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7 Evaluation of Final Reports of Individual Studies INTRODUCTION THE STRUCTURE OF THE ecological monitoring program was segmented, rather than integrated, in that the research teams worked independently of each other. The purpose of ]:TTRI's solicitation in March 1982 was to attract sub- contracting researchers to develop and conduct separate ecological monitoring studies that would determine whether low-level, long-term electric and mag- netic fields (EMFs) and gradients produced by the ELF communications sys- tem would affect vegetation or wildlife in and near the system area or other- wise result in changes in individual organisms or their communities. Eventu- ally physiologic, developmental, behavioral, and ecological aspects of predom- inant organisms in upland, riverine, and wetland habitats near the Navy's ELF transmitting facilities were monitored for possible effects of EMFs produced by the Navy's antennas. The organisms and ecological relationships selected for monitoring were chosen because they were judged to be important to their ecosystems and to be of interest to local residents (Zapotosky and Gauger 1993~. The monitoring program studies were designed to compare data collected at control sites with data collected at treatment sites. As discussed in Chapter 2, the paired sites were intended to have matched environmental factors but to be dissimilar in the magnitude of their exposure to the 76-Hz EMFs gener 32

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EVALUATION OF FINAL REPORTS 33 ated by the communications system antennas. Sites exposed to those 76-Hz EMFs were established by locating treatment sites near or within the rights-of- way for the antennas; control sites had to be far enough from the communica- tions system that EMF intensities resulting from antennas would be substan- tially lower than those at treatment sites. However, control sites had to be close enough to have environmental factors similar to those of their matched treatment sites. Siting criteria called for intensities of the 76-Hz EMFs at treatment sites to be at least 10 times those at control sites, for intensities of the 76-Hz EMFs at treatment sites to be at least 10 times those of the 60-Hz EMFs at treatment sites and control sites, and for intensities of the 60-Hz EMFs at treatment sites to be within 10 times those at control sites (Haradem et al. 19941. This chapter presents evaluations of the ~ ~ final ecological reports on the following topics: wetlands, slime mold, Wisconsin birds, Michigan birds, small vertebrates, litter decomposition and microflora, upland flora, aquatic ecosystems, pollinating insects, soil arthropods and earthworms, and soil amebas. The committee combined its discussion of Wisconsin and Michigan birds in this chapter. The committee used the following criteria to evaluate the reports: adherence to the original project proposal, adequacy of selection of species and response variables, adequacy of experiment design and imple- mentation (including biologic and ecological sampling techniques, physical measurements, and statistical power), responsiveness to reviewers' comments while studies were being conducted, presentation of results (including consid- eration of alternative analyses or hypotheses and interpretation), and appropri- ateness of conclusions (including validity and uncertainties). The committee found that the various criteria did not warrant the same amount of discussion for each study. WETLANDS PROJECT PROPOSAE The authors of the wetlands final report (Guntenspergen et al. 1989) pointed out that wetlands in the upper Midwest are sensitive ecosystems and are common near the ELF communications system sites, especially the Wis- consin location. They pointed out that past studies on effects of ELF EMFs indicated that plant membranes might be affected by electromagnetic radiation.

