Jennifer A. Jay

Mercury cycling in surface water, pore water and sediments of Mugu Lagoon, CA, USA.

Mugu Lagoon is an estuary in southern California, listed as impaired for mercury. In 2005, we examined mercury cycling at ten sites within at most four habitats. In surface water (unfiltered and filtered) and pore water, the concentration of total mercury was correlated with methylmercury levels (R2=0.29, 0.26, 0.27, respectively, p<0.05), in contrast to sediments, where organic matter and reduced iron levels were most correlated with methylmercury content (R2=0.37, 0.26, respectively, p<0.05). Interestingly, levels for percent methylmercury of total mercury in sediments were higher than typical values for estuarine sediments (average 5.4%, range 0.024-38%, n=59), while pore water methylmercury Kd values were also high (average 3.1, range 2.0-4.2l kg(-1), n=39), and the estimated methylmercury flux from sediments was low (average 1.7, range 0.14-5.3ng m(-2) day(-1), n=19). Mercury levels in predatory fish tissue at Mugu are >0.3ppm, suggesting biogeochemical controls on methylmercury mobility do not completely mitigate methylmercury uptake through the food web.

The Effect of Wildfire on Soil Mercury Concentrations in Southern California Watersheds

Mercury (Hg) stored in vegetation and soils is known to be released to the atmosphere during wildfires, increasing atmospheric stores and altering terrestrial budgets. Increased erosion and transport of sediments is well-documented in burned watersheds, both immediately post-fire and as the watershed recovers; however, understanding post-fire mobilization of soil Hg within burned watersheds remains elusive. The goal of the current study is to better understand the impact of wildfire on soil-bound Hg during the immediate post-fire period as well as during recovery, in order to assess the potential for sediment-driven transport to and within surface waters in burned watersheds. Soils were collected from three southern California watersheds of similar vegetation and soil characteristics that experienced wildfire. Sampling in one of these watersheds was extended for several seasons (1.5 years) in order to investigate temporal changes in soil Hg concentrations. Laboratory analysis included bulk soil total Hg concentrations and total organic carbon of burned and unburned samples. Soils were also fractionated into a subset of grain sizes with analysis of Hg on each fraction. Low Hg concentrations were observed in surface soils immediately post-fire. Accumulation of Hg coincident with moderate vegetative recovery was observed in the burned surface soils 1 year following the fire, and mobilization was also noted during the second winter (rainy) season. Hg concentrations were highest in the fine-grained fraction of unburned soils; however, in the burned soils, the distribution of soil-bound Hg was less influenced by grain size. The accelerated accumulation of Hg observed in the burned soils, along with the elevated risk of erosion, could result in increased delivery of organic- or particulate-bound Hg to surface waters in post-fire systems.

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation.

Covalently linked immunomagnetic separation/adenosine triphosphate technique (Cov-IMS/ATP) enables rapid, in-field detection and quantification of Escherichia coli and Enterococcus spp. in freshwater and marine environments

Aims:  Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody–bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems.

Long-term monitoring of molecular markers can distinguish different seasonal patterns of fecal indicating bacteria sources

Elevated levels of fecal indicator bacteria (FIB) have been observed at Topanga Beach, CA, USA. To identify the FIB sources, a microbial source tracking study using a dog-, a gull- and two human-associated molecular markers was conducted at 10 sites over 21 months. Historical data suggest that episodic discharge from the lagoon at the mouth of Topanga Creek is the main source of bacteria to the beach. A decline in creek FIB/markers downstream from upper watershed development and a sharp increase in FIB/markers at the lagoon sites suggest sources are local to the lagoon. At the lagoon and beach, human markers are detected sporadically, dog marker peaks in abundance mid-winter, and gull marker is chronically elevated. Varied seasonal patterns of FIB and source markers were identified showing the importance of applying a suite of markers over long-term spatial and temporal sampling to identify a complex combination of sources of contamination.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

