabolites geosmin, 2-methylisoborneol (MIB), and microcystin using multiple years of data from 4 reservoirs located in the Midwestern United States. We also examined the relationship between water column concentrations of chemically oxidized (NO3) and reduced (NH3) nitrogen, the NO3:NH3 ratio, cyanobacterial biovolume, and associated secondary metabolites. We found that the cyanobacterial secondary metabolites geosmin, MIB, and microcystin primarily occurred when the TN:TP ratio was <30:1 (by mass), likely due to higher cyanobacterial biovolumes at lower TN:TP ratios. We also found that relative cyanobacterial biovolume was inversely related to the NO3:NH3 ratio. Both N2- and non-N2-fixing cyanobacteria seemed to produce secondary metabolites and had higher concentrations per unit biovolume when NO3:NH3 ratios were relatively low. Our data thus are consistent with the hypothesis that lower TN:TP ratios favor cyanobacterial dominance and also suggest that relatively low NO3:NH3 ratios provide conditions that may favor the production of cyanobacterial secondary metabolites. Our data further suggest that increases in the absolute concentrations of TP or NH3 (or both), causing decreases in TN:TP and NO3:NH3 ratios, respectively, may stimulate cyanobacteria having the metabolic ability to produce geosmin, MIB, or microcystins. Future studies should address how the NO3:NH3 ratio affects phytoplankton community structure and occurrence and production of cyanobacterial secondary metabolites.

Elucidation of Taste- and Odor-Producing Bacteria and Toxigenic Cyanobacteria in a Midwestern Drinking Water Supply Reservoir by Shotgun Metagenomic Analysis

ABSTRACT While commonplace in clinical settings, DNA-based assays for identification or enumeration of drinking water pathogens and other biological contaminants remain widely unadopted by the monitoring community. In this study, shotgun metagenomics was used to identify taste-and-odor producers and toxin-producing cyanobacteria over a 2-year period in a drinking water reservoir. The sequencing data implicated several cyanobacteria, including Anabaena spp., Microcystis spp., and an unresolved member of the order Oscillatoriales as the likely principal producers of geosmin, microcystin, and 2-methylisoborneol (MIB), respectively. To further demonstrate this, quantitative PCR (qPCR) assays targeting geosmin-producing Anabaena and microcystin-producing Microcystis were utilized, and these data were fitted using generalized linear models and compared with routine monitoring data, including microscopic cell counts, sonde-based physicochemical analyses, and assays of all inorganic and organic nitrogen and phosphorus forms and fractions. The qPCR assays explained the greatest variation in observed geosmin (adjusted R 2 = 0.71) and microcystin (adjusted R 2 = 0.84) concentrations over the study period, highlighting their potential for routine monitoring applications. The origin of the monoterpene cyclase required for MIB biosynthesis was putatively linked to a periphytic cyanobacterial mat attached to the concrete drinking water inflow structure. We conclude that shotgun metagenomics can be used to identify microbial agents involved in water quality deterioration and to guide PCR assay selection or design for routine monitoring purposes. Finally, we offer estimates of microbial diversity and metagenomic coverage of our data sets for reference to others wishing to apply shotgun metagenomics to other lacustrine systems. IMPORTANCE Cyanobacterial toxins and microbial taste-and-odor compounds are a growing concern for drinking water utilities reliant upon surface water resources. Specific identification of the microorganism(s) responsible for water quality degradation is often complicated by the presence of co-occurring taxa capable of producing these undesirable metabolites. Here we present a framework for how shotgun metagenomics can be used to definitively identify problematic microorganisms and how these data can guide the development of rapid genetic assays for routine monitoring purposes.

Preliminary evaluation of an in vivo fluorometer to quantify algal periphyton biomass and community composition

Abstract The bbe-Moldaenke BenthoTorch (BT) is an in vivo fluorometer designed to quantify algal biomass and community composition in benthic environments. The BT quantifies total algal biomass via chlorophyll a (Chl-a) concentration and may differentiate among cyanobacteria, green algae, and diatoms based on pigment fluorescence. To evaluate how BT measurements of periphytic algal biomass (as Chl-a) compared with an ethanol extraction laboratory analysis, we collected BT- and laboratory-measured Chl-a data from 6 stream sites in the Indian Creek basin, Johnson County, Kansas, during August and September 2012. BT-measured Chl-a concentrations were positively related to laboratory-measured concentrations (R2 = 0.47); sites with abundant filamentous algae had weaker relations (R2 = 0.27). Additionally, on a single sample date, we used the BT to determine periphyton biomass and community composition upstream and downstream from 2 wastewater treatment facilities (WWTF) that discharge into Indian Creek. We found that algal biomass increased immediately downstream from the WWTF discharge then slowly decreased as distance from the WWTF increased. Changes in periphyton community structure also occurred; however, there were discrepancies between BT- and laboratory-measured community composition data. Most notably, cyanobacteria were present at all sites based on BT measurements but were present at only one site based on laboratory-analyzed samples. Overall, we found that the BT compared reasonably well with laboratory methods for relative patterns in Chl-a but not as well with absolute Chl-a concentrations. Future studies need to test the BT over a wider range of Chl-a concentrations, in colored waters, and across various periphyton assemblages.