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34 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM Therefore, they proposed to look for changes in plant competitive ability through measurement of plant or ecosystem functions that were related to membrane functions. Possible changes included leaf diffusion resistance, foliar nutrient content, changes in functions related to transport of water and nutri- ents across membranes, and decomposition by microorganisms, all of which depend heavily on secretion and adsorption through membranes. The basic null hypothesis of the wetland-monitoring project was that ELF EMFs resulting from the operation of the Navy antenna have no effect on selected ecosystem variables. SYSTEM, SITE, AND SPECIES SELECTION Much of the initial, pilot-study year of this project was spent in establish- ing study sites and testing methods. Stearns et al (1982) described five north- ern wetland vegetation types in Wisconsin: northern conifer swamp, shrub wetland, emergent marsh, northern sedge meadow, and open bog. Because the northern conifer swamp was common near the ELF location in Wisconsin and offered all life forms-including trees, shrubs, herbs and nonvascular plants it was chosen as the ecosystem type for monitoring. The authors of the proposal recognized the heterogeneity of the region near the ELF antenna, as well as the heterogeneity among stands of the same type of wetland. That created a problem in site selection but was addressed through identification of sites with close similarities in vegetational composi- tion and environmental characteristics. The former was determined through use of contingency tables and similarity indexes (such as Sorensen's Index), and the latter through measurement of soil-water temperature and pH, cation concentrations, and redox potential. in general, it was thought that sites with similar vegetation would have similar, but certainly not identical, environ- ments. More than 200 potential sites were selected from aerial photographs; these were reduced to SO sites for priority-setting, and then 15 sites were selected for measurement of 60- and 76-Hz EMFs in potential study plots, one per site. Eleven sites were eventually chosen for initial summer (1983) stud- ~es. Location of "similar" sites was based on relative EMF intensity. Electric-field strength measured by lITR] was used to establish intensity gradi- ents; these were called background, intermediate, antenna, and ground to correspond to location of sites relative to ELF facilities. No true control could be established, because the ELF communications system was already function

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EVALUATION OF FINAL REPORTS 35 ing, so the background sites were considered as control sites. In most cases, three sites were used for each exposure scenario, but only two ground sites were used within one large peatiand. Although the selection of study sites satisfied the criteria established by IITR} and a gradient of exposure from treatment sites to control sites existed, the three treatment sites varied among themselves in terms of EMF intensity. For example, 1987 field measurements showed that the plots within the antenna treatment sites ranged from 0.053 to 0.196 V/m in electric-field intensity and from 6.! to 19.S mG in magnetic- f~eld intensity. Selection of the northern conifer forest wetland allowed use of tree, shrub, and herb species, when appropriate, for determination of response variables associated with plants. Final experimental species were not selected until 1985. Labrador tea (Ledum groeniandicum) was selected as the primary species for measuring stomata! resistance after other species were tried; for instance, the leaf anatomy of black spruce (Picea mariana) made stomata! measurement difficult. Labrador tea is a common shrub throughout the conifer wetlands of Wisconsin. Labrador tea leaves replaced pure cellulose sheets in the decomposition studies for testing of "natural plant materials" and to im- prove within-site measurement consistency. Black spruce, Labrador tea, the shrub leather leaf (Chamaedaphne calyculata), and the herb false solomon's seal (Smilacina trifolia) were used for foliar nutrient content; this permitted comparisons across life forms. Nitrogen fixation studies, initially on alder and then on moss and peat, were dropped during the study period. RESPONSE VARIABLES Response variables chosen for the wetland studies and used throughout the study period all were related to membrane-associated functions. The choice was based on a National Research Council (NRC 1977) report and other stud- ies on effects of EMFs that indicated that biologic and ecological responses to EMFs were most likely in functions associated with membranes. Stomatal resistance was chosen as a response variable because it is associated with water transport across membranes and all vascular plants could potentially be influ- enced through this leaf function. Nutrient content of leaves was examined because it is closely related to nutrient transport across membranes in root cells. Decomposition was examined because it is associated with across-mem- brane secretion of enzymes by microorganisms and adsorption of decomposed cellulose and other organic compounds. Other processes could have been

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36 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM examined, such as growth rates or species composition changes, but they were not, because they were considered likely to result in too much variability within sites and were considered to be long-term response variables. Nitrogen fixation was examined for a couple of years, but that effort was eventually dropped from the program because the method was not reliable. EXPERIMENT DESIGN The wetland monitoring study was conducted from 1983 through 1987. The first year was used for selecting sites, developing and evaluating protocol, and beginning preliminary sampling. After experimentation with a rectangular plot design in the first year in which all the experimental measurements were made in a regular array throughout the plot, the study team settled on a 70 x 15-m rectangular plot for all experiments, oriented with the long axis parallel to the closest antenna. Six square subplots were designated in the rectangular plot with centers (where shallow groundwater wells were placed) 10 m apart. All measurements of response variables were taken within these subplots; all selected species were within each subplot. Environmental data were collected monthly from May to September (the frostfree period). ELF EMFs were measured once a year and assumed to be constant over the year. The antennas were capable of operating at full strength from 1985 through 1987, when most of the established protocols were in place; however, the antennas were not on full-time during this period, and it is not certain to what extent the off periods were taken into account in the study. That might be important for response variables that respond instanta- neously or in a short term. Biologic Sampling The final report presented three biologic measurements used to determine possible effects of ELF EMFs on wetland ecosystems in the vicinity of the Wisconsin transmitter facility (Guntenspergen et al. 1989~: stomata! resistance, foliar nutrients, and decomposition. Stomatal Resistance The primary results of stoma/al-resistance tests were based on responses of Labrador tea, a common species in every study bog. Other species were tested both because of ease of measurement and because

