Letharia vulpina, lichen, WHW, lichens, background levels, JSW, collection, Jedediah Smith Wilderness, Winegar Hole Wilderness, lichen communities, plot, Air Quality, elemental content, Co Cr Cu Fe Hg, abundance, specimens, dry samples, target species, United States Forest Service, Clearinghouse website, Usnea lapponica, Grand Targhee Ski Resort, elemental analysis, National Forests, Ozone Air Pollution, Pacific Northwest Region Air Program, National Forest, Leigh Lakes Basin, Sulfur dioxide, collected, inconspicuous species, Jedediah Smith Wilderness Area, Siuslaw National Forest Supervisor, Yale University Press, Lichens of British Columbia, British Columbia, Oregon State University Press, L. Geiser, nitrogen deposition, lichen species, Biological Trace Element Research, Journal of Radioanalytical and Nuclear Chemistry, United States Forest Service National Lichen & Air Quality Database, Biogeochemistry of Lichens & Mosses, Wilderness areas, Forest Service, Teton County ID, epiphytic lichens, Doug Glavich, The Clean Air Act, J. O. Sickman
Biomonitoring Air Quality with Lichens in the Jedediah Smith Wilderness and the Winegar Hole Wilderness, Caribou-Targhee NF Jill Grenon (USFS R1) April 2008
Air quality monitoring in Wilderness areas: The Clean Air Act
requires the Forest Service to protect air quality related values (AQRV) in Class I Wilderness Areas (those wilderness areas in existence as of August 7, 1977 that are larger than 5,000 acres). Non-Class I Wilderness areas also have management goals identified in the 1964 Wilderness Act. A large piece of Wilderness air protection stems from the Prevention of Significant Deterioration (PSD) provisions of The Clean Air Act. State legislation and PSD regulations determine how the air quality regulatory process is actually conducted state by state. More information can be found at: http://www.epa.gov/air/oaq_caa.html/. Lichens as air quality monitors: Lichens have many attractive qualities as passive monitors of ecosystem health. Monitoring air quality via lichens is relatively inexpensive and economically feasible. Lichens are potentially long lived, are visible at any time of the year, accumulate pollutants throughout the year, may show physical signs of air pollution damage, and have wide geographical ranges, which allow for comparisons with other parts of the region and world. Many lichens are extremely vulnerable to air pollution, especially acidifying sulfur and nitrogen compounds, and fertilizing agents. Ozone appears to be much less damaging to lichen communities in natural conditions
(McCune 1988, Lorenzini et al. 2003, Ruoss & Vonarburg 1995). Lichen communities in polluted environments typically have low diversity, though the abundance of pollution-tolerant species may be relatively high. Elemental content of epiphytic lichens (those growing off the ground in shrubs and trees) has commonly been used as a method to assess atmospheric pollutants (Blett et al. 2003, Geiser and Neitlich 2007, Geiser 2004, Jovon and Carlberg 2006). Lichens lack a waxy cuticle (commonly found in plants) and absorb nutrients and pollutants from wet and dry atmospheric deposition, this characteristic makes lichens useful biologic indicators of air quality. The neon yellow Letharia vulpina and the pale mint green Usnea lapponica are two epiphytic macrolichens that are wide spread within the Caribou-Targhee National Forest and are also commonly used in air quality studies throughout the west. These were the two lichens chosen for elemental analysis in this report.
OBJECTIVES: 1) Establish 6 plots that can easily be replicated during reevaluation visits (Three plots in areas sampled from 1998, three plots in new areas which have never been sampled). 2) Collect, identify, and document the lichen communities in the six plots, include species name and abundance rating. 3) Collect two different 21 gram samples of lichen tissue for elemental analysis from each plot (Collect replicates every few plots). 4) Evaluate overall lichen community composition, compare communities from 1998 to 2008, and include air quality zone ratings 5) Report the elemental content of all established plots and compare to known elemental background levels and levels previously analyzed in 1998. 6) Discuss the air quality of the Jedediah Smith Wilderness and the Winegar Hole Wilderness. 7) Make recommendations based on findings. Plot Location overview for the Jedediah Smith Wilderness (JSW) and the Winegar Hole Wilderness (WHW): In 1998 Larry St. Clair a professor of Botany at Brigham Young
University conducted an air quality assessment in three areas of the Jedediah Smith Wilderness in the CaribouTarghee National Forest using lichens as primary indicators through community analysis and tissue elemental analysis. St. Clair found the JSW area to have clean air as concentrations of all critical pollutants were below background levels and lichen communities were diverse and healthy-looking (St. Clair, L. 2000). St. Clair recommended reevaluation of the three areas evaluated in 1998 and an addition of three more sites in the JSW. In 2008 replicable plots were established in the previously studied and recommended areas. The initial three areas assessed in 1998 were: 1) South Fork of Darby Creek (near trailhead) JSW (DAR033-01) 2) Bitch Creek Trail (near trailhead) JSW (BI-002-01) 3) South Leigh Lakes Basin JSW (LELAKE-01) In addition three plots were added 2008: 1) Bitch Creek Trail (approx. 1.36 miles from trailhead) JSW (BI-002-02) 2) North East of Grand Targhee ski resort JSW (TAR-01) 3) Winegar Hole Wilderness - across from Squirrel Meadows Ranch Road. (WHW-01) The additional plot up Bitch Creek Trail (BI-002-02) was added to compare the lichen community and elemental content to the BI-002-01 plot which lies near the trailhead and a dirt road. The plot behind Grand Targhee ski resort (TAR-01) was added because the resort is planning growth, will be under construction for the next few years, and the wind
blows predominantly from the sites of construction into the wilderness. No previous air quality monitoring exists for the WHW, which is part of the reason WHW-01 was added. Also of concern in the WHW is increasing truck traffic on a dirt road that borders the wilderness and a local gravel pit. Methods: Community analysis: Each plot is a 0.378 hectare circle (114 ft radius); the plot design and methodology follow Geiser (2004) a modified version
taken from the FIA Field Methods Guide: http://fia.fs.fed.us/library/field-guides-methods-proc/docs/2007/p3_40_sec10_10_2007.pdf (also see appendix II). Once the plot boundaries were established with measuring tape and flagging, the epiphytic lichen community was sampled for a maximum of 2 hours. Sampling stopped after 45 minutes if all microhabitats and substrates had been sampled and no new species were found within a ten minute timelapse. Each lichen species collected was given an abundance number (see results). Data about the plot location, elevation, conditions, and vegetation were recorded. Lichens collected for community analysis were identified by Doug Glavich (certified lichenologist, R6) with the exception of two plots: Targhee (TAR-01) and WHW (WHW01) which were identified by the author of this report. Questionable lichens were sent to Doug Glavich for ID confirmation. Elemental analysis: To analyze lichen tissue for elemental analysis twenty-one grams each of Letharia vulpina and Usnea lapponica were collected using sterile methods (Geiser 2004, appendix II) into Kapak bags. In 1998 St Clair analyzed a third lichen, Rhizoplaca melanophthalma, but suspected that the habitat substrate (rock) elevated the elemental outcome. Because of this R. melanophthalma was not analyzed in this report. If Usnea lapponica was not available for collection at a plot, then two samples of Letharia vulpina were collected. Every few plots replicate samples were collected for tissue analysis. Plot names, the lichen species collected at each plot and corresponding abbreviations used throughout this report are listed below in Table 1.
Table 1. Plot names and abbreviations along with the lichen species collected for tissue
analysis from each plot and their abbreviated lichen codes.
Abbr eviated Site Name
Lichens Collected for Elemental Analysis
Abbr eviated Lichen Codes
South Fork of Darby Creek
Letharia vulpina Usnea lapponica
Bitch Creek Trailhead
Letharia vulpina Usnea lapponica
Bitch Creek Trail BI-002-02 Usnea lapponica
Leigh Lake Basin
Letharia vulpina Letharia vulpina
NE of Grand Targhee ski resort
Letharia vulpina Letharia vulpina
Winegar Hole Wilderness
Letharia vulpina Usnea lapponica Usnea lapponica
WHLET WHUSN1 WHUSN2
All debris, necrotic thalli, and mixed species that were not the target species
were removed from the lichen tissue collected for elemental analysis. Samples were then sent to the University of Minnesota Research Analytical Lab for analysis. The lab used ICPAES method to analyze elemental concentrations in parts per million (ppm) of aluminum (Al), boron (B), barium (Ba), beryllium (Be), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), potassium (K), Lithium (Li), magnesium (Mg), manganese (Mn), Molybdenum (Mo), nitrogen (N), sodium (Na), nickel (Ni), phosphorus (P), lead (Pb), Rubidium (Rb), silica (Si), strontium (Sr), titanium (Ti), vanadium (V) and zinc (Zn). The above elements were chosen following Geiser (2004) based on "likelihood of detectable enrichment from anthropogenic sources". Mercury (Hg) was analyzed by atomic absorption spectrophotometry conducted on a mercuryMonitor Elemental Mercury Detector (see Appendix III). Total sulfur (S), nitrogen (N) and ash were analyzed for percent content (see Appendix III). Background levels: Lichen tissue analysis from the new and old sites was compared to known or calculated background levels of elemental content. Background level concentrations for L. vulpina elemental content (except for Hg) were available through The United State
s Forest Service National Lichen and Air Quality Database and Clearinghouse website: http://gis.nacse.org/lichenair/index.php?page=cleansite). These calculations are based on the 97.5% quantile of 159 samples taken in R6. A conservative background level for Hg was calculated based on 75% quantile of available sites. The background levels for L. vulpina in R6 can be used because the habitat and environmental conditions for Letharia vulpina from R6 and the N. Rockies is comparable. For more details on background N levels see Jovon and Carlberg (2007).