Experimental additions of aluminum sulfate and ammonium nitrate to in situ mesocosms to reduce cyanobacterial biovolume and microcystin concentration

Abstract Recent studies suggest that nitrogen additions to increase the total nitrogen:total phosphorus (TN:TP) ratio may reduce cyanobacterial biovolume and microcystin concentration in reservoirs. In systems where TP is >100 μg/L, however, nitrogen additions to increase the TN:TP ratio could cause ammonia, nitrate, or nitrite toxicity to terrestrial and aquatic organisms. Reducing phosphorus via aluminum sulfate (alum) may be needed prior to nitrogen additions aimed at increasing the TN:TP ratio. We experimentally tested this sequential management approach in large in situ mesocosms (70.7 m3) to examine effects on cyanobacteria and microcystin concentration. Because alum removes nutrients and most seston from the water column, alum treatment reduced both TN and TP, leaving post-treatment TN:TP ratios similar to pre-treatment ratios. Cyanobacterial biovolume was reduced after alum addition, but the percent composition (i.e., relative) cyanobacterial abundance remained unchanged. A single ammonium nitrate (nitrogen) addition increased the TN:TP ratio 7-fold. After the TN:TP ratio was >50 (by weight), cyanobacterial biovolume and abundance were reduced, and chrysophyte and cryptophyte biovolume and abundance increased compared to the alum treatment. Microcystin was not detectable until the TN:TP ratio was <50. Although both treatments reduced cyanobacteria, only the nitrogen treatment seemed to stimulate energy flow from primary producers to zooplankton, which suggests that combining alum and nitrogen treatments may be a viable in-lake management strategy to reduce cyanobacteria and possibly microcystin concentrations in high-phosphorus systems. Additional studies are needed to define best management practices before combined alum and nitrogen additions are implemented as a reservoir management strategy.

Predicting cyanobacterial abundance, microcystin, and geosmin in a eutrophic drinking-water reservoir using a 14-year dataset

ABSTRACT Harris TD, Graham JL. 2017. Predicting cyanobacterial abundance, microcystin, and geosmin in a eutrophic drinking-water reservoir using a 14-year dataset. Lake Reserve Manage. 33:32-48. Cyanobacterial blooms degrade water quality in drinking water supply reservoirs by producing toxic and taste-and-odor causing secondary metabolites, which ultimately cause public health concerns and lead to increased treatment costs for water utilities. There have been numerous attempts to create models that predict cyanobacteria and their secondary metabolites, most using linear models; however, linear models are limited by assumptions about the data and have had limited success as predictive tools. Thus, lake and reservoir managers need improved modeling techniques that can accurately predict large bloom events that have the highest impact on recreational activities and drinking-water treatment processes. In this study, we compared 12 unique linear and nonlinear regression modeling techniques to predict cyanobacterial abundance and the cyanobacterial secondary metabolites microcystin and geosmin using 14 years of physiochemical water quality data collected from Cheney Reservoir, Kansas. Support vector machine (SVM), random forest (RF), boosted tree (BT), and Cubist modeling techniques were the most predictive of the compared modeling approaches. SVM, RF, and BT modeling techniques were able to successfully predict cyanobacterial abundance, microcystin, and geosmin concentrations <60,000 cells/mL, 2.5 µg/L, and 20 ng/L, respectively. Only Cubist modeling predicted maxima concentrations of cyanobacteria and geosmin; no modeling technique was able to predict maxima microcystin concentrations. Because maxima concentrations are a primary concern for lake and reservoir managers, Cubist modeling may help predict the largest and most noxious concentrations of cyanobacteria and their secondary metabolites.

Quantifying suspended sediment loads delivered to Cheney Reservoir, Kansas: Temporal patterns and management implications

Cheney Reservoir, constructed during 1962 to 1965, is the primary water supply for the city of Wichita, the largest city in Kansas. Sediment is an important concern for the reservoir as it degrades water quality and progressively decreases water storage capacity. Long-term data collection provided a unique opportunity to estimate the annual suspended sediment loads for the entire history of the reservoir. To quantify and characterize sediment loading to Cheney Reservoir, discrete suspended sediment samples and continuously measured streamflow data were collected from the North Fork Ninnescah River, the primary inflow to Cheney Reservoir, over a 48-year period. Continuous turbidity data also were collected over a 15-year period. These data were used together to develop simple linear regression models to compute continuous suspended sediment concentrations and loads from 1966 to 2013. The inclusion of turbidity as an additional explanatory variable with streamflow improved regression model diagnostics and increased the amount of variability in suspended sediment concentration explained by 14%. Using suspended sediment concentration from the streamflow-only model, the average annual suspended sediment load was 102,517 t (113,006 tn) and ranged from 4,826 t (5,320 tn) in 1966 to 967,569 t (1,066,562 tn) in 1979. The sediment load in 1979 accounted for about 20% of the total load over the 48-year history of the reservoir and 92% of the 1979 sediment load occurred in one 24-hour period during a 1% annual exceedance probability flow event (104-year flood). Nearly 60% of the reservoir sediment load during the 48-year study period occurred in 5 years with extreme flow events (9% to 1% annual exceedance probability, or 11- to 104-year flood events). A substantial portion (41%) of sediment was transported to the reservoir during five storm events spanning only eight 24-hour periods during 1966 to 2013. Annual suspended sediment load estimates based on streamflow were, on average, within ±20% of estimates based on streamflow and turbidity combined. Results demonstrate that large suspended sediment loads are delivered to Cheney Reservoir in very short time periods, indicating that sediment management plans eventually must address large, infrequent inflow events to be effective.