2002 Progress Report: Multi-level Indicators of Ecosystem Integrity in Alpine Lakes of the Sierra Nevada

EPA Grant Number: R827643
Title: Multi-level Indicators of Ecosystem Integrity in Alpine Lakes of the Sierra Nevada
Investigators: Oris, James T. , Bailer, A. John , Guttman, Sheldon I. , Miller, Glenn C. , Reuter, John E.
Institution: Miami University , University of California - Davis , University of Nevada - Reno
EPA Project Officer: Hahn, Intaek
Project Period: September 13, 1999 through September 12, 2002 (Extended to September 12, 2003)
Project Period Covered by this Report: September 13, 2001 through September 12, 2002
Project Amount: $894,627
RFA: Ecological Indicators (1999) RFA Text |  Recipients Lists
Research Category: Ecosystems , Ecological Indicators/Assessment/Restoration

Objective:

The overall objective of this research project is to develop protocols for environmental assessments of alpine lakes in the Sierra Nevada with a range of human impacts. These assessments will be conducted over the range of levels of biological organization (molecular to ecosystem), utilizing currently available assessment techniques with the addition of two new ecological indicators. The use of population genetics analysis as a response indicator and the use of molecular biomarkers of exposure to contaminants as a diagnostic indicator are proposed for incorporation into monitoring and assessment programs for surface waters. These indicators will provide critical information concerning the status of population diversity and stability and concerning the exposure to nonpersistent, nonbioaccumulative contaminants. This is information that is missing from current monitoring and assessment protocols.

Progress Summary:

Miami University, University of Nevada at Reno, and University of California at Davis, Tahoe Research Group continued to participate in the U.S. Environmental Protection Agency (EPA) sponsored project, Multi-Level Indicators of Ecosystem Integrity in Alpine Lakes of the Sierra Nevada during the summer of 2002. Field work and laboratory analyses were similar to previous years and followed the guidelines set forth in the Environmental Monitoring and Assessment Program Surface Waters Field Operations Manual for Lakes (EPA/620/R-07/001). Deviations in sampling procedures and laboratory analysis occurred when the physical features of the water body or sample collections did not lend themselves to prescribed methods. The descriptions of alternative methods and their justification will be described in each appropriate section with detailed methods appearing in the final project report.

During the summer 2002 field season, five sites were completely surveyed, representing four separate lakes. Marlette Lake, on the eastern crest of the Lake Tahoe basin, NV, was visited twice in 2002. The other lakes were sampled only once and included Boca Reservoir and Donner Lake in Nevada County, CA, and Lower Twin Lake in Mono County, CA. In addition, during the summer of 2002, we resampled lakes for which data were missing or analysis indicated a need for additional data.

Physical Water Quality Measurements. At each of the water bodies visited during the summer of 2002, a physical water quality profile was conducted at the index location over the deepest part of the lake. Measurements of temperature, dissolved oxygen (DO), conductivity, and pH were collected from the surface to the bottom of the water body, or a maximum depth of 60 m, using a YSI multiparameter Water Quality Monitor (610DM and 600XL sonde). Two water quality profiles were conducted at Marlette Lake to evaluate changes in the physical characteristics of the water column during the summer season. The first profile was conducted on June 17, and the second was conducted on August 7.

Water Sample Collections. Water samples were collected from two depths (0.5 m below surface and 1 m above the bottom) at the index location at each of the lakes visited during the 2002 field season. Samples to be analyzed for nutrients (total phosphorus, soluble reactive phosphorus [SRP], nitrate [NO3], and ammonia [NH4]) and chlorophyll-a were transported to the Tahoe Research Group laboratory in Tahoe City. Chlorophyll samples were filtered within 24 hours of collection and stored frozen for later analysis. Lake samples collected for dissolved organic carbon (DOC), mercury (Hg), metals, anions, cations, chlorinated hydrocarbons, ortho-phosphates, and polycyclic aromatic hydrocarbons were delivered to the University of Nevada at Reno. DOC samples were filtered within 24 hours of collection.