Two different background levels of Usnea lapponica were calculated from two separate studies areas; Medicine bow-Routt and White River National Forests (Jackson et.al 1996) and Grand Teton National Park
(NPElement at: http://www.nwhc.usgs.gov/our_research/np_element.jsp). Background levels were calculated using 97.5% quantiles of the cleanest sites. For the study done in the Medicine Bow Routt and White River NF, the Mt. Zirkel Wilderness and the Rabbit Ears Pass sites were excluded from background level calculations because they were considered possible polluted sites. Usnea sp. analyzed from Grand Teton NP was not identified to species. However, only two Usnea sp. have been identified in the park, Usnea lapponica and Usnea substerilis, the former is much more abundant and most likely contributed to the bulk of the tissue collection. We are assuming elemental analysis would not be thrown off in the case of mixed collections because both species share the same genera and growth form. All background levels of lichens used for this report were analyzed with ICP-MS or ICP AES techniques. In 1998 the lichen elemental content for the JSW was analyzed with PIXE (Duflou et al. 1987). RESULTS: Epiphytic lichens species and abundance ratings for each plot: Below is the list of lichen species found at each plot and their corresponding abundance rating. Bolded names indicate species not previously found at that site (doesn't apply to new plots). * Usnea lapponica was misidentified as Usnea subfloridana in 1998 (St Clair, personal comm. 2009). Abundance Rating Description: 1 Rare < 3 individuals/colonies in area 2 Uncommon 4-10 individuals or colonies in area 3 Common 10-40 individuals or colonies in area 4 Very Common >40 individuals or colonies in area but less than half of the boles and branches are covered by the species. Choose one: 4-1 Individuals/colonies are few (between 40-80) and widely scattered around the area 4-2 The lichen is restricted to one or two small areas in the area, usually on just a handful of trees or shrubs. The total number of individuals or colonies is >40. 4-3 Many trees or shrubs have up to 20 individuals or colonies. 4-4 Many trees or shrubs have more than 20 individuals or colonies. 4-5 More than half the trees or shrubs have up to 20 individuals or colonies. 4-6 More than half the trees or shrubs have more than 20 individuals or colonies. 5 Abundant More than half of the available substrate is covered by the subject species.
Darby Creek: DAR-033-01 Alectoria sarmentosa (1) Bryoria fremontii (3) Byroria fuscescens (4-4) Bryoria lanestris (2) Bryoria pseudofuscescens (4-2) Candelaria concolor (3) Hypogymnia imshaugii (1) Letharia vulpina (4-2) Melanelia exasperatula (4-4) Parmelia sulcata (3) Parmeliopsis ambigua (4-5) Parmeliopsis hyperoptera (1) Physcia adscendens (4-4) Physcia aipolia (2) Usnea lapponica (4-6) Usnea scabrata (3) Usnea sp. (3) Usnea cf. rigida (may be rare apotheciate U. lapponica) (3) Xanthomendoza fulva (1) Bitch Creek Trail: BI-002-01 Bryoria fremontii (4-1) Bryoria fuscescens (4-6) Bryoria pseudofuscescens (2) Candelaria concolor (4-1) Hypogymnia austeroides (1) Hypogymnia imshaugii (2) Letharia vulpina (4-4) Melanelia elegantula (4-6) Melanelia subolivacea (4-1) Parmelia sulcata (4-1) Physcia adscendens (4-3) Usnea lapponica (4-6) Xanthoria polycarpa (3) BI-002-02 Bryoria fremontii (4-4) Bryoria fuscescens (4-4) Bryoria pseudofuscescens (2) Candelaria concolor (4-4) Hypogymnia austeroides (2) Hypogymnia physodes (1) Hypogymnia tubulosa (1)
Letharia vulpina (4-1) Melanelia elegantula (4-3) Melanelia exasperatula (4-2) Melanelia subolivacea (4-3) Parmelia sulcata (4-4) Parmeliopsis ambigua (1) Parmeliopsis hyperoptera (1) Physcia adscendens (4-6) Usnea lapponica (4-4) Usnea sp. (could be U. rigida or a rare form of apotheciate U. lapponica) (1) Xanthoria polycarpa (4-1) South Leigh Lakes Basin: LELakes01 Bryoria sp. (1) Candelaria concolor (3) Letharia columbiana (1) Letharia vulpina (4-2) Melanelia exasperatula (4-6) Parmeliopsis ambigua (4-2) Physcia adscendenss (3) Xanthomendoza fulva (4-3) NE of Grand Targhee Ski Resort TAR-01 Bryoria fremontii (4-6) Bryoria fuscescens (4-6) Bryoria implexa (3) Candelaria concolor (4-4) Letharia columbiana (2) Letharia vulpina (4-4) Melanelia elegantula (2) Melanelia exasperatula (4-6) Physcia adscendens (4-3) Usnea lapponica (4-4) Xanthomendoza fulva (4-4) Winegar Hole Wilderness WHW-01 Bryoria fremonii (3) Bryoria fuscescens (4-6) Bryoria pseudofuscescens (2) Candelaria concolor (4-6) Letharia vulpina (4-4) Melanelia elegantula (4-4) Melanelia subolivacea (4-6)
Parmelia sulcata (1) Physcia adscendens (4-4) Usnea lapponica (4-6) Xanthomendoza fulva (4-4) Xanthoria polycarpa (4-4)
Table 2. 2008 elemental analysis of Usnea lapponica from the JSW and WHW, WY. Samples from 1998 represent values from the original analysis. MB-R and WR N.F. = the Medicine Bow- Routt and the White River National Forests. Background levels of elements were calculated from the 97.5% quantiles of clean sites. Elements are presented in parts per million (ppm) unless stated otherwise. Dup = duplicate sample done in the lab for quality control
Element analyzed Total Nitrogen (%) Ash* (%) Total Sulfur ppm Nitrate-N (ppm) Al As B Ba Be Ca Cd Co Cr Cu Fe K Li Mg Mn Mo Na Ni P Pb Rb Si Sr Ti V Zn
MB-R and WR NF backgrnd levels 1.182 4.145 0.150 550.800 1.183 16.933 46.573 8695.250 0.615 0.500 0.758 3.330 389.125 4843.000 0.585 881.725 226.000 0.370 95.050 0.800 1968.750 8.000 807.025 28.373 14.203 1.300 38.158
Teton NP backgrnd levels 8.788 0.101 12887.5 409.625 0.675 8.788 39.888 8210.000 0.388 0.200 0.775 2.988 307.250 4420.000 853.625 181.375 0.200 158.375 1.100 1380.000 3.875 696.125 20.231 19.838 0.887 30.712
B1USN 1.52 2.71 0.084 17.7 351.170 3.046 13.801 0.020 5730.300 0.671 0.780 0.990 3.121 274.850 4199.100 0.379 1245.300 180.310 0.540 115.400 1.018 1107.800 3.520 8.860 496.290 13.914 16.497 0.647 43.748
Dup USNSUB USN1
337.790 3.103 14.038 0.020 6031.800 0.722 0.780 1.129 3.202 260.610 4346.700 0.393 1261.000 183.450 0.540 132.950 1.338 1154.700 3.520 8.860 558.610 14.447 15.747 1.002 44.058
0.077 600 2 41 4200 11.5 1.83 3.2 610 3700 104 1.07 750 3.6 5.5 3500 15 83 1.83 39
1.27 2.79 0.070 12.4 278.560 2.361 51.891 0.020 7834.400 0.500 0.780 1.116 2.678 202.080 4064.500 0.371 977.530 199.480 0.540 119.650 0.850 894.460 3.520 8.860 445.770 19.674 13.060 1.037 37.607
1.40 2.62 0.076 15.0 332.220 2.600 33.625 0.020 7253.800 0.473 0.780 0.924 3.018 246.450 3844.300 0.376 881.610 215.120 0.540 107.030 0.925 880.220 3.520 8.860 565.550 16.344 15.639 1.208 40.866
Table 2. continued. 1994
MB-R and WR NF backgrnd levels
Total Nitrogen (%) Ash* (%) Total Sulfur ppm Nitrate-N (ppm) Al As B Ba Be Ca Cd Co Cr Cu Fe K Li Mg Mn Mo Na Ni P Pb Rb Si Sr Ti V Zn
1.182 4.145 0.150 550.800 1.183 16.933 46.573 8695.250 0.615 0.500 0.758 3.330 389.125 4843.000 0.585 881.725 226.000 0.370 95.050 0.800 1968.750 8.000 807.025 28.373 14.203 1.300 38.158
1995 Teton NP backgrnd levels 8.788 0.101 12887.5 409.625 0.675 8.788 39.888 8210.000 0.388 0.200 0.775 2.988 307.250 4420.000 853.625 181.375 0.200 158.375 1.100 1380.000 3.875 696.125 20.231 19.838 0.887 30.712
1.14 2.92 0.068 17.0 242.710 2.693 23.576 0.020 9046.600 0.368 0.780 0.870 2.847 186.520 4230.500 0.405 1161.000 74.360 0.540 101.850 0.640 867.990 3.520 8.860 359.110 13.811 10.675 1.114 46.003
0.076 320 1.63 37 5800 3.2 1.7 3.3 300 3100 43 1 600 4.7 3.5 1160 10.9 44 1.7 28
1.55 4.77 0.082 40.6 378.670 2.963 45.826 0.020 12177.000 0.348 0.780 1.176 2.903 314.470 2966.400 0.328 971.860 92.278 0.540 142.910 0.990 608.120 3.520 8.860 497.450 26.584 18.218 0.320 36.842
1.85 3.75 0.088 70.1 452.150 3.769 28.527 0.020 8051.800 0.313 0.780 1.076 3.322 379.950 3051.000 0.391 957.730 71.605 0.540 167.700 1.243 746.320 3.520 8.860 591.490 17.129 20.407 0.359 39.216
Table 3. . 2008 elemental analysis of Letharia vulpina from the JSW and WHW, WY.
