Superfund Research Program
Methylmercury Production and Fate in Response to Multiple Environmental Factors
Project Leader: Celia Y. Chen
Grant Number: P42ES007373
Funding Period: 2000-2021
Final Progress Reports
Forty-eight states in the USA currently issue public advisories to limit fish consumption to reduce human exposure to mercury and other toxins. Project 7 studies the mechanisms driving lake-to-lake variation in metal burdens (Hg, Cd, Zn, As, and Pb) of aquatic organisms focusing on the accumulation and movement of metals in lower trophic levels. The project investigator’s working hypothesis is that a large fraction of the difference in metal levels in fish from different lakes arises from fundamental differences in their food webs (e.g. different lakes have different assemblages of algae, microscopic zooplankton, larger insects and fish). Drs. Folt and Chen along with their research teams seek to determine mechanisms underlying movement of five potentially toxic metals (As, Hg and MeHg, Zn, Cd, and Pb) through aquatic food webs into fish as a primary conduit for human exposure to metals. Their recent studies include: 1) comparison of pelagic and littoral zone food webs as conduit habitats for metal trophic transfer using field and modeling approaches; 2) laboratory investigation of bioaccumulation of Hg in the lowest portions of the planktonic food web using specialized stable metal isotope techniques; 3) field investigation of the effects of drawdown and productivity on Hg bioaccumulation in reservoir food webs; 4) laboratory studies of effects of metal toxicity on population and genomic responses in the model organism, Daphnia pulex.
Field Studies. A central hypothesis of the researchers work is that fish foraging in particular habitats (“conduit habitats”) build up more metal due to predictable differences in metal burdens characteristic of those conduits. Fish feed in different habitats over their lifetime and therefore, derive contaminants from different prey types. The researchers conducted a field study to compare the metal bioaccumulation and transfer in pelagic vs. littoral food webs in Post Pond, NH on 5 sampling dates in the spring, summer, and fall. They are in the process of analyzing these samples for As, Hg, MeHg, Cd, Zn, and Pb. They also collaborated with the Vermont Department of Environmental Conservation to conduct a comparative analysis of four field datasets in Northeastern lakes to identify the most universal predictors for Hg concentrations in fish. The researchers found that in addition to physical/chemical variables (pH, alkalinity, lake size), landuse (human disturbance) and ecological variables (plankton density) are also important correlates of Hg bioaccumulation in fish. They also are collaborating with ecologists at the Biodiversity Research Institute in Maine to investigate the Hg trophic transfer in reservoirs. Due to the existing evidence that flooding and drawdown in reservoirs is correlated with enhanced Hg bioaccumulation in fish, the teams are investigating the effect of different drawdown levels and schedules across a nutrient enrichment gradient on metal trophic transfer in pelagic food webs. Sixteen reservoirs were sampled for fish, zooplankton, and loon blood and feathers in July and in September. Zooplankton samples are currently being analyzed by the Trace Element Analysis (TEA) Core Facility. Finally, the investigators have been working on the Hg speciation of different zooplankton taxa to compare their potential as keystone conduits for metal transfer in different food webs. Specifically, they are using newly developed Hg speciation techniques that involve simultaneously measuring inorganic mercury and methylmercury from a single sample at extremely low levels.
Metal data from field studies in Post Pond described above will be used to further parameterize a bioenergetics model for Hg uptake in fish that feed in pelagic vs. littoral zone habitats. The researchers used the University of Wisconsin’s “Fish Bioenergetics 3.0” model (1997) to develop predictions for fish metal accrual based on their foraging on different prey and in different (littoral vs. pelagic) habitats. They find: 1) Littoral zone prey have lower mass-specific metal burdens than pelagic zooplankton. 2) Foraging on a mixed habitat diet reduces the accrual of some metals in fish tissue. They parameterized the Wisconsin model using their preliminary data and constructed four fish diets (a) littoral alone as a mixture of the littoral prey based on an extensive study of fish diets in Post Pond, NH (b) pelagic alone 1 as 75% cladoceran, 25% copepod, (c) pelagic alone 2 as 75% copepod, 25% cladoceran, (d) mixed habitat as a 50:50 mix of the pelagic and the littoral diet. They compared predictions from the growth model for pumpkinseed sunfish body burdens across the four diets when fish reached 3.3 grams (about 1 year growth). Mercury burdens were greatest in fish grown on cladoceran dominated diets. The fish growth model also includes other important prey characteristics that affect fish growth rates (e.g. energy density of the prey, assimilation efficiencies, etc.)