Marlette Lake and Donner Lake were sampled during each field season. Each lake was visited within the same 2-week period, each year, to minimize water quality and biological differences that develop through the summer months. This allowed for year-to-year comparisons. Additionally, Marlette Lake was sampled twice during the summer of 2002, to evaluate changes that occur with seasonal climatic conditions.

The annual nutrient sampling of Marlette Lake showed little variation in concentrations of phosphorus in both the shallow and deep water. NO3 and NH4 concentrations showed a greater degree of annual change. It is suspected that the difference in NH4 concentration is due to the timing of the spring snow melt. The winter of 2000-2001 was one of the driest on record for the Northeastern Sierra, while the winter of 2001-2002 provided about 75 percent of the normal precipitation. It is likely that Marlette Lake became free of ice much earlier in the past 2 years than it did in 2000. Therefore, although the lake was sampled during the same time period each year, biological activity and algal production may have been at a significantly different stage in the annual cycle.

Evidence that Marlette Lake experiences large, annual changes in nutrient concentration can be seen in the comparison of the June and August sampling events that took place during 2002. Deep samples of phosphorus and NH4 increased dramatically during the summer months. This is likely the result of nutrient release from the sediments, which occurs under anoxic conditions. The process is known as internal fertilization and can lead to diminished water clarity.

At Donner Lake, the total phosphorus concentrations measured in the shallow and deep water during the summer of 2000 were more than twice as those measured in 2001 and 2002. NO3 and NH4 concentrations remained consistent the first 2 years, but showed a decrease in 2002. The chlorophyll concentrations remained similar for the first 2 years, suggesting that nitrogen is a limiting nutrient for algae growth in Donner Lake. If this is the case, one would expect chlorophyll-a levels to be lower for the current sampling year. These samples are stored at the TRG laboratory and will be analyzed this winter.

Benthic Sample Collections. No benthic sediment samples were collected during the 2002 field season. Analysis of samples collected in previous years failed to produce enough organisms for statistical analysis. An alternative method of assessing benthic fauna was developed using a hand-held D-net. Samples were collected at each study lake and processed onsite.

To replace benthic samples, we resampled all lakes' sites from all years using a semiquantitative dipnet technique. Ten areas at each site were chosen to be representative of all habitats at a particular lake. Each area was sampled using a dip net from the sediment surface to the water surface in water up to 1 m in depth. Standard dip net sweeps (2-m sweeps) were taken, and invertebrates were removed to fixation jars until at least 100 organisms were collected or a maximum of 15 sweeps was conducted.

Organisms currently are being identified to species and will be used in place of the originally scheduled invertebrate assemblage information.

Sediment Core Collection and Analysis. Sediment cores were collected from Marlette Lake and Lower Twin Lake during the 2002 field season. Several attempts were made to collect a core from Boca Reservoir. The core continually struck hard substrate and did not return a sample. This problem was experienced in other reservoirs during this study. It is suspected that sediments might be swept clear in the deepest location of the water body, when water is withdrawn. Cores were returned to the TRG laboratory, where they were sectioned into 1-cm segments, frozen, and stored for later analysis. The top and bottom section will be analyzed for diatom assemblage.

Zooplankton Collections. Lakes visited during the 2002 summer field season were sampled for zooplankton assemblage with both an 80-mm and 202-mm mesh zooplankton net. Samples were collected from 1 m off the bottom to the surface. Captured zooplankton were condensed in the nets and preserved. All samples have been sent out for analysis. Preliminary results from the first 2 years of the study indicate a dominance of cyclopoid copepods and cladocerans (Bosmina and Daphnia) in the 80-micron net samples. As much as 50 percent of the organisms identified in the fine mesh nets (202 micron) are copepod nauplii. A more detailed breakdown is underway and should be available soon.

Several of the study lakes (Fallen Leaf Lake, Donner Lake, and Lake Tahoe) are known to contain mysid shrimp (Mysis relicta). This species has not shown up in the zooplankton samples, most likely due to diurnal sampling and the species negative phototaxic response. Mysis shrimp can play an important role in the ecology of aquatic systems.