Samples from 1998 represent values from the original analysis. Background levels of
elements (except Hg, see methods) were taken from the United States Forest Service
National Lichen and Air Quality Database and Clearinghouse website
(http://gis.nacse.org/lichenair/index.php?page=cleansite). Elements are presented in parts per
million (ppm) unless stated otherwise. Dup = duplicate sample done in the lab for quality
L. vulpina background levels
2008 WHLET Dup
Nitrate-N-ppm Ash* (%) Total Sulfur (%) Total Nitrogen (%) Al B Ba Be Ca Cd Co Cr Cu Fe Hg (ppb) K Li Mg Mn Mo Na Ni P Pb Rb Si Sr Ti V Zn
2.6 0.08 1.03 614 3.5 52.1 0.04 7325 0.4 0.4 2.1 14.9 472 781 3243 0.4 833 408.5 0.3 134 2.7 1147 7 53 728 31.7 56 1.5 43.6
0.80 273.050 1.575 20.360 0.020 12300.000 0.651 0.780 0.809 1.847 194.220 383.910 3624.000 0.389 970.350 75.156 0.540 116.190 0.640 970.490 3.520 8.860 431.610 16.472 12.404 0.761 46.833
180 43 5200.00 25 1.64 2.2 210 3500.00 42 0.62 780 4.4 5.3 920 17 33 5.2 33
1.36 477.090 2.718 13.002 0.020 6407.200 0.755 0.780 1.062 2.769 373.090 320.780 4004.400 0.578 1310.500 187.420 0.540 123.630 1.004 884.710 3.520 8.860 443.480 14.412 24.991 1.794 48.098
810 52 7600 4.9 1.99 3.5 820 2800 70 1.24 640 7.2 4.6 3300 18 130 1.99 37
0.93 443.780 1.697 25.680 0.020 4951.100 0.688 0.780 1.000 1.983 316.210 249.570 3476.500 0.554 944.440 95.254 0.540 104.690 0.851 672.500 3.520 8.860 561.920 12.281 21.612 1.884 45.485
1.35 604.190 2.232 10.251 0.020 6007.200 0.487 0.780 1.433 2.305 485.830 255.730 2251.900 0.448 742.040 40.635 0.540 132.180 1.072 465.390 3.520 8.860 596.160 8.374 28.477 0.540 39.563
554.340 2.191 9.780 0.020 5829.400 0.439 0.780 1.302 2.225 443.750 2134.600 0.405 711.510 38.720 0.540 119.520 0.991 447.650 3.520 8.860 606.610 8.042 25.054 0.320 37.832
Table 3. continued.
L. vulpina background levels
Nitrate-Nppm Ash* (%) Total Sulfur (%) Total Nitrogen (%) Al B Ba Be Ca Cd Co Cr Cu Fe Hg (ppb) K Li Mg Mn Mo Na Ni P Pb Rb Si Sr Ti V Zn
2.6 0.08 1.03 614 3.5 52.1 0.04 7325 0.4 0.4 2.1 14.9 472 781 3243 0.4 833 408.5 0.3 134 2.7 1147 7 53 728 31.7 56 1.5 43.6
11.9 5.25 0.090 1.11 572.140 2.362 14.420 0.020 13549.000 0.431 0.780 1.030 2.233 430.750 349.980 2616.800 0.500 839.740 63.498 0.540 127.200 0.802 576.750 3.661 8.860 475.940 11.225 22.745 0.320 28.944
2008 TARLE1 Dup 441.300 1.985 13.759 0.020 12481.000 0.342 0.780 0.852 1.959 329.730 2457.000 0.404 779.220 61.545 0.540 118.380 0.820 531.140 3.520 8.860 354.310 10.506 18.271 0.320 26.725
2008 TARLE2 9.4 6.42 0.070 1.03 244.720 1.726 10.056 0.020 19212.000 0.436 0.780 0.666 1.477 176.830 2387.700 0.245 644.300 56.352 0.540 108.790 0.640 515.930 3.520 8.860 522.710 11.318 9.406 0.320 32.737
2008 LALE2 Dup
1998 L. LAKESLETVUL
1.53 680.290 2.591 14.210 0.020 7622.000 0.454 0.780 1.259 2.878 522.680 452.130 2729.800 0.423 909.760 115.330 0.540 180.450 0.640 717.660 4.380 8.860 246.840 11.826 28.777 0.911 34.015
1.35 655.260 3.028 13.163 0.020 7294.400 0.504 0.780 1.039 2.883 504.200 2778.700 0.465 848.870 111.210 0.540 195.050 0.640 729.330 5.394 8.860 448.730 12.445 25.484 0.784 29.870
639.930 2.988 12.413 0.020 6695.900 0.495 0.780 1.124 2.740 493.950 2624.300 0.404 789.060 102.440 0.540 191.910 0.849 690.680 4.815 8.860 379.610 11.401 26.233 0.670 28.597
0.079 410 31 4600 3.9 1.41 2.8 500 3200 200 0.67 540 3.7 7.9 2000 9.1 83 1.41 38
Nitrophytes and Acidophytes: Geiser and Neitlich (2007) have created six "air quality zones" that lichens exist in (based on Indicator Species Assessment and Percentiles): Best, Good, Fair, Degraded, Poor, and Worst. Lichens that exist in Poor and Worst are considered pollution tolerant whereas lichens that exist in Best are the most pollution sensitive. Different lichen species have different tolerance levels for environmental change such as increases in pollution deposition and pH change. Lichens that can tolerate an increasing pH change are known as Nitrophytes. Nitrophytes are often found to inhabit the "Worst
and Poor" airsheds. Lichens that inhabit acidic substrate are called Acidophytes, acidophytes are typically pollution sensitive and most often found in the "Best and Good" airsheds. The following is a list of nitrophytes, acidophytes, and neutrophytes that were found in the JSW and WHW; the list is based on research from the Pacific North West (Jovon 2008, Geiser and Neitlich 2007).
More details about individual air pollution sensitivity ratings for species can be found at The United States Forest Service National Lichen & Air Quality Database and Clearinghouse website: http://gis.nacse.org/lichenair/index.php?page=sensitivity.
Table 4. A list of the nitrophytes, acidophytes, and neutrophytes lichens found in the JSW and WHW and the corresponding air quality zone these lichens are associated with.
Air quality Zone
Potential acidophytes Bryoria fremontii Bryoria fuscescens Bryoria pseudofuscescens Hypogymnia imshaugii Hypogymnia physodes Hypogymnia tubulosa Parmeliopsis ambigua Parmeliopsis hyperopta Usnea lapponica Usnea scabrata
Best Best Best Good Fair Fair Best Best Poor Best
Potential neutrophytes Melanelia elegantula Melanelia exasperatula Melanelia subolivacea Parmelia sulcata
Worst Worst Worst Fair
Discussion: Lichen Community Composition The major difference in lichen composition between 1998 and 2008 is the presence and increased observations of Candelaria concolor, Physcia adscendens, Physcia aipolia, Xanthomendoza fallax, and Xanthoria polycarpa, all of which are known nitrophytes (Table 4., Jovon 2008.). C. concolor and P. adscendens were found at every plot in 2008 whereas in 1998 no sites reported C. concolor and only one site (South Fork of Darby Creek) recorded P. adscendens present. Because no replicable plots were established in 1998 it is more difficult to compare lichen communities between the two years. The species mentioned above maybe small and inconspicuous, but it is hard to believe they were completely overlooked in 1998. Many of the nitrophytes listed above scored high enough abundance ratings and should have been noticed in the first round of community surveys. Nitrophytes thrive in areas with increased pH and nitrogen levels, and often are found to dominate some of the "Worst" air quality zones. The abundance and diversity of nitrophytes found in all plots in 2008 does not, however, signify degraded airsheds in the JSW and WHW. Nitrophyte presence may be explained by the surrounding geology which is karst limestone. Limestone is a known base and may raise the pH of lichen inhabited substrate or directly deposit dust on the lichen thalli. Aside from the presence of nitrophytes many acidophytes that occur in the "Best" air quality zones were abundant on the plots, namely different Bryoria species. The only plot where a high abundance rating of Bryoria didn't occur was the South Leigh Lakes Basin plot. Leigh Lakes Basin is a very high elevation, exposed site, where most lichens grow out of arms reach due to deep winter snow packs. The abundance of Bryoria sp. at this site may have been underestimated due to tree branch access or harsh environmental factors may prohibit Bryoria growth. Another sensitive indicator of air quality Parmeliopsis ambigua was found at the Leigh Lakes Basin plot, this suggests the lack of Bryoria found is not an air quality issue. The presence and abundance of sensitive indicator species at each plot along with diverse lichen communities suggests the air quality zone in the JSW and WHW is in the "Good to Best" category. Lichen species listed from the "Best" air quality zones (Table 4.) should be monitored for decreases in abundance and diversity. Nitrophytes should be monitored for increases in diversity and abundance. Elemental Analysis: When comparing elemental content of Letharia vulpina in Region 2 to background levels from Region 6, we realize that environmental factors may cause differences in tissue analysis outcomes; for instance Sodium (Na) tends to be higher in Oregon and Washington because of increased precipitation (Geiser L. per. comm.), whereas N and S are naturally elevated in drier sites due to lack of precipitation (Jackson et.al 1996, Jovon and Carlberg 2007, Geiser L. per. comm.).