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EVALUATION OF FINAL REPORTS 37 of response to different light levels. Spruce and smilacina were dropped be- cause of measurement difficulties. The equipment used for measurement of stomata! resistance was a null-balance diffusive-resistance porometer, which measures the rate of water-vapor diffusion through the stomata. It is standard equipment for such measurement, and the method is easily replicated if need- ed. Stomatal resistance was measured in leather leaf and Labrador tea under different light intensities. Labrador tea, least responsive to differences in light especially at low intensities, was selected as the species for testing the re- sponse of plant stomata! resistance to ELF EMFs. That choice essentially eliminated light as one of the independent environmental variables that might have to be considered a covariate in later statistical analyses. Selection of only one species, however, implied the assumption that all other species would respond to ELF EMFs in a similar fashion. if different species had different stoma/al-resistance responses to light levels, might this indicate different re- sponses to other external variables? That is partially addressed by the results, which include data on leather leaf, which was dropped as a consequence of tests on light intensities. Measurements were made during four periods at all I! study sites in August and September 1986 and 1987. Measurements were made over several days in each sample period, and there was an attempt to stratify measurements to cover all environmental variables, especially light intensity and cloud cover, for the background, intermediate, antenna, and ground sites. The number of samples taken was doubled between 1986 and 1987 to allow for resolving 20% differences in means at p = 0.05 with an 80% probability. The increase in samples increased variability in the measurements of external environmental conditions. That situation is commonly found in field sampling. An increase in sampling frequency might reduce the statistical significance of the results because it increases variability in sample measurements. Foliar Nutrients Changes in foliar nutrient concentrations in plants grow- ing in a relatively nutrient-poor environment were considered a possible indi- cator of the condition of various plant biochemical pathways. The initial spe- cies for foliar-nutrient sampling were a shrub (leather leaf), an herb (smilaci- na), two sedges, and a tree (black spruce). Labrador tea was substituted for the sedges because destructive harvesting of these sedge species might have damaged their limited populations. Sampling periods were based on phenol- ogy; thus, herbs were sampled earlier in the season than shrubs. Sample size was increased several times over the early years of the study to improve the

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38 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM power of the statistical analysis, eventually reaching 396 (6 x 6 x ~ i) sam- ples per species per sample date. Only current-year foliar tissue was collected. Standard methods were used for preparation and analysis for calcium, magne- sium, and potassium. Samples from 1987 were also analyzed for manganese and phosphorus. For quality control, National Bureau of Standards (NBS) standards were analyzed with field samples, and spikes of known-cation stan- dard solutions were added to field and NBS samples. Decomposition Two approaches were used for decomposition studies. The first used pure cellulose as a substrate, and the second used Labrador tea leaves. The use of pure cellulose was intended to provide a uniform substrate for decomposition. If preweighed samples of cellulose were placed in a f~ber- glass bag and inserted vertically into the peat, less variation was expected among all samples. However, the cellulose became soft, adhered to the bag, and could not be retrieved fully for posttreatment weighing. Labrador tea leaves were used instead of cellulose. Bags with about the same amount of leaf material were mixed and randomly selected for placement in the bogs. The use of Labrador tea leaves resulted in less within-group variance. The decomposition bags were allowed several months for incubation in situ. The bags were retrieved, and the cellulose or leaf samples were removed, cleaned of foreign material, dried, and weighed. Weight loss indicated decom- position. The duration of incubation varied from 4 to 12 months. Environmental Characteristics The primary environmental characteristics measured during the study, excluding ELF-EMF exposure levels, were those of the interstitial water with- in each wetland site. The characteristics of the bog water were considered to influence decomposition rates and root activity. Shallow groundwater wells were placed in the center of each of the six subplots in the study plot at each site. Water-quality measures were depth to water table, depth to anaerobic zone, reduction-oxidation potential, specific conductance, temperature, pH, and calcium, magnesium, and potassium contents. Dissolved organic carbon was measured in 1984 only. Water samples were prepared with standard methods. No data in the wetlands report indicate regular measurement of ambient temperature, rainfall, or other external climatologic conditions. Measurements