Fish Assemblage Sampling. Scientific gill nets were deployed in each of the new lakes visited during the 2002 summer field season to determine the resident fish assemblage. Nets were set on the bottom forming a curtain to a height of 2 m. The depths of the net sets varied between lakes, but were set to sample both above and below the thermocline. As in previous years, the gillnet sets proved to be the most effective means of capturing fish species.

Baited minnow traps have been successful in capturing the native, nongame fish species and crayfish. Traps were set on the bottom in depths ranging from near the surface through the thermocline. Traps were left to fish overnight. Both adults and juvenile life stages of fish were susceptible to capture by this gear type. Although known to inhabit several of the water bodies studied, the Piute sculpin (Cotus beldingi) has yet to be represented in the fish catch. This native species is very difficult to capture with traditional scientific sampling gear, yet it can be an important prey species for larger predators.

All of the lakes sampled during the two field seasons have cumulatively produced 16 fish species. Seven of these are considered game fish species and eight are nongame species. Only five species captured are native to the Lahontan Basin and include Lahontan redside shiners (Richardsonius egregious), Lahontan speckled dace (Rhinichthys osculus), Tahoe sucker (Catostomus tahoensis), Tui chub (Gila bicolor), and Mountain whitefish (Prosopium williamsoni). Flesh samples from fish representing the top of the food chain at each of the study lakes have been sent to the University of California at Davis for Hg analysis. Preliminary results indicate that some top predators, greater than 1.5 kg, have the potential to accumulate Hg in concentrations at or above California state health guidelines, while individuals of the same species, less than 700 g total body weight have low concentrations. Furthermore, the data indicate that Hg accumulation is not directly linked to fish size, but may be a function of species diet. Very large lake trout (Salvelinus namaycush) (> 7 kg weight) had Hg levels below 0.2 ppb, while brown trout (Salmo trutta) (< 2 kg weight) had concentrations greater than 1.2 ppb. Rainbow trout (Oncorhynchus mykiss) collected from Marlette Lake had the lowest concentrations of Hg. The exact mechanism for Hg accumulation within the food chain of the Sierra lakes will require further investigation.

Molecular Biomarkers of Environmental Stress. Biological indicators of waterborne contaminants in rainbow trout consisted of measuring changes in transcriptional gene expression (messenger ribonucleic acid [mRNA]). Total mRNA was analyzed for five genes in liver and three genes in gill of 25 fish exposed in situ for 48 hours in each of 16 lakes exhibiting a range of habitat impairment. The habitat impairment range (from degraded to near pristine) was based on approximately 1,100 metrics that defined three categories: (1) water chemistry; (2) human structural impact to shoreline and riparian zones; and (3) recreational use of shoreline, surface water, and riparian zones. The objective of incorporating molecular analysis of rainbow trout tissue in an ecosystem analysis of lake water quality was to link potential degradation of alpine lake habitat quality to changes in physiological health of a representative alpine aquatic vertebrate. Analyses of habitats of the 16 lakes revealed distinct separation of lakes into disturbance groups; lakes such as Eagle Lake and Castle Lake had low levels of anthropomorphic nutrient deposition, low degradation of riparian zones, and little or no recreational activity. Lakes such as Donner Lake and Topaz Lake suffered disturbed riparian, shoreline, and surface water degradation and were significantly higher in disturbance among the 16 lake sites. The link between gene expression in fish and physical metrics of habitat disturbance was shown with a positive correlation between changes in the genes CYP1A1, metallothionein, and activin and the presence of high levels of recreational activity (e.g., motorized boating activity). Recreational activity was the main associative factor in explaining significant differences in gene expression among all 16 lake sites.

In addition to the standard studies at each lake, the following studies were conducted or completed: (1) development and testing of oligonucleotide primer for p53 gene in rainbow trout; (2) completed running molecular biomarker samples collected in 2001-2002; (3) completed third and final field sample collection season; and (4) completed July 4th Donner Lake and Tahoe City experiments investigating temporal induction of CYP1A1 biomarker.