Sulfur dioxide: Sulfur dioxide (SO2), an acidifying agent, is well known to have detrimental effects on lichen communities. In many parts of the U.S. sulfur dioxide is not the threat it once was to lichen communities, due to decreasing emissions in recent years. Fossil fuel combustion, vehicle exhaust, paper manufacturing, and industries produce SO2. Sulfur dioxide (SO2) is naturally found in the environment at low concentrations. St. Clair (2000) recognized the Sulfur threshold level for lichens at 0.2%. The United States Forest Service National Lichen & Air Quality Database and Clearinghouse (http://gis.nacse.org/lichenair/index.php?page=cleansite) documents background S % level for Letharia vulpina to be 0.08. Background levels of Usnea lapponica from the Mount Zikel Wilderness study were calculated to be 0.15%. All lichen tissue collected in the Caribou-Targhee National Forest both in 1998 and 2008 were below the 0.15% level. However, lichen tissue collected from Letharia vulpina at Leigh Lakes Basin (LALE1 and LALE2), Targhee Ski Resort (TARLE1), and WHW (WHLET) were above the 0.08% level, with the highest S content coming from Leigh Lakes Basin (0.145 and 0.125). The level of Sulfur at Leigh Lakes Basin 1998 was recorded as 0.079%. The difference in S between 1998 and 2008 may be due to differences in lab technique or environmental conditions and should be paid attention during the next round of surveys. Nitrogen Unlike Sulfur, inputs of fixed nitrogen into ecosystems of the United States have doubled since 1961 due mainly to agricultural application of nitrogen fertilizer
s, combustion of fossil fuels, and industry (Howarth et al. 2002). Nitrogen deposition occurs in three main forms HNO3, NH3, and NOx. Ammonium nitrate (NH4NO3) is a major component of the fine particulate matter and is likely active in altering the lichen communities when present. Ammonia (NH3) and Nitric acid (HNO3) deposition has been documented to cause a shift in lichen communities (Riddell et. al 2007, van Herk et al. 2003, van Dobben & ter Braak 1999, Wolseley & Pryor 1999). No previous studies have acknowledged N content (% or ppm) of lichens in the JSW or WHW. Background levels of %N in lichens for Letharia vulpina have been estimated at 1.03 and for Usnea lapponica 1.182. Percent Nitrogen was slightly above background levels at most plots but not elevated enough to warrant concern. WHW-01, BI-002-01, and LELAKES-01 had the highest % N content. WHW-01 and BI-002-01 are both close to dirt roads, LELAKES-01 is a high and dry elevation site and also had the highest S %. Parts per million (ppm) of N were established for all sites and can be used as a comparative record for future research. Mercury In 1990 The Clean Air Act acknowledged Mercury (Hg) as a highly toxic pollutant that needed to be controlled. By 1999 the US industrial emissions of Hg dropped by 45% (from 1990), from 220 tons to 115 tons per year (see USEPA http://www.epa.gov/mercury/control_emissions/emissions.htm). Anthropogenic Hg emission sources in the U.S. come from coal-fired electric power plants (40%), industrial
boilers (10%), burning hazardous waste (5%), and chlorine production (10%) (EPA: 1999 National Emissions Inventory). Hg had not previously been analyzed in the JSW and WHW. A conservative calculation found a background Hg level in dry sites to be 781 ppb. Leigh Lake Basin had the highest concentration of Hg at 452.13 ppb. All Hg levels in the JSW and WHW were found to be well below the calculated background level. These established Hg levels can now be used for future research in the JSW and WHW. ICP-AES elements: None of the elements analyzed for ICP were elevated to a level of concern. However there were patterns of slight elevation above background levels. Mg was slightly elevated at all sites for all samples. Zn and Cd were elevated at most of the sites for both sample species. Cr was elevated in all the Usnea lapponica samples but none of the Letharia vulpina samples. Ca was elevated at DAR033-01, TAR-01, and WHW-01. Fe was elevated in L. vulpina samples at LELAKE-01, WHW-01, and TAR-01. Al was elevated in all the LELAKE-01 samples. The elevations in the elements analyzed with ICP may be due to the limestone and volcanic geology of the JSW and WHW. Mg, Zn, Cr, Ca, Fe, Al, K, Na, and V are all elements found in limestone (Dim et.al 1991). BI-002-01 had slightly higher concentrations of metals than BI-002-02 which may be explained by location. BI-002-01 is right alongside a gravel road and is more exposed to disturbance and dust than BI-00202. Many trace elements
at LELAKE-01 were elevated especially Al. Overall Conclusions: Many nitrophytic lichen species were found in 2008 that were not present 1998; this may signify a transitioning lichen community. However, sensitive lichen species were found in abundance at every plot. The lichen diversity and abundance were typical for the geographic area and indicates healthy lichen communities and a clean airshed. Slightly elevated N and S were present but expected due to the lack of summer precipitation in the JSW and WHW. Hg (ppb) levels were all below background levels. None of the ICP analyzed elements were alarmingly above background levels. The slight elevations noted may be influenced by the limestone geology. The South Leigh Lakes Basin did have many elements slightly raised above background levels. Due to its subalpine nature and naturally low biotic activity, LELAKE-01 may be the most sensitive plot to pollution deposition (Fenn et al. 2003a, 2003b). A gradient was noticed between the two Bitch Creek Trail sites where higher concentrations of metals existed at BI002-01 nearer to the gravel road. Overall the lichen community analysis and elemental analysis indicates the air quality of the JSW and WHW airsheds was clean in 1998 and remains so as of 2008. However, because the JSW and WHW are located downwind from Teton Valley airquality monitoring should continue. Concerns for the future health of the JSW and WHW
airsheds include but are not limited to the rapid growth in Teton County, wood burning stoves, pesticide use, increased traffic, and long distance transport. Teton County in ID currently supports 8, 349 residents (2007) (http://quickfacts.census.gov/qfd/states/16/16081.html), in 2000 it was estimated that about 17% of existing household use wood burning stoves as primary heat source
in the winter (http://factfinder.census.gov/servlet/QTTable?_bm=y&qr_name=DEC_2000_SF3_U_DP4&-ds_name=DEC_2000_SF3_U&-_lang=en&_sse=on&-geo_id=05000US16081). Recommendations: 1) Due to a possible changing community and slightly elevated chemistry, resample lichen communities and lichen tissue analysis at the known plot locations in the next 5 -8 years with special attention paid to N concentrations. 2) Monitor diversity and abundance or decline of nitrophytes and sensitive acidophytes, especially note any decline in Bryoria sp. diversity or abundance. 3) Closely note S, N, Al, Cr, and Zn concentrations at Leigh Lakes Basin during the next sampling period
. 4) Arsenic was not measured in 2008. It was slightly elevated in 1998 at one site and should be reanalyzed during the next round of sampling. 5) Due to construction and possible increase in traffic closely monitor changes in TAR01 and WHW-01. 6) Add bulk deposition samplers or passive monitors near designated plots to calibrate and substantiate the lichen data. Acknowledgements: Thanks to Chad Grossenburg (USFS R4) for instigating and overseeing the continuation of this project and for creation of the WHW map. Rob Marin (USFS R4) for GIS work in the field, the creation of all the maps except WHW, and for collecting most of the lichen tissue for elemental analysis. Linda Geiser (USFS R6) for all her advice on the elemental analysis interpretation. Doug Glavich (USFS R6) for lichen identification and advice. Larry St. Clair (Bingham Young University) for his previous work and advice. And finally, thanks to the Caribou-Targhee National Forest for funding this project. References: Air Quality Monitoring in Wilderness, EPA: http://www.epa.gov/air/ Blett, T., L. Geiser, & E. Porter. 2003. Air pollution-related lichen monitoring in National Parks, Forests, and Refuges: Guidelines for studies intended for regulatory and management purposes. USDA National Park Service Air Resources Division and US Fish & Wildlife Service Air Quality Branch, USDA Forest
Service. NPS D2202. Dim L.A., Adetunji J., Okujeni C.D., Elegba S.B., Agaja S.A. 1991. Instrumental Neutron-Activation analysis of limestone and associated calcite samples from Abakaliki,
Lower Benue Trough, Nigeria. Journal of Radioanalytical and Nuclear Chemistry. 148(1): 145-153. Duflou H., Maenhaut W., and DeReuck J. 1987. Application of PIXE analysis to the study of regional distribution of trace elements in normal human brain
tissue. Biological Trace Element Research. 13:1. EPA Emissions Inventory: 1999 National Emissions Inventory EPA Mercury website: http://www.epa.gov/mercury/control_emissions/emissions.htm Fenn, M. E., J. S. Baron, E. B. Allen, H. M. Rueth, K. R. Nydick, L. Geiser, W. D. Bowman, J. O. Sickman, T. Meixner, D. W. Johnson, & P. Neitlich. 2003a. Ecological effects of nitrogen deposition in the western United States. Bioscience(53): 404-420. Fenn, M. E., M. A. Poth, A. Bytnerowicz, J. O. Sickman, and B. Takemoto. 2003b. Effects of ozone, nitrogen deposition, and other stressors on montane ecosystems in the Sierra Nevada
. in A. e. a. Bytnerowicz, editor. Ozone Air Pollution in the Sierra Nevada. Geiser, L., Neitlich, P.N. 2007. Air pollution and climate gradients in western Oregon and Washington indicated by epiphytic macrolichens. Environmental Pollution
. 145: 203218. Geiser L. per. comm. 2009. Ecologist. US Forest Service, Pacific Northwest Region Air Program, Corvallis, OR 97339-1148. Geiser, L. 2004. Manual for Monitoring Air Quality Using Lichens on National Forests of the Pacific Northwest. USDA-Forest Service Pacific Northwest Region Technical Paper, R6-NR-AQ-TP-1-04. 126 pp. Howarth R.W., Boyer E.W., Pabich W.J., Galloway J.N. 2002. Nitrogen use in The United States from 1961-2000 and potential future trends. Ambio 31: 88-96. Jackson L.L., Geiser L., Blett T., Gries C., Haddow D. 1996. Biogeochemistry of Lichens & Mosses in and near Mt. Zirkel Wilderness, Routt National Forest, Colorado: Influences of coal-fired power plant emissions. Open-file Report, USGS. Jovon S., 2008. Lichen bioindication of biodiversity, air quality, and climate: Baseline results from monitoring in Washington, Oregon, and California. USDA-FS, PNW Research Station. General Technical report
: PNW-GTR-737. Jovon S. and Carlberg T. 2007. Nitrogen content of Letharia vulpina tissue from forests of the Sierra Nevada, California: Geographic patterns and relationship to ammonia estimates and climate. Environ Monit Assess. 129: 243-251.