Influences of Zero-Valent Sulfur on Mercury Methylation in Bacterial Cocultures

The speciation of mercury (Hg) is a major determinant of its methylation rate by sulfate-reducing bacteria (SRB), considered the primary methylators. Under anoxic conditions, sulfur (S) cycling may have a significant influence on Hg complexation and methylation, by influencing both SRB activity and the pool of available reduced S ligands, as the presence of zero-valent sulfur (S(0)) in sulfidic water results in the formation of polysulfides. While SRB frequently coexist with S-oxidizing bacteria in natural environments, the effect that these organisms may have on methylation by SRB is not understood. In this study, we investigate the role of S(0) in methylation by SRB monocultures and cocultures with phototrophic green or purple S-oxidizing bacteria. In the coculture experiments, the presence of S-oxidizers was found to increase Hg methylation rates, apparently by maintaining favorable chemical speciation in the environment. The measured Hg methylation rates were in accord with predictions based on geochemical modeling of speciation. In SRB monoculture experiments conducted in the presence and absence of S(0), the data showed that at limited total Hg, the presence of polysulfides resulted in decreased Hg methylation, presumably by causing a decrease in the most bioavailable Hg–sulfide complexes. These results indicate that models of Hg speciation and methylation in the environment should include a detailed investigation of S redox speciation.

Virulence Genes among Enterococcus faecalis and Enterococcus faecium Isolated from Coastal Beaches and Human and Nonhuman Sources in Southern California and Puerto Rico

Most Enterococcus faecalis and E. faecium are harmless to humans; however, strains harboring virulence genes, including esp, gelE, cylA, asa1, and hyl, have been associated with human infections. E. faecalis and E. faecium are present in beach waters worldwide, yet little is known about their virulence potential. Here, multiplex PCR was used to compare the distribution of virulence genes among E. faecalis and E. faecium isolated from beaches in Southern California and Puerto Rico to isolates from potential sources including humans, animals, birds, and plants. All five virulence genes were found in E. faecalis and E. faecium from beach water, mostly among E. faecalis. gelE was the most common among isolates from all source types. There was a lower incidence of asa1, esp, cylA, and hyl genes among isolates from beach water, sewage, septage, urban runoff, sea wrack, and eelgrass as compared to human isolates, indicating that virulent strains of E. faecalis and E. faecium may not be widely disseminated at beaches. A higher frequency of asa1 and esp among E. faecalis from dogs and of asa1 among birds (mostly seagull) suggests that further studies on the distribution and virulence potential of strains carrying these genes may be warranted.

Investigation of mercury methylation pathways in biofilm versus planktonic cultures of Desulfovibrio desulfuricans.

Biofilms can methylate mercury (Hg) at higher rates than unattached bacteria and are increasingly recognized as important Hg methylation sites in the environment. Our previous study showed that methylation rates in biofilm cultures were up to 1 order of magnitude greater than those in planktonic cultures of a sulfate-reducing bacterium. To probe whether the differential Hg methylation rates resulted from metabolic differences between these two cultures, Hg methylation assays following molybdate or chloroform inhibition (a specific inhibitor of the acetyl-CoA pathway) were conducted on biofilm and planktonic cultures of Desulfovibrio desulfuricans strains M8 and ND132. Molybdate was as effective in inhibiting Hg methylation as well as growth in both planktonic and biofilm cultures. The addition of chloroform only impacted Hg methylation in biofilm cultures, suggesting that different pathways are used for methylation in biofilm compared to planktonic cultures. To investigate this further, expression of the cooS gene, which encodes for carbon monoxide dehydrogenase, a key enzyme in the acetyl-CoA pathway, was compared in biofilm and planktonic cultures of ND132. Biofilm cultures showed up to 4 times higher expression of cooS than planktonic cultures. On the basis of these results, the acetyl-CoA pathway appears to play an important role in methylation in biofilm cultures of this organism, possibly by supplying the methyl group to Hg methylating enzymes; methylation in planktonic cultures appears to be independent of this pathway. This observation has important implications, particularly in developing reliable models to predict Hg methylation rates in different environments and perhaps eventually in being able to control this undesirable chemical transformation.