Population Genetic Assessments

Population Sampling. During the summer of 2002, populations of three species were sampled from each lake. Successful collections are indicated by year for each lake and species in Table 1. Unsuccessful collections may have resulted either because the species was not present or because the collection attempts were unsuccessful. For fishes and crayfish, if possible, at least 30 individuals were collected from more than two stations on each lake. All samples are being held in –80°C freezers at Miami University or in alcohol. Enzyme electrophoresis has been conducted on crayfish, Lahontan redside, and speckled dace samples. DNA extractions for amplified fragment length polymorphism (AFLP) analyses have been completed for all redside, dace, and crayfish samples. Quantification and normalization of DNA concentrations has been completed for the three aforementioned species in preparation for polymerase chain reaction (PCR). AFLP optimization for each species is currently underway. Amphipods were not sampled in 2002 because analyses of 2000 samples suggested the presence of multiple cryptic species, which confounded the data and made this species unsuitable as a bioindicator of anthropogenic influence at these sites.

Table 1. Collections for Each Lake and Species as Indicated, by Year

 
Signal Crayfish
Lahontan Redside Shiner
Speckled Dace
Amphipods
Marlette
00, 01, 02
-
00, 01, 02
00, 01
Stampede
00
-
-
00, 01
Castle
-
-
-
-
Eagle
-
-
-
00, 01
Spaulding
00
-
-
00
Prosser
00
00, 02
-
00
Upper Angora
-
01
01
*
Gold
00
00, 01, 02
-
00, 01
Sand Harbor
01
01
01
-
Jackson Meadow
01
01
01, 02
-
Topaz
01
-
-
00, 01
Fallen Leaf
01
01
01, 02
*
Upper Twin
01, 02
-
-
-
Tahoe Keys
00
00
-
00, 01
Donner
00, 01
00, 01, 02
01, 02
00, 01
Tahoe City
01
01
01
-
Cascade
01
01
01
-
Independence
02
02
-
-
Lower Twin
02
02
-
-
Boca
02
02
-
-
Spaulding
02
-
-
-
Key: 00 = year 2000 collection, 01 = year 2001 collection, and 02 = year 2002 collection.
*Amphipods were not found at the lake, but were collected at nearby sites.


Signal Crayfish. Crayfish samples collected from each lake for each year have been prepared for allozyme and AFLP analyses. At least 20 enzymatic loci were surveyed for genetic variability. Three variable enzymatic loci were identified, and analyses have been conducted on all individuals, up to 40 per lake or site where possible. Preliminary data analyses have been completed, and results revealed limited genetic structure within and among populations, with some regional patterns in allelic distributions. Genetic distance was not related to geographic distance, and neither allelic frequencies nor genetic diversity appeared to be related to hydrologic regime or human impact. The lack of a strong relationship between genetic and geographic patterns suggests that the observed genetic structure results from a mechanism such as dispersal by human transport of crayfish as bait, rather than from natural dispersal via stream corridors. Additional analyses using AFLPs and microsatellites currently in progress may be useful in discerning patterns not apparent in our survey of enzymatic loci. DNA extractions have been completed, and the AFLP-PCR optimization on the crayfish samples is in progress.

Lahontan Redside Shiner. Allozyme analyses for four polymorphic loci have been completed on samples from each lake for each year sampled. DNA extractions have been completed for up to 36 samples from each lake with resident redside populations. The DNA samples have been normalized and prepared for mass PCR amplification. Currently, PCR is being optimized for AFLP analysis of the shiner samples.

Data analyses were conducted on 2000 and 2001 samples, but have not been completed on 2002 samples. Allozyme genotype frequency data were analyzed using the computer software TFPGA (Tools for Population Genetic Analysis, Miller 1997). Genotype frequencies met the expectations of Hardy-Weinberg equilibrium for all samples. Allele frequencies differed significantly among sites at all four variable loci surveyed (exact test for population differentiation, P <0.05). Seventeen of 36 pairwise exact tests among samples from all lakes (excluding Gold, due to low sample size) indicated significant differences in allele frequencies (pooled loci) using an adjusted P-value of 0.0013. Allele frequencies of populations from the three sites on Lake Tahoe (Tahoe City Marina, Sand Harbor, and Tahoe Keys) were not significantly different. Allele frequencies in Donner Lake fish differed significantly from all other sites (P <0.001), except Tahoe Keys (P <0.003). Heterozygosity ranged from 0.25 to 0.35, with the lowest heterozygosity at Tahoe Keys and Upper Angora, and the highest heterozygosity at Sand Harbor and Cascade.