Lorenzini G, Landi U, Loppi S, et al. 2003. Lichen distribution and bioindicator tobacco plants give discordant response: A Case study
from Italy. Environmental Monitoring and Assessment. 82 (3): 243-264. McCune, B. 1988. Lichen communities along O3 and SO2 gradients in Indianapolis. Bryologist 91:223-228. NPElement: A Database of Lichen Elemental Concentrations in the U.S. National Parks http://www.nwhc.usgs.gov/our_research/np_element.jsp Riddell J., Nash T.H. III, Padgett P. 2008. The effect of HNO3 gas on the lichen Ramalina menziesii. Flora 203: 47-54. Ruoss, E. and C. Vonarburg. 1995. Lichen diversity and ozone impact in rural areas
of central Switzerland. Cryptogamic Botany 5:252-263. St. Clair, L. L. and Porter, L.D. 2000. Establishment of a Lichen Biomonitoring Program and Baseline for Jedediah Smith Wilderness Area of the Traghee National Forest, Wyoming. Brigham Young University
, Provo, UT. Teton County ID Quick Facts: (http://quickfacts.census.gov/qfd/states/16/16081.html) The United States Forest Service National Lichen & Air Quality Database and Clearinghouse (http://gis.nacse.org/lichenair/index.php?page=cleansite) University of Minnesota Research Analytical Lab: http://ral.cfans.umn.edu/index.htm U.S. Census, Teton County ID. 2000. Housing Characteristics http://factfinder.census.gov/servlet/QTTable?_bm=y&qr_name=DEC_2000_SF3_U_DP4&-ds_name=DEC_2000_SF3_U&-_lang=en&_sse=on&-geo_id=05000US16081 van Dobben H.F. and ter Braak C.J.F. 1999. Ranking of epiphytic lichen sensitivity to air pollution using survey data: a comparison of indicator scales. Lichenologist 31:27-39. Van Herk, C. M., E. A. M. Mathijssen-Spiekman, and D. de Zwart. 2003. Long distance nitrogen air pollution effects on lichens in Europe. Lichenologist 35: 347-359. Wolseley P.A. and Pryor K.V. 1999. The potential of epiphytic twig communities on Quercus petraea in a Welsh woodland site (Tycanol) for evaluating environmental changes. Lichenologist 31: 41-61.
PLOT DIRECTIONS AND MAPS:
Table 5. summarizes information that may be helpful for repeated lichen tissue
collection. ABBI = Abies bifolia, PIEN = Picea engelmanii, PIAL = Pinus albicaulis,
PICO = Pinus contorta, and PSME = Pseudotsuga menziesii.
Substrate collected from
DAR033-01 Letharia vulpina
PIEN and PSME branches and boles
BI-002-01 Letharia vulpina
PICO and ABBI branches and boles
BI-002-02 Usnea lapponica
PICO, PSME, and ABBI branches and boles
LELAKE01 Letharia vulpina Letharia vulpina
Snag/lignin, PIEN, and PIAB branches and boles
Letharia vulpina Letharia vulpina
PIEN and ABBI branches and boles
WHW-01 Usnea lapponica
Snag, PIEN, and PICO Branches and boles
Leigh Lake Basin 7/31/2008 Plot ID #: LeLake-01 UTMs: Map: Granite Basin Directions: From Grand Targhee Ski Resort take the lift or hike up to trail 025. When trail 025 junctions with trail 023 continue up the ridge (East) ~5500 ft. to knoll marked "x" ats 9570 ft. on the Granite Basin 1:24,000. map. The Jump off point is slightly S. of lakes (but you can see some of the lower lakes). Drop off ridge ~170ft N, NW to the first clump of trees. Plot center is the Huge Pinus albicaulis. There are not many trees E of plot center, most trees are N and W of plot center. Plot is WSW of lowest lake. Plot Discription: This high alpine plot lies at 9400ft on the lower 1/3 of a north-facing slope. Snow was still present on plot or had just melted. The ground was mostly duff with light vegetation primarily of grasses and Western Springbeauty (Claytonia lanceolata). Abies bifolia was predominant tree, intermixed were dead or dying Pinus albicaulis and large Picea engelmannii. Snow level is high in the winter making it hard to reach lichens in the summer. Lichen abundance was high, but for the most part out of reach. Diversity is probably more rich than reported through community sampling. It would be best to collect from this plot following a storm when freshly fallen branches may litter the ground.
Lichen collected: Letharia vulpina, 32g, dry Letharia vulpina (replicate), 32g, dry Lichens were collected predominately from the SW corner of the plot on lignin. South Fork Darby Creek 7/29/2008 Plot ID #: DAR033-01 UTMs: Map: Mt Bannon Directions: From the trail head of Darby Creek trail 033 walk 900ft down path to the first user split trail that sandwiches a tree. Turn S. off trail and walk 80ft to three trees >20" DBH. The trees line the N side of a dry creek bed (may not be dry depending on when visit). Plot center is the middle Picea engelmannii. Plot Discription: Darby Creek Trail runs through the N. end of the plot which is flat. S of plot center hill slopes at 25 % and is N. facing. The plot is a mixture of large and pole/saplings. There are 300+ year old Doug fir on plot, but they are not considered old growth due to beetle kill. Picea engelmannii dominates the plot with Abies bifolia and Pseudotsuga menziesii mixed in. The underbrush is thick with many high bushes--low visibility. Abundance of Usnea and Melanelia is very high. Lichen collected: Usnea, 27g, Dry Letharia vulpina, 22g, Dry Lichens collected over whole plot area. Bitch Creek/Coyote Meadows 7/30/2008 Plot ID #: BI002-01 UTMs: Map: Hominy Peak Directions: From the Center of the parking lot sign walk S. down the road (24 paces). Walk E. at 91 ° for 125 ft. Plot center is a 13" PICO on the bench before the plot slopes down to a field SE with a horse trail running through it. Plot Description: Open PICO stand with LOIN and ABBI interspersed. Lots of grasses and flowering plants carpeting the floor, no duff/rocks/dirt spots. Few dead snags. Road 265 runs just W. of the plot~ 10 feet from plot edge. Trails border N and S sides of plot. Lichen collected: Usnea, 28 g, dry Letharia vulpina, 22g, dry Collected over the whole plot. Bitch Creek 7/30/2008
Plot ID #: BI 002-02 UTMs: Map: Rammel Mountain Directions: Take 265 to Coyote Meadows/Bitch Creek Trail head. Follow the Bitch Creek Trail approx. 7140 ft. Go past the Wilderness sign, immediately after crossing the first creek (Bitch Creek) continue to follow creek instead of trail NE at 50 ° (on the South side of creek). Plot center is the first Abies Bifolia you come to. Plot Description: Large old Doug fir and Subalpine fir near the creek. The few Doug firs are dead or dying, probably due to bark beetle. The slopes on both sides of the creek are predominately second or third growth Lodgepole pine with a few ABBI mixed in. Most of the lichen diversity is found on the older trees near the creek. High lichen abundance near creek, not so much on the PICO filled slopes of the plot. The undergrowth is lush near the creek with lots of grasses. Lichens collected: Usnea, 26g, dry Usnea replicate, 26g, dry Letharia vulpina, 19g, dry Lichens were collected along the creek. Letharia was collected off plot. Grand Targhee Ski Resort 9/16/2008 Plot ID #: TAR-01 UTMs: zone: 12 Northing: 4849457 Easting: 504720 Map: Directions: From the base of Grand Targhee Ski Resort take the Blackfoot Return Cat Track to the boundary with the Jedediah Smith Wilderness. Stay on Contour and walk approx. 1,900 feet East SE to plot center (see map). Plot Description: Nice open large Engelmann Spruce Forest with Sub-alpine firs mixed in. Steep north-facing side hill, with dense ground layer of asters, box elderwood, and grasses. Uphill portion of plot is really open. Predominant lichens are Melanelia spp. which covered most branches, xanthoria spp., usnea spp., and candelaria spp. were also abundant. Xan. and Can. abundance could be due to limestone bedrock. Most of the lichens are high and out of reach due to snow levels in the winter. Lichens collected: Letharia vulpina, 18g, dry Letharia, vulpina, 18g, dry replicate Lichens were collected along the bottom half of the plot on PIEN, ABBI BR/BO. Winegar Hole Wilderness 9/17/2008 Plot ID #: WHW-01 UTMs: zone: 12 Northing: 4879916.80 Easting: 499421.93
Map: Sheep Falls Directions: From the Junction of highway 32 and Ashton-Flagg Ranch rd. head East approx. 22.5 miles to the junction of Ashton-Flagg Ranch rd. and the Squirrel Meadow Ranch Rd. This is the jump-off point. Walk NNE of the junction approx. 1,040 feet to plot center. Plot Description: Plot center is a 28" DBH Engelmann Spruce tree. This plot is a fairly open Engelmann Spruce and Lodgepole pine forest
. Few nice large Engelmann Spruce and Cottonwoods. Lots of bear and deer signs. A field borders NW/W of plot center. Plot is flat with lots of grasses and understory forbes. Bryoria, Usnea, Melanelia, and Xanthoria are the dominant lichens. Lichens collected: Letharia vulpina, 17g, dry Usnea spp, 20g, dry Usnea spp. 18g, dry replicate Lichens were collected over the whole plot and into the field East of plot from PIEN, PICO, and snags.