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EVALUATION OF FINAL REPORTS 39 of water quality, light intensity and leaf temperature made during various sampling periods appear to have been considered sufficient for evaluating the influence of the external conditions. Statistical Methods Statistical methods were chosen to test the null hypothesis that ELF EMFs resulting from the operation of the Navy antenna have no effect on selected ecosystem variables. Researchers primarily used nested analysis-of- variance (ANOVA) models to examine treatment and control groups. Stepwise-multiple-regression models were used to explain the variance in the dependent biologic variables (stomata! resistance, foliar nutrients, and decom- position rates) on the basis of environmental variables (such as water-quality data) and ELF-EMF data. Significance levels (p=0.05) of the two models were compared. In a few instances, the models did not agree, and lack of significance within one mode! was selected as the appropriate test of the re- sponse variable (i.e., it was not significantly influenced by ELF EMFs). Reviewers of the project over the period of the study sometimes questioned the use of stepwise multiple regression because of the interdependence of the variables, even those considered independent. In ANOVA, the variables were usually considered covariates; in multiple regression, they were treated as independent. Treating variables in this manner is a common practice in eco- logical studies, the understanding being that no variables in an ecosystem are truly independent. The use of stepwise regression attempts to alleviate this concern. To show the relationship of environmental variables to decomposition rates, all environmental data collected during the incubation period were sub- jected to principal-components analysis (PCA), an approach that reduces the number of independent variables to a few "composite" variables. The princi- pal components representing environmental data were then regressed (in step- wise fashion) against decomposition rates. Several times during the study period, the number of samples was in- creased to increase the power of the statistical analysis. That was done mostly for the foliar nutrient analysis. Other levels of sampling were considered sufficient, after preliminary studies, to achieve the established level of confi- dence at a 0.05 significance level.

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40 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM Quality Assurance and Quality Control Quality assurance and quality control, especially as related to chemical analysis, were addressed in the methodology discussion of the final report. Methods selected for stomata! resistance were standard and repeatable. Decomposition-rate methods were straightforward. The research design estab- lished for this study helped to avoid pseudoreplication and allowed sound ANOVA of data from study sites. Exposure Assessment UTR} provided ELF-EMF exposure data at the background, intermediate, antenna, and ground sites. ITTR] measured ELF EMFs annually at each sam- ple plot at the study locations. The 1987 annual report (Guntenspergen et al. 1988) made it obvious that the investigators took into account the spatial EMF gradient resulting from antenna operation and used it in regression analyses. The ELF communications system was not operated continuously; study sites were therefore not consistently exposed to ELF EMFs. Different por- tions of the antennas were turned on and off several times each day, with varying modulations, frequencies, current intensities, and phase angles. The Navy provided ITTR} and researchers with detailed logs of antenna activity. The final wetlands report does not indicate whether the antennas were on or off during field measurements or whether information on the antenna operation was used in data analyses. Response variables would likely have varied in their sensitivity to antenna operations. For example, annual decomposition rates could be related to the annual EMF measurements, but stomata! resistance might have an immediate cellular response to external factors, such as light, temperature, and ELF EMFs. Use of short-term response variables, such as stomata! resistance, could have created analytic difficulties. Field measurements of these very short-term response variables would have had to be timed in coordination with antenna operations to ensure measurement of possible EMF influences that might not be observable when the antenna is off. In 1987, stomata! resistance was measured in August, the same month that I]:TRI measured exposure levels at each study plot. There is some evi- dence in the 1987 annual report (p. 17) that the researchers examined the on- off status of the Wisconsin transmitting facility relative to their field sampling.