These results indicate that gene flow among Lahontan redside populations is limited and that demographic and environmental processes (such as genetic drift, founder effects, and natural selection) have resulted in significant genetic differentiation of populations. These results also alleviate any concern that human transport of fishes among lakes has resulted in complete homogenization of populations. Statistical analyses of relationships among allele and/or genotype frequencies and environmental variables are in progress. The allozyme results also will be analyzed in relation to the results of AFLP-PCR amplification of the same samples.

Speckled Dace. Speckled dace were collected at as many sites as possible in 2001 and 2002 because they were abundant in many lakes and were present in Marlette Lake, where redsides were not. In all, we collected dace in eight different lakes, taking into account the potential for spatial and temporal variation of the genetic structure of the dace populations. Populations were collected at several locations in the lake if adequate populations of the dace were present. When possible, specimens were collected from the same locations during both the 2001 and 2002 field seasons. Enzyme electrophoresis has been completed for up to 40 fish per lake, and preliminary analysis suggests that significant differentiation exists among samples. Analysis of the DNA from the samples will provide additional insight into genetic diversity levels found within the dace populations.

Integration of Studies Using a Geographic Information System (GIS) Framework. The primary objective of this portion of the project was to develop a multilevel index of ecological integrity using several physical, chemical, biological, and anthropogenic variables. This was accomplished in three phases. First, during a 3-year time span, several parameters describing ambient water chemistry, shoreline habitat, and human impact were measured at 16 sites on 14 alpine lakes, surrounding and including Lake Tahoe, CA/NV. Lakes were selected along a presumed human impact gradient. In addition, 25 caged rainbow trout were exposed to surface water at each site for 48 hours to act as sentinel organisms from which molecular biomarker responses could be measured. Spatial data from each site then were integrated into GIS and additional variables describing human impact and lake/watershed morphometry were measured.

Phase 2 involved introducing all variables from each group of measurements (water chemistry, shoreline habitat, human impact, molecular biomarkers, lake/watershed morphometry) into a data reduction process. Principle Component Analysis (PCA) was used to elucidate the most informative measures of ecosystem integrity.

In phase 3, the variables found to be most important from PCA were used to rank each site from high to low integrity. The sums of the ranks of each variable within a group were used to generate an overall "group rank." The mean "group rank" was used as a final index of ecological integrity and the standard deviation of the mean group rank was used as an indicator of variability across variable groups.

The results from this study contribute to the overall understanding of anthropogenic impacts on alpine lake ecosystems and will ultimately assist in refining alpine lake management strategies.

Future Activities:

We have completed data collection from all sites and have begun final analyses of the data. Collections of invertebrates will be repeated from 2002 during the summer of 2003 to have repeated samples for all sites. Data analysis and report generation will occur during the summer of 2003, and a final report will be produced during September 2003.

Journal Articles:

No journal articles submitted with this report: View all 25 publications for this project

Supplemental Keywords:

water, sediments, ecological effects, animal, cellular, population, enzymes, stressor, genetic polymorphisms, susceptibility, chemicals, PAH, heavy metals, pesticides, toxics, ecosystem indicators, aquatic, integrated assessment, ecology, genetics, Environmental Monitoring and Assessment Program, EMAP, measurement methods, EPA Region 9, exposure, biomarkers, limnology, RNA, enzymes., RFA, Scientific Discipline, Geographic Area, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Contaminated Sediments, Genetics, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecological Risk Assessment, Biology, West Coast, EPA Region, Mercury, Ecological Indicators, anthropogenic stresses, EMAP, molecular genetics, Region 9, ecological exposure, sediment, contaminated sediment, metal release, biomonitoring, ecosystem indicators, aquatic ecosystems, contaminant impact, DNA, population-based, Sierra Nevada, genetic differentiation, fish

Relevant Websites:

http://zoology.muohio.edu/oris/tahoe/ Exit

Progress and Final Reports:

Original Abstract
  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report