Appendix II How to collect lichen tissue for elemental analysis and methods for lichen community analysis. *Information that was not relevant to perform a revisit on the Jedediah Smith Wilderness (JSW) and the Winegar Hole Wilderness WHW) was deleted, a few comments were adjusted to fit the methods used for lichen community analysis and tissue collection on the JSW and WHW. TISSUE COLLECTION AND element analysis
METHODS Element content refers to the concentration (percent or ppm dry weight) of selected elements. Elements are selected for analysis based on the likelihood of detectable enrichment from anthropogenic sources. ICP-AES, or preferably ICP-MS, analysis provides a suite of elements for a set cost. Typically, this includes aluminum (Al), boron (B), barium (Ba), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na), nickel (Ni), phosphorus (P), lead (Pb), silica (Si), strontium (Sr), titanium (Ti), vanadium (V) and zinc (Zn). Total sulfur (S) and nitrogen (N) are measured by separate methods, and concentrations of these elements provide the best estimate of the exposure of lichens to harmful pollutants. Because of their environmental toxicity, mercury (Hg) and arsenic (As) are important elements to monitor, but they are much more expensive to analyze, and extra care must be taken in the field and laboratory to avoid volatilization leading to low recovery. Each sample for element analysis must contain only one lichen species and be free of visible dirt, bark and other surface grit. Because lichen species differ in their ability to accumulate elements and because measurements must be comparable across national forest boundaries, only a few regionally common species are used for tissue analysis. The elemental content of selected lichen species establishes site baselines for toxic elements and determines regional toxic profiles. Lichen elemental content is clearly indicative of key assessment questions, especially those concerning contamination of natural resources
. Because a suite of elements is measured, it is sometimes possible to identify the different sources of contamination such as fossil fuels, saltwater aerosols and agricultural dusts (e.g. Sloof 1995, Reis et al. 1996). Elemental content is determined by various methods. Typically, a 10 g, cleaned and dried, composite sample can adequately represent the mean element content at a plot. Samples are handcleaned of debris, oven dried, then ground. A bulk collection of thoroughly dried and ground material, if stored in the dark, can be used to assess laboratory precision over a period of about ten years. Because lichens hydrate readily, and hydrated material can decompose, storage of the bulk collection in the freezer is preferable to storage at room temperature. Recently, it has become possible to acquire standardized lichen materials (SRMs) (see section 2.38). Lichen SRMs are highly desirable because some important elements, such as sulfur and nitrogen, can be considerably lower in lichen samples than in plant SRMs. Sample collection procedure What to collect
Collect > 20 grams each of two target lichens, dry weight. Dusty, gritty, discolored, or decaying material should be avoided. Collect replicate samples as described in the quality control section below. Where to collect The target species should be collected from a minimum of 6 different locations near or within the plot. Lichens attached to tree branches, shrubs or tree boles, in the litter, or on fallen branches, may be used. Exceptions are target species in the genera Alectoria, Bryoria and Usnea. These deteriorate quickly on the forest floor and should not be collected from litter or fallen branches. In non-forested areas, Xanthoparmelia cumberlandia may be collected from rocks. Replicates and repeat collections should be made from the same host species and types of substrate locations, in roughly the same proportions. Collections should be made near or on the plot but not more than 1 km (.65 miles) away from the plot perimeter--approximately 20% of the distance to the next plot. Lichens should not be collected within 35 m of any road. Off-plot collecting will increase the probability of finding a given target species at each collection site. How to collect Most samples can and should be collected with the fingers. Non-powdered vinyl gloves are worn to prevent contamination of the samples. While wearing gloves the field crew should not touch anything brought with them onto the plot except the Kapak bag. Unused Kapak bags should be stored in a clean zip-loc plastic bag. New gloves will be used at each plot and replaced if they become torn or contaminated. A clean, stainless steel knife may be used to collect target species, e.g. Xanthoparmelia, that are tightly adhered to the substrate. Keep the designated collecting knife in a separate, clean, plastic bag and wash it after several uses with soap and water. Snow and ice remove lichens from tree boles and in some areas, particularly high elevations in the Cascades, lichens may be above arm's reach. In these cases, it may be a good idea to carry a pole length tree pruner to saw off small branches with high target lichen cover. The samples should be collected as clean and free from bark and other foreign surface material as is practical and will be cleaned carefully later in the office. Place samples in metalized polyester Kapak bags and weigh on a 100 g Pesola spring scale. If the lichens are dry, the sample and bag together should weigh > 28 g. If the lichens are wet, the bag should weigh more than 100 g and adequacy of the sample size should be judged by volume rather than weight. After enough material has been collected, press out excess air, fold the open edge of the bag over three times, and carefully seal with waterproof, removable, laboratory tape. The bag should be airtight. What to record The following information should be recorded directly on the Kapak bag, and also on the field data card. Write on the bag with an indelible marker: 1. Plot number
2. Date 3. Substrate(s): Host species name and substrate location in order by the amount of sample in the bag from that substrate. E.g. "Pinus contorta branches, Pinus ponderosa branches and boles" would indicate that the sample weight collected primarily from P. contorta branches and in lesser amounts from P. ponderosa branches and boles. 4. Target species acronym 5. Collector's initials 6. Moisture status of sample at the time of collection: dry, damp or wet. Target Species Two species will be collected at each monitoring site from the following list of target species. Whenever possible, one of the target species should be Platismatia glauca. Target species are grouped below by desirability. Target species acronyms are given in parentheses. Most Preferred Platismatia glauca (Plagla), collect whenever possible Preferred Alectoria sarmentosa (Alesar) Evernia prunastri (Evepru) Hypogymnia enteromorpha (Hypent) Hypogymnia imshaugii (Hypims) Hypogymnia inactiva (Hypina) Letharia vulpina (Letvul) Good Bryoria fremontii (Bryfre) Letharia columbiana (Letcol) Sphaerophorus globosus (Sphglo) Acceptable if no other target species are present Isothecium myosuroides (Isomyo)--moss, do not collect from litter Lobaria oregana (Lobore) Lobaria pulmonaria (Lobpul) Neckera douglasii (Necdou)--moss, do not collect from litter Usnea (Usnea)--shrubby species only If a moss is collected, the second target species must be a lichen. Sample preservation and storage On the plot, sealed, airtight, sample bags should be placed in a shady location or inside a daypack so they do not overheat while the remaining work on the plot is performed. Damp or wet samples should be air dried as quickly as possible, preferably the same day, by spreading onto clean 100% acid free blotter paper laid over a flat surface covered with clean plastic wrap. Label blotters so that sample identity is retained. Lichens and mosses should not be air-dried in areas subject to contamination (e.g. near cooking areas, roads, or in rooms where organic solvents are used, dust levels are high, or smoking is permitted). Kapak bags can be dried by crimping them open and leaving them upright, or
by hanging them open on a line with a clothespin. As soon as they are dry, place the lichens back in the dried Kapak bags and carefully reseal. Sealed bags should be airtight. Dry lichens make a crunchy sound when the Kapak bag is squeezed; if the contents feel soft, they are probably damp. Specimens must be thoroughly air dried to avoid fungal decay. Store dry, samples in a clean, dry, dark place (bags are not opaque). Sample delivery After the first two plots are completed, specimens will be mailed or brought to the lichen specialist to allow immediate feedback to the field crews concerning specimen quality and quantity. Thereafter, the bags will be delivered biweekly or monthly to the program coordinator. Bags should be packed closely, but without excessive crushing, in a sturdy cardboard box. Bags from several plots can be mailed in the same box. A packing list should be kept by the field crew specifying the plot number, Forest, species, field replicate number, and mailing date of each sample (see "Forms" section). Common problems and solutions Problem: None, or only one, of the target species is present in sufficient quantities for collection, even if the sampling area is expanded to the 1 km maximum sampling radius. Solution: In this case, no samples, or only one sample should be collected. Do not substitute nontarget species. Problem: Platismatia glauca is present, but it would be faster to collect other target species. Solution: Limit collection time to 1.5 hours and collect other target species while looking for P.glauca. Although 20 grams is the desired sample size, if the material is clean, dry, and in good condition, field sample size as low as 12 grams may be useable. Samples that weigh < 8 grams after cleaning in the office are not usually sent to the analytical laboratory. Problem: More than 1 hour has been spent collecting but sample weight is still very low. Solution: Get help from other crew member
s or switch to a more easily collected target species. Usually it's a good idea to stop collecting after two hours and process the collection that has been made. The decision to send the sample to the laboratory will be made in the office after the sample is cleaned. Problem: All the sample material came from one or two trees. Solution: This is not acceptable. The material must evenly represent at least six locations. Expand the area of collection up to the maximum size allowed. If material is still too scarce, collect a different species or collect nothing. Problem: Lichens were collected wet, it is still raining by evening, and the field crew is camping. Solution: Drying the lichens is still important to prevent fungal decay. If the distance is reasonable, go to the nearest district office to dry the lichens. Alternatively, store the samples in a cooler with ice up to two days, then air dry the lichens in a tent or in clean mesh bags on a clothes line as soon as conditions improve. Avoid use of heating devices to dry samples. Equipment and supplies Non-Consumable
1. Pesola spring scale, 100 g. 2. Reference samples of target species (provided or approved by the lichen specialist). 3. Locking-blade (ca. 4" blade), cleaned regularly with soap and water and stored in a new Ziploc bag after washing. 4. Poled tree pruner, useful for high elevation plots where deep snows create a high "lichenline"(didn't use in the Jedediah Smith Wilderness, but would be helpful at plots LELAKE-01 and TAR-01). Consumable 1. Black Water
proof markers, such as "Sharpies", for writing on specimen bags. 2. 4 x 7" metalized polyester bags (sold by Kapak Corp., 5305 Parkdale Drive, Minneapolis, MN 55416, 1-800-527-2557 Product #60-4B-IM)). Two bags are needed for most plots, one for each species. Bring extra bags for field replicates. 3. Rolls of laboratory tape, ѕ" or 1" wide. Tape should adhere to wet surfaces and be removable. Masking tape and cellophane tapes are difficult to remove and are not recommended. 4. Disposable vinyl gloves, not powdered, one pair of gloves per plot. 5. New, gallon size Ziploc bags. Store gloves and Kapak bags in separate Ziploc bags, placed inside a third Ziploc bag with the sharpie, scale and laboratory tape. 6. Roll of clear plastic wrap. 7. 100% cotton herbarium blotter sheets (11.5 X 16"), folded in half and stored in a clean Ziploc bag. Replace as they become visibly stained or smudged (for drying wet lichen samples). Interferences This method may be used in any season or weather condition. Normally, the tissue collection season is May 15-Oct 15, that is, after the winter rains have ceased and before autumn rains have begun. Lichens collected during the rainy season typically have lower concentrations of mobile elements like S, N, K, Na, therefore mid to late summer is the ideal time to capture maximum pollutant loading. The method requires careful discrimination among species in the field and should not be performed in poor light. If the field crew observes or smells smoke from forest fire, field burning, or other types of combustion during collection, this should be noted on the field data card. Because of the potential for lead and other heavy metal contamination, no smoking is allowed on the plot or in the vicinity of the plot during the visit. Safety Only minor hazards are associated with the method. Care should be used when removing lichen specimens with a knife. A locking-blade or fixed-blade knife is best. Trees should not be climbed to procure specimens. Quality control and performance standards Only people who have successfully completed lichen training should collect the lichen elemental samples. Data quality will be measured at several times. The following components of data quality will be evaluated: Precision
Precision, or repeat measurement error, is determined in several ways: 1. Revisits by the field crews who re-sample one plot within one month of its initial sampling. Collections are made for the same target species sampled in the previous visit. 2. Field replicates are made at every fifth plot for each species. After collection for the first bag is completed, collection should be made into a second bag. The purpose of the field replicate is to assess variability in elemental content on the site due to the collection method. If each collection contains a representative selection of lichens on the plot, and both samples are in good condition, there should be little variability in element content between the two collections. Field replicates should only be collected at sites where material is plentiful to avoid creating a bias in which the first bag contains samples that are in better condition than the second bag. 3. Other components of precision are determined during lab analysis, by using various kinds of quality control samples (standards, splits, blanks--see section 2.4). The data quality objective (DQO) for precision is a coefficient of variation of 15%. Accuracy Accuracy is determined in the laboratory by analyzing reference samples with known elemental content. The DQO for precision is a coefficient of variation of 15%. Completeness Completeness is the proportion of plots that will yield usable data. The DQO for completeness is 90%. The most important aspect of quality control for completeness is ensuring that the lichen elemental samples are adequate, not decomposed, and being received by the program coordinator. The field crew should ensure specimen quality by periodically calling the program coordinator to verify shipments and soliciting comments and suggestions on the quality of the specimens. Specialist procedures The samples will be processed first by the field crew who air-dry any samples that were damp or wet at the time of collection. The dry samples are then mailed or hand-carried to the program coordinator. The program coordinator processes all samples by 1) checking to see that each sample is thoroughly air dried, 2) verifying the identity of the species contained in the sample, 3) cleaning the sample so that it contains only one species, and 4) assigning a unique sample number to each sample bag. Data from sample bags are entered in a computerized database. The samples are then randomized, and assigned a consecutive laboratory ID number. The coordinator mails the samples to the analytical laboratory where they are processed and analyzed in order by laboratory ID number. Between 1993 and 2001 this laboratory was the Research Analytical Laboratory, Dept. of Soil Science
, 135 Crops Research Bldg., University of Minnesota, St. Paul, MN 55108, Attn.: Roger Eliason, (612) 625-9211. LICHEN COMMUNITY METHODS The purpose of the lichen community indicator is to use lichen species and communities as biomonitors of change in air quality, climate change
, and/or change in the structure of the forest community. Lichen communities are good indicators of air quality, particularly long-term averages of sulfur dioxide concentrations. Other pollutants that alter natural lichen communities include sulfur and nitrogen-based acid deposition, nitrogen fertilizers, fluorine and, possibly, ozone and other oxidants (see Section 1.32).