Laboratory Studies. The project investigators also are conducting a major research project to link genomic responses with population-level outcomes and establish predictive biomarkers for the extent to which aquatic biota from different lakes are likely to be susceptible to metal contamination. Genomic biomarker development has been conducted collaboratively with Project 2 and the University of Indiana to identify patterns of gene expression in D. pulex, exposed to metals (i.e., As, Cd, Zn). The investigators are testing the general hypothesis that the specificity of genomic and population level responses will differ between different metals, exposure times, and doses and that these responses can be used in a proactive manner to protect human health and the environment. They have already constructed several full-length cDNA libraries from organisms maintained under control and manipulated conditions (metals, UV, temperature, predator kairomones, etc.). Screening for nonredundancy yielded ~50,000 cDNAs that will be sequenced in winter 2005 by the Joint Genome Institute. Microarrays will be printed that contain the full complement of unique cDNAs identified from this effort. In preparation for large-scale printing of high-content, high-quality arrays, 1st generation Daphnia microarrays were constructed with 100 well-characterized elements to optimize and standardize print conditions and hybridization techniques. The investigators are currently on their 2nd generation microarray. This represents 3552 unique elements that have been arrayed in tandem, as well as 240 blanks and spiking standards. These ‘blind’ arrays were developed to allow for identification of differentially expressed genes, but require post-hoc sequencing to identify genes.
Initial investigations were conducted to determine the genomic response of Daphnia pulex to arsenic and cadmium. These metals were selected for study in part because of their differences in toxic mechanisms. To discover differential gene expression patterns under metal stress, Daphnia were acutely (48-h) exposed to incipient concentrations (LC10) of each metal (20 µg Cd/L and 1384 µg As/L). RNA was isolated from metal exposed animals (both treatments) and from their genetic clones under standard (reference) conditions. Each treatment and reference sample was labeled indirectly using aminoallyl-dNTP coupling and alternate Alexa Fluor dyes (555, 647). The researchers replicated experiments measured the relative amounts of gene transcripts between both conditions. Statistical analyses revealed that a small number of genes were differentially expressed in the two metal treatments (2-5%). However, arsenic and cadmium produced almost entirely different transcriptional responses as there was very little overlap (5%) between these two groups. These blind arrays allowed for identification of differentially expressed genes prior to sequencing, but required post-hoc analysis to identify genes. As such, these identified probes are being sequenced and differential responses validated by real-time (quantitative) PCR. Future studies will focus on chronic/sub-lethal exposures, linking genomic response and demographic outcome with the ultimate goal of establishing predictive biomarkers that could be employed to determine the susceptibility of natural populations to metals.
Investigations also were conducted to explore generational effects of exposure history on acute and chronic responses of Daphnia pulex to metals. Selective pressures from pollution often result in acquisition of tolerance, which can manifest in individuals/clones as physiological modifications (acclimation) and over time restructure natural populations via genetic adaptation. However, little attention has been given to understanding mechanisms and costs associated with tolerance. For these studies, three metal-acclimated populations of D. pulex were developed by continuous exposure to cadmium concentrations ranging from 0.25 to 2.5 µg /L for >45 generations (~2 yr) in the laboratory. Acute (48-h) toxicity tests revealed that cadmium acclimated Daphnia exhibit increased tolerance (higher LC50) following exposure to cadmium, zinc, and silver but were not cross-tolerant to arsenic. These studies suggest that altered uptake is not responsible for metal tolerant phenotypes, since cadmium/zinc and silver are accumulated by different pathways. Rather they hint at a role of the metal binding protein, metallothionein (MT) since these metals are known potent inducers of MT, whereas, arsenic is not known to stimulate MT expression. Chronic studies (21-d) revealed that in the presence of cadmium (1 and 2.5 µg /L), metal acclimated populations had greater cumulative reproduction, reproduction per adult, and rates of net reproductive output (R0) compared to naïve populations that were not pre-exposed to elevated concentrations of metals. However, these patterns were reversed in control conditions as naïve outperformed metal-acclimated Daphnia, suggesting there is a physiological cost associated with tolerance. Multi-generation tests revealed that metal tolerance was rapidly lost (≤ 2 generations) following removal from metal. Collectively, these results demonstrate the importance of considering tolerance when evaluating effects of chemical stressors on aquatic organisms. Future work will focus on defining adaptive limits of metal acclimation and identifying the molecular pathways responsible for increased metal tolerance.
Finally, the team investigated the trophic transfer of Hg in the lower planktonic food web that represents the trophic levels that are potentially the most critical in the biomagnification of Hg but the least studied. In collaboration with the TEA Core and a visiting scientist from the University of Liege (Belgium), the researchers conducted experiments to examine the bioaccumulation and transfer of inorganic and MeHg by Daphnia, rotifers, ciliates, and bacteria. Due the challenges of working with such small organisms, the team compared mass-specific uptake of enriched stable isotopes of inorganic (HgI) and monomethylmercury (199Hg2+ and Me201Hg spikes) using novel techniques developed in the TEA Core. Experiments were conducted using two different algal species (Chlamydomonas reinhardi, Ankistrodesmus falcatus), a ciliate (Tetryahymena sp.) and a bacterium (E, coli) to compare uptake of Hg by Daphnia pulex and Brachionus calyciflorus and to also measure bioaccumulation of Hg in Daphnia via ingestion of the rotifer. Experimental samples are currently being analyzed.