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EVALUATION OF FINAL REPORTS 41 If these measurements occurred at the same time, the exposure assessment of this study took into account the need to measure short-term responses while the antenna was actually on. The final report does not address the coordination of field measurements with antenna activity. If the measurements were not coordinated, the use of a short-term response variable, such as stomata! resis- tance, is questionable. RESPONSE TO REVIEW The researchers received comments from reviewers starting with the 1983 annual report (Stearns et al. 1984~. Common comments were related to site characterization, use of specific response variables and timing of measure- ments, and level of sampling for adequate statistical analysis. The study design was modified to accommodate some of the critics' comments; others were addressed through explanations of why changes were not made. For example, a criticism of site selection was lack of extensive soil data. The researchers characterized similar sites as those with similar peat sub- strates, but then emphasized measurement of interstitial water at each site as more appropriate for studying substrate composition. The use of stomata! conductance as a response variable was acceptable to the reviewers, but they were concerned that because this variable is so closely influenced by light and temperature, it needed to be measured at the same time of day under similar conditions of light and temperature to yield useful comparisons. The researchers selected the species with the least re- sponse to light variation because it was impossible to make all measurements at the same time of day or even on the same day. The use of foliar composition was questioned because it is not considered appropriate for determining soil nutrient availability in agricultural systems. The researchers pointed out, however, that foliar composition is commonly used in natural ecosystems as an indicator of plant condition and therefore was appropriate for this study. Although the final statistical approaches did not fulfill all the requirements of the reviewers, the study design was altered in some cases to increase sample size. Inappropriate uses of statistical terms, pointed out by reviewers, also were corrected in the final report. In most cases, the investigators did respond to reviewers' comments, thus producing an improved final document.

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42 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM PRESENTATION OF RESULTS Alternative Hypotheses Results were presented in different forms: graphical presentations with bar graphs (often showing standard errors), tables with nested ANOVA analy- ses, and tables with stepwise-multiple-regression analyses. The initial proposal suggested that ANOVA would be the appropriate statistical approach to test the null hypothesis of no difference between or within treatment and control sites. Development of data sets also demonstrated to the researchers that there was a need to attempt to explain the variances of the biologic responses be- tween and within treatments, in addition to assessing them. Although no alter- native hypotheses were presented, use of stepwise multiple regression to assess the significance of independent environmental variables indicates possible consideration of an additional hypothesis that variances in dependent biologic responses are explained no more by natural external environmental factors than by ELF EMFs. Interpretation Interpretation of results was based primarily on comparison of the two statistical approaches to the empirical data. If the ANOVA models indicated that there was no greater difference between study sites than within study sites at the 0.05 significance level, the researchers interpreted that as conclusive. They checked their interpretation by applying the multiple-regression models that used environmental characteristics (sometimes considering biologic vari- ables as independent, such as leaf nutrients for stoma/al-resistance compari- sons) and ELF-EMF characteristics (in most cases in a PCA form). In many cases, neither environmental nor ELF-EMF principal components accounted for much of the variance of the response variable. in a few cases, a signifi- cant correlation for a response variable was found using the regression mode! that was not found using the ANOVA model. The researchers interpreted the mode! showing no significant correlation to be correct. That raises the question of the level of confidence selected to demonstrate statistical significance of the ANOVA and multiple-regression models. For this study, a significance level of 0.05 was chosen. Considering the variability of the ecosystems being stud- ied, this is probably appropriate, although many statisticians might consider it no more than an indication that the variance of the results depends on chance.