The following lichen community survey methods employed by our program were developed under the auspices of the USDA-Forest Service Forest Health Monitoring Program and are described in the FIA Field Methods Guide (http://fia.fs.fed.us/library.htm#Manuals). A few differences exist between the protocol that follows and the FIA Field Methods Guide. Our abundance rating has more categories than FHM, but can be collapsed to FHM ratings; substrates are recorded; and it is permissible to collect lichens below 0.5 meter on woody substrates east of the Cascade crest as long as they are tree and shrub-dwelling epiphytes and not terricolous or rotting wood species. The objectives of this task are to determine the presence and abundance of macrolichen species on woody plants (using a 34.7 m [114 ft] radius plot) and to collect samples to be mailed to the lichen specialist(s). The method has three parts, performed at the same time: 1. Make a collection of voucher specimens for identification by a specialist, the collection representing the species diversity of macrolichens on the plot as fully as possible. The population to be sampled consists of all macrolichens occurring on woody plants, excluding the 0.5 m basal portions of trees and shrubs (west side of the Cascade crest only). Fallen branches are included in the sampling. 2. Estimate the abundance of each species. Note that the crew member responsible for this task need not be able to accurately assign species names to the lichens (that is done later by a specialist), but must be able to make distinctions among species. 3. Record the substrate from which the lichen was collected. For woody substrates, record the species and location (i.e., branches, bole, limbs, etc.). Procedure 1. The area to be sampled (henceforth the "lichen plot") is a circular area with 34.7 m (114ft) radius. The area of the lichen plot is 3782 m2 = 0.378 ha = 0.935 acres. 2. Sampling continues for a maximum of two hours or until 10 minutes elapse with no additional species recorded. At least 30 minutes must be spent searching the plot, even if very few lichens are present. 3. A reconnaissance walk through the lichen plot should be taken to locate lichen epiphytes on woody plants, collect voucher samples and assign abundances. The following method is suggested: Begin at approximately 30 m (100 ft) due north from plot center, measuring with the eye to the limiting boundary of 114 ft. and continue to the right in a sinuous manner 90°. (The plot should be flagged every 90° along the perimeter). The same procedure is followed around the rest of the plot. If time allows, a second circuit of the plot can be made, searching for spots which were not visited in the first pass. 4. Lichen species with fruticose and foliose (i.e. macrolichen) growth forms will be collected. 5. Woody plants (all trees and shrubs >0.5 m tall) within the lichen plot will be inspected for lichen species. Fallen and reachable branches will also be inspected. 6. Care should be taken to inspect the full range of substrates and microhabitats available: shaded and exposed, conifers and hardwoods, fallen upper branches and lower branches, large shrubs and trees in particular topographic positions (for example, checking in draws or ravines of an otherwise uniform slope, so long as it occurs within the lichen plot).
Rotten logs, stumps, or other semi-permanent features of the forest floor should NOT be sampled. 7. Abundance ratings. Relative abundance within the lichen plot will be recorded. Relative abundance for each species is estimated as follows. Choose the highest rating that is true in Table 2. below 8. A sample of each putative species will be collected and placed in a paper packet labeled with plot number, collector's initials, forest acronym, substrate and relative abundance. The abundance rating can be revised as collection proceeds or given at the end of the collection period. Any relevant comments are recorded on the outside of the packet under "Remarks". For more details, see section 2.52 below. Before leaving the plot all packets should be checked to make sure that, as a minimum, plot number, abundance and substrate has been recorded on every packet. They should then be alphabetized by genus and species (if known) and sequential packet numbers assigned, beginning with "1". 9. How to handle uncertainties: The field crew will frequently have uncertainties about the classification of an organism. The following rules for the field crew are designed to put the onus of the responsibility for classification on the specialist, not the field crew. a. When in doubt, assume it is a lichen. b. When the growth form is in doubt, assume it is a macrolichen. c. When species distinctions are in doubt, assume that two different forms are different species. The purpose of these rules is to encourage the field crew to make as many distinctions in the field as possible. The specialist can later adjust the data by excluding specimens that are not macrolichens and by combining forms that were considered separately by the field crew but are actually the same species. Sample collection, preservation, and storage Optimally, palm-sized (about 5 cm in diameter) samples of fruticose and foliose growth forms are collected. These growth forms include all species that are three-dimensional or flat and lobed. Even minute fruticose and lobate forms should be included. Squamulose species and Cladonia squamules lacking upright stalks should not be included. Table 2. Abundance ratings for lichen community surveys Code Abundance Description 1 Rare < 3 individuals/colonies in area 2 Uncommon 4-10 individuals or colonies in area 3 Common 10-40 individuals or colonies in area 4 Very Common >40 individuals or colonies in area but less than half of the boles and branches are covered by the species. Choose one: 4-1 Individuals/colonies are few (between 40-80) and widely scattered around the area 4-2 The lichen is restricted to one or two small areas in the area, usually on just a handful of trees or shrubs. The total number of individuals or colonies is >40. 4-3 Many trees or shrubs have up to 20 individuals or colonies. 4-4 Many trees or shrubs have more than 20 individuals or colonies. 4-5 More than half the trees or shrubs have up to 20 individuals or colonies.
4-6 More than half the trees or shrubs have more than 20 individuals or colonies. 5 Abundant More than half of the available substrate is covered by the subject species. These codes correspond to the FHM lichen indicator codes as follows: 1 = FHM 1; 2 and 3 = FHM 2; 3 to 4-4 = FHM 3; 4-5 to 5 = FHM 4. In some cases, a small sample should be obtained because of the scarcity of the species. However, if the abundance rating is > 3, the sample should be generous. Large samples containing multiple individuals simplify the identification process and demonstrate that the collector was able to distinguish the species from look-alikes, improving confidence in the assigned abundance code. Collecting large samples also improves the likelihood of picking up inconspicuous species that may not be distinguishable in the field. These can be recorded by the lichen specialist in the office. Species in the genera Usnea and Bryoria are most difficult to distinguish in the field and large samples nearly always contain a mix of species within a packet. If species are present in equal amounts, the abundance code may unusable. For these genera, the best strategy is to carefully learn characters that differentiate species and collect smaller samples in multiple packets. Before leaving the plot, each specimen will be placed in a separate folded paper packet and labeled as follows: 1. Plot number (use FIA Plot ID code for on-frame plots). 2. Ocular abundance code (can be revised as collection proceeds and the observer becomes more familiar with the plot). 3. Substrate. 4. Occasionally there will be more than one species on a given bark sample. If there is any chance of ambiguity about which species in the packet corresponds with the abundance rating, a descriptive clarifying phrase, such as "the white one" or "the sorediate one", will be written on the packet. Packets will be labeled with an indelible marker. If the packets are damp, a soft pencil (No. 2 or softer) can be used. The lichen worksheet and all of the specimen packets from a given plot will be placed into a paper or Ziploc bag with the plot number, collectors initials, and date recorded on the outside of the bag and the top folded down or sealed. The bags should be stored in a dry place until delivery to the specialist. Specimens must be thoroughly air dried to avoid fungal decay. If specimens were wet when collected, the individual packets should be spread out and dried inside or in the sun as soon as possible. If temperatures are above room temperature, wet lichens are likely to mold within 2-3 days. Sample delivery After the first two plots are completed, the specimens will be mailed or brought to the program coordinator. This allows the coordinator to provide immediate feedback to the field crews concerning specimen quality and quantity. Thereafter, the packets can be delivered biweekly or monthly. Packets should be packed closely, but without excessive crushing, in sturdy cardboard boxes. Packets from several plots can be mailed in the same
box. The field crew should save a running packing list (see "Forms" section) specifying the CVS plot numbers, Forest, and date mailed. Any notes of possible use to the lichen specialist should be sent with the packets. Equipment and supplies Consumable 1. Folded labeled paper packets (can be made by recycling one-sided office paper). Carry 40 packets per plot west of the Cascade crest, and 25-30 packets for plots east of the Cascade crest. 2. Black waterproof markers for writing plot numbers and abundance data on paper or plastic bags. 3. Larger brown paper bags (16.5 x 9.5 " or similar size), or gallon-sized re-sealable clear plastic bags, one per plot. 4. Soft pencils (No. 2 or softer) and indelible pens. 5. 6 mailing forms (if going to mail all specimens together, only need one or two forms) 6. 60 field data cards per Forest (only need one data card per plot, so adjust accordingly). Non-Consumable 1. Locking-blade or fixed-blade knife (ca. 4" blade) with belt sheath. 2. 14-20x hand lens (Bausch and Lomb Hastings Triplet is recommended). 3. Guides for lichen identification: a. Brodo, I., S. D. Sharnoff and S. Sharnoff. 2001. Lichens of North America
. Yale University
Press, New Haven, CT. b. Goward, T. 1999. The Lichens of British Columbia
. Part 2. Fruticose Species. British Columbia Ministry of Forests Research Program. c. Goward, T. McCune, B. and Meidinger, T. 1994. Lichens of British Columbia. Part 1. Foliose lichens. British Columbia Ministry of Forests Research Program. d. McCune, B. and L. Geiser. 1997. Macrolichens of the Pacific Northwest. Oregon State University Press. See Bruce McCune's website for updated keys: http://oregonstate.edu/~mccuneb/getkeys.htm. e. McCune, B. and T. Goward. 1995. Macrolichens of the Northern Rocky Mountains. Mad River Press, Eureka, CA. 4. Hand pruners (useful for collecting small branch segments). 5. 1" wide chisel (useful for collecting samples from tough-barked hardwoods, a sheath can be made from a piece of cardboard and strapping tape). 6. Clipboard (for field data forms). Interferences and safety This method may be used in any season or weather condition. Because careful discrimination among species in the field is required, the method should not be performed within an hour of sunset or sunrise, or during dark, rainy conditions. Only minor hazards are associated with the method. Care should be used when removing lichen specimens with a knife or chisel. A locking-blade or fixed-blade knife is best. Trees should not be climbed to procure specimens.
Quality control and performance standards Only people who have completed lichen training and have been certified should collect the lichen community data. Procedure for processing specimens 1. Receive boxes of specimens in the mail or directly from the field crew. 2. Open the boxes immediately and check for damp lichens. If some are damp, thoroughly air-dry them. 3. Identify the contents of each bag by species. In the case of mixed collections or multiple collections of the same species, see the special instructions below. 4. Enter the list of species identifications, along with plot numbers, substrates and abundances for all identifications in a computerized spreadsheet. 5. Prepare voucher specimens. Select individuals for herbarium specimens such that, ideally, each species is represented by about three specimens from each national forest. These specimens should be stored in standard, labeled herbarium packets. In all cases, the label data should include the plot number and the date of collection. Vouchers are currently stored in the Siuslaw National Forest herbarium. 6. Store packets (with lichens!) for future reference. Packets are currently stored in the Siuslaw National Forest Supervisor's Office storeroom.
Appendix III University of Minnesota Research Analytical Lab Methodology (taken directly from the website: http://ral.cfans.umn.edu/analyses.htm)
Sample drying and grinding
Moist plant samples are dried at 65°C until crisp. Samples are then passed through a stainless steel grinder with 20 mesh sieve and mixed thoroughly. Ground samples can be stored at room temperature, but must be redried at 65°C for 2 hours and cooled in a desiccator before weighing for analysis.
Jones, Jr. J.B. and V.W. Case. 1990. Sample, handling, and analyzing plant tissue samples. p. 389-427. Westerman, R.L. ed. 1990. Soil Testing and Plant Analysis. 3rd Edition. SSSA, Inc. Madison, WI.
Kjeldahl nitrogen, semi-micro
and then measuring the NH4+ concentration. To accomplish this, 0.150 g of dry, ground
plant material is digested in 3.5 mL conc. H2SO4 with 1.5 g K2SO4 and 7.5 mg
Selenium. [The K2SO4 and Selenium are in tablet form available from Tecator Inc., PO
Box 405, Herndon, VA 22070]. This mixture is placed in an electrically heated aluminum
block at 400°C and digested for 1 hour. The ammonium formed is determined
colorimetrically by reaction with salicylate in the presence of hypochlorite and
nitroprusside to form an emerald green complex. Color intensity is measured on a
Technicon AutoAnalyzer at 660 nm. The method converts only partial amounts of nitrate,
thus samples high in nitrates must be pretreated with salicylic acid to ensure complete
Horneck, D. A. and R. O. Miller. 1998. Determination of Total Nitrogen in Plant Tissue. p. 75-83. In Y. P. Karla (ed.) Handbook of Reference Methods for Plant Analysis. CRC Press, Boca Raton, FL.
Colorimetric Analysis: Isaac, R. A. and W. C. Johnson. 1976. Determination of total nitrogen in plant tissue. J. Assoc. Off. Anal. Chem. 59:98-100.
Nitrates are extracted by shaking 200 mg of dried plant material with 30 mL 2% acetic acid
solution for 30 min. Add 0.85 cc of prewashed charcoal to each sample and continue shaking for 5 additional minutes. Samples are centrifuged or filtered through a
Whatman No. 42 filter paper. Nitrate concentrations in the filtrate are determined colorimetrically by the cadmium reduction method. In this method, nitrate is reduced to nitrite in a copperized cadmium column. The nitrite ions react with sulfanilamide under acidic conditions to form a diazo compound. This couples with N-1Napthylethylenediamine dihydrochloride to form a reddish purple azo dye which is measured at 520 nm on an Alpkem Rapid Flow Analyzer. Extraction: Gavlak, R.G., D.A. Horneck, and R.O. Miller. 1993. Recommended soil and plant tissue reference methods for the western states. Miller, R. O. and J. Kotuby-Amacher. 1994. Extractable nitrate, ortho-phosphate, and chloride of botanical materials. p. 47-48. Western states agricultural laboratory exchange program suggested soil and plant analytical methods. Analysis: RFA Methodology. 1986. Nitrate/Nitrate A303-S171. Astoria-Pacific International PO Box 830, Clackamas, OR 97015 Also: O-I Analytical (Alpkem Division) PO Box 9010, 151 Graham Road, College Station, TX 77842. Total nitrogen (Dumas method) This technique uses a LECO FP-528 Nitrogen Analyzer to determine total N in plant materials
. A 150-200 mg sample is weighed into a gel capsule and dropped into an 850o C furnace purged with O2 gas. The combustion products of CO2, H2O and NOx are filtered, cooled by a thermoelectric cooler to condense most of the water, and collected in a large ballast. A 3 cc aliquot of the ballast combustion products is integrated into a He carrier stream and passed through: 1.) a hot copper column where the O2 is removed and the NOx gasses are converted to N2 2.) a reagent tube which scrubs the CO2 and remaining H2O from the stream. The N2 content is then measured by a thermal conductivity
cell against a He background and the result displayed as weight percentage of nitrogen. Simone, H.A., J.B. Jones, Jr., D.A. Smitties, and C.G. Hussey. 1994. A comparison of analytical methods for nitrogen analysis in plant tissues. Commun. Soil Sci. Plant Anal., 25(7&8), 943-954. Matejovic, I. 1995. Total nitrogen in plant material determined by means of dry combustion: A possible alterntive to determination by Kjedahl digestion. Commun. Soil. Sci. Plant Anal. 26(13&14), 2217-2229. Total Sulfur A 0.100-0.150 g sample is weighed into a ceramic boat and covered with Com-Cat Accelerator (LECO trade name for tungsten oxide compound). The boat is placed in a furnace at a temperature of 1350°C in an oxygen rich atmosphere. Total Sulfur is
determined by infrared absorption of evolved sulfur dioxide upon the dry combustion on a LECO Sulfur Determinator, Model No. S144-DR. (LECO Corporation. 3000 Lakeview Dr. St Joseph, MI 49085) Multi-element, ICP-dry ash method Ca, Mg, Na, K, P, Fe, Al, Mn, Cu, Zn, B, Cd, Cr, Ni, Pb, Mo, Co, Rb, Li, Sr, Ba, Be, V, Ti, and Si are determined simultaneously by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). A 500 - 1000 mg sample of dried plant material is weighed into a 20 mL high form silica crucible and dry ashed at 485°C for 10 - 12 hours. (Crucibles are covered during the ashing as a precaution against contamination.) The ash is equilibrated with 5 mL of 20% HCl at room temperature for Ѕ hour. Then 5 mL of deionized water is added, gently swirled and allowed to settle for 3 hours. The solution is decanted into 15 ml plastic disposable tubes for direct determination by ICP-AES. Upon special request the dry ash can be refluxed in 20% HCl to improve the recovery of Fe, Al, and Cr. (Also see the website: http://ral.cfans.umn.edu/plant.htm) Dry Ash Munter, R. C. and R. A. Grande. 1981. Plant tissue and soil extract analysis by ICP-AES. in R. M. Barnes (ed.) Developments in atomic plasma spectrochemical analysis. p.653673. Heydon and Son, Philadelphia, PA
. Munter, R.C., T.L. Halverson, and R.D. Anderson. 1984. Quality assurance for plant tissue analysis by ICP-AES. Comm. Soil Sci. Plant Anal. 15(15):1285-1322. Analysis Fassel, V.A., and R.N. Kniseley. Nov. 1974. Inductively Coupled Plasma Optical Emission Spectroscopy. Anal. Chem. 46 (13):1110A-1120A. Dahlquist, R.L. and J.W. Knoll. 1978. Inductively Coupled Plasma-Atomic Emission Spectrometry: Analysis of biological material
s and soils for major trace, and ultra-trace elements. Appl. Spectroscopy 32:1-30. ICP: ARL (Fisons) Model 3560 ICP-AES Ref. Thermo Instrument Systems Inc. (Fisons Instruments Inc. Division) Waltham, MA. /Mercury, Total A 0.25-0.50 g sample of dried plant material is digested with 2 mL H2O2 and 0.5 mL HNO3 in a microwave digestion vessel for 4 min. at 296 watts and 8 min. at 565 watts. The microwave digestate is further digested for 2 h with 0.25 M H2SO4, 5% potassium permanganate and 5% potassium persulfate in a 95oC hot water bath. After reduction with stannous chloride, the Hg is quantified by the cold vapor technique using atomic absorption spectrophotometry on a mercuryMonitor Elemental Mercury Detector.