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~ 00 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM only the first three foraging trips were measured. Observer identity was used as a variable in the ANOVA mode! to test for bias. Because sex ratios can vary with nest diameters and depths, data not standardized for these variables were excluded from the analysis. Exposure Assessment L Measurements of actual ELF-EMF exposures are reported for the treat- ment and control sites, with maximums and minimums given for one of the two treatment sites for June through August. The 76-Hz magnetic-flux densi- ties at the treatment site were 330 times stronger than at the control site. Exposure varied substantially between treatment sites, within treatment sites, and among years. The two treatment sites differed, in cumulative gauss- hours, by a factor of 2 (Strickler and Scriber 1994, Fig. 13~. Nest hutches differed within a treatment site by up to a factor of 100. Pretreatment years included low to 50% power (1983-19881. Mobile adult bees are subject to different exposures, but developing progeny are stationary and should have a consistent exposure, although this obviously varied between nests, sites, and years. The researchers argue that this variation in ELF EMFs is unlikely to be Important because the bees will show a threshold response to the large expo- sure differences between treatment and control sites. However, if the re- sponses are dose-dependent, this variation could confound the results. If there is a humped response curve (maximum at some intermediate level), ELF-EMF effects would be missed in the analysis. RESPONSE TO REVIEW Reviewers' suggestions to focus the initial study were heeded and con- tributed greatly to the success of this research. Other suggestions about meth- ods, such as shielding overwintering progeny from electric fields indoors and including observer bias as a variable, were also followed. Two major suggestions were not followed, for reasons that are not en- tirely clear. One was to use a BACT analysis with covariates. The other was to increase replication by placing fewer trap nests at many sites. The original design had four sites (with no replicates), but two of these were dropped. Given that ELF EMFs become ambient at 1.6 km, one reviewer suggested that

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EVALUATION OFFINALREPORTS lO] control and treatment sites could be closer than 48 km apart, which could reduce some of the other environmental differences between sites. increased replicates and reduced variation could have allowed for stronger conclusions about the results. PRESENTATION OF RESULTS The authors are forthright about problems, such as unreliable data, which were eliminated from the analysis, and other confounding variables. A strength of this report is the clarity of discussion about problems and alterna- tives. Alternative hypotheses were often used to dismiss results that indicated potential ELF-EMF effects. The researchers' arguments are reasonable and soundly based on the data, but opposing arguments could be made for real ELF-EMF effects in most cases. Of eight parameters analyzed for one or both species, five showed statistically significant ELF-EMF effects for one or the other species, but only one, overwintering mortality, is interpreted as a possi- ble real ELF-EMF effect and this effect is regarded as ambiguous. Statistically significant effects on three parameters (cell length, leaf num- ber, and nest orientation) are interpreted as being caused by factors other than ELF EMFS. Cell lengths became more similar after full antenna operation for one species. The authors argue that ELF EMFS were not likely to have pre- vented the reduction in cell length (0.2 mm of ~ ~ . ~ mm) at the treatment site. Leaf number also became more similar after the antenna began operating at full power for one species; that is the opposite of what is expected if ELF EMFS are detrimental. The authors also argue that this difference (0.5 leaf/ cell) is trivial and unlikely to affect fitness or population growth. Nest orien- tation was argued as reflecting differences in local flower availability or shad- ing, rather than ELF-EMF exposure. Effects on nest thickness were not statistically significant, but the authors neted that the effect would have had to be very large to be detected with their test (hence, they dismissed this as a strong conclusion). They also cautioned against accepting the null hypothesis for trip duration and sex ratios, because of low power; that is commendable. In contrast, they accept the null hypothe- sis of no effects on offspring weight because statistical power was good. The ELF-EMF effect on overwintering mortality was statistically signifi- cant for one species. Mortality was lower at the treatment site at low antenna power but increased to the level of the control site after full antenna operation.

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~ 02 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM The authors dismiss this as an ELF-EMF effect because mortality became increasingly similar and because of protocol changes, parasitism, and small sample sizes. However, mortality did increase after full antenna operation. Overwintering mortality was also found to differ significantly with ELF- EMF exposure in a nest-transplant experiment. Nests occupied at a treatment site but moved and overwintered at control site showed lower mortality than nests occupied and overwintered at the treatment site. In addition, the mortal- ity of transplanted nests was similar to that of the nests occupied and left at the control site. Although that is strong evidence for ELF-EMF effects, the au- thors argue that the effect is unlikely to be caused by ELF-EMF exposure. The sources of mortality were not emphasized in the researchers' report, because the sources were difficult to determine. For example, the researchers could not separate prepupal mortality in winter from that in summer, fall, or spring. They speculate that weather probably was important for much of the prepupal mortality. Because prepupal mortality varied greatly between years and sites, they decided to test the potential effects of ELF-EMFs on mortality after a bee had survived to the prepupal stage. That would reduce the varia- tion caused by site and weather differences. For this study, a major parasite was the cuckoo bee, Coelioxys, which is also a megachilid and could not be distinguished from the host in the prepupal stage. Therefore, both parasites and hosts are included in the mortality data. They argue that hosts and para- sites would probably have been similarly affected by ELF EMFs so this should not distort the results or interpretations. Overall, the authors conclude that ELF-EMF impacts are absent or at most minimal. CONCLUSIONS Validity Two major problems in the design seriously reduce the ability to detect statistically significant ELF-EMF effects: low replication (pseudoreplication) and confounding variables. The treatment and control sites initially differed in many factors, including flower resources, which also probably varied inde- pendently over time. Otherwise, the details of the study were well designed and well executed. Great care was taken in standardizing variables and in maintaining data qual- ity. A more appropriate statistical analysis could have been used, but it is

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EVALUATION OF FINAL REPORTS 103 doubtful that different conclusions would be reached. Problems are discussed at length. Uncertainties The authors' conclusion that ELF-EMF effects are absent or minimal is uncertain because of the weak ability to detect effects. That results in a great- er likelihood of accepting a false null hypothesis (type II error) than of reject- ing a true null hypothesis (type T error or false positive). A type Il error would also be likely if the response curves were dose-dependent and either monotonic or hump-shaped. Therefore, the fact that the authors did find statistically significant effects requires careful consideration. The finding of increased overwintering mortality in two independent experiments makes an especially strong case for the existence of statistically significant ELF-EMF effects. The researchers' conclusion that ELF-EMF effects are absent or minimal might reflect the low power of the tests rather than the reality of no effects. Real effects would likely have been difficult to detect because of the small sample sizes and high variation in many factors. Therefore, the conclusion of "no effects" might, in fact, be based on the acceptance of a false null hypothe- sis (type TT error). A type lI error would also be likely if the response were dose~ependent and showed a monotonic or hump-shaped relationship to dose. To their credit, they discuss their reasons at length, but good reasons could be advanced for rejecting the null hypothesis in most cases. They also argue that the few statistically significant effects are small and would have little impact on populations. That is an erroneous argument because small differences over a long time can produce large changes in population sizes. Summary The authors' final conclusion that ELF-EMF effects are absent or mini- mal is questionable. The authors' explanations are inadequate for discounting potential ELF-EMF effects and for accepting the null hypothesis of no effect. Given the weak ability of the experiment design to detect ELF-EMF effects, any significant effects should be given careful consideration. Similar argu- ments were made by one reviewer of the final report who strongly feels that ELF-EMF effects were clearly demonstrated in this study. More independent

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1 04 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM replicates would help to determine whether the statistically significant effects are caused by ELF EMFs or by some other factor. SOIL ARTHROPODS AND EARTHWORMS PROJECT PROPOSAL Soil macrofauna, such as arthropods and earthworms, control many of the decomposition processes critical to ecosystem function. It makes sense to examine the abundances, activity, and demographics of dominant soil animals. This study has six parts: soil and litter arthropod censuses, analyses of surface-active arthropod activity via pitfall traps, analyses of earthworm popu- lations sampled by square cores, analyses of growth and reproduction rates of earthworms incubated in soil bags, analyses of litter inputs sampled by litter traps, and analyses of litter-decomposition rates measured in litterbags. SPECIES AND SYSTEM SELECTION Soil and Litter Arthropods The species chosen for analysis were essentially the numerically dominant mites and collembolans (springtails) found in soil cores and litter samples. With no reason to choose a particular species, it is sensible to use the domi- nant species, because their frequency of occurrence makes them conducive to statistical analysis, compared with organisms that occur in only a few samples. Surface-Active Arthropods The researchers selected species on the basis of their abundance and commonness, which is reasonable. it would also have been desirable to pick species on the basis of relative uniformity in distribution over the areas of interest, so as to diminish the statistical problems of place-to-place and time- to-time variations. in addition, arthropods that actively forage on the soil surface could exhibit sublethal effects because of environmental perturbations (through altered behavior), and their activity patterns are potentially good indicators of ELF-EMF effects.

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EVALUATION OF FINAL REPORTS Earthworm Field Populations 105 The researchers examined all nine earthworm species found at the treat- ment or control sites. Because they looked at all species, there was nothing arbitrary in the analysis, and the thoroughness is commendable. Earthworm Growth and Reproduction in Incubation Bags In addition to counting animals, it is a good idea to look for per capita differences in reproductive rates or growth. Using such demographic charac- teristics can yield far more sensitive indicators of ELF-EMF effects than waiting for population densities to reflect differences due to the activation of the antenna. Litter Inputs One can imagine ELF-EMFs influencing tree phenology, leaf production, and leaf abscission in a way that could alter litter inputs into forest soils. The measurement of litter inputs represents a sound research decision. Litter Decomposition Litter decay is certainly an appropriate system process to examine, espe- cially in the context of concordant measures of earthworm and other macro- fauna associated with decomposition. SEEECTION OF RESPONSE VARIABEES Most of the response variables involved counts or densities of individuals by species, an unassailable focus for analysis. Measures of community struc- ture, such as diversity and evenness indexes, were also examined; these aggre- gate indexes are difficult to interpret, and any changes in them would be impossible to understand without also analyzing effects species by species. However, these community indexes play a minor role in the analyses and are

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~ 06 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM distracting only in that they add length to an already confusingly long docu- ment. A few response variables deserve comment. For the earthworm compo- nent, the vertical distribution of worms was examined as a possible indicator of changes in behavior or habits. The age distribution of worms and the size and cocoon production of earthworms were examined as functions of year (preoperational versus operational antenna years). in the earthwo~n~-incuba- tion experiments, the growth and reproductive status of "enclosed" earthworms were followed as a function of antenna operation and previous exposure. Because earthworms can live several years and might require 3 or more years to reach maturity, the attention to growth and reproductive rates in the earth- worm component of the study is commendable; these demographic rates should be more sensitive than absolute population numbers (which could re- flect long time lags and history). Leaf litter inputs were assessed by lumping grams of dry weight per square meter for basswood, maple, and all other species. Litter decomposition was assessed on the basis of the percentage of initial mass remaining. Mass loss is a crude measure of decomposition, and it would have been far better if a more-direct measurement of nutrient release had been obtained. EXPERIMENT DESIGN Biologic and Ecological Sampling Techniques The field design involved a single control site and a single treatment site for all components of the research. Replication is therefore impossible, and the only appropriate statistical analysis uses a before-and-after, control-and- impact (BACT) approach. Although the absence of replication might be un- avoidable, some aspects of the rationale are dubious. First, the two sites were not comparable: they had strikingly different earthworm and arthropod fauna. Second, for the earthworm-incubation experiments, the experiment design involved moving earthworms to a control site at which they did not occur naturally in any abundance (compared with either the treatment site or the sites from which they were taken). Thus, the experiment could be interpreted as an investigation of the effects on a species' growth and reproduction of trans- planting it out of its habitat. The detailed studies of earthworm size, age, and reproductive structure were confined to species that occurred only at the treat- ment site. That makes it virtually impossible to distinguish possible effects of antenna activation from effects of other temporally varying factors.

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EVALUATION OF FINAL REPORTS Physical and Chemical Measurements 107 ELF EMFs were measured, and it was well demonstrated that the treat- ment site exhibited ELF-EMF levels at least 10 times those at the control site. The sites fulfilled TTTRI's other criteria as well. Statistical Methods The studies had four severe statistical flaws. First, when the BAC! analysis was used, the appropriateness of its assumptions of no serial autocorrelation and of additivity was never evaluated. Because the control and treatment sites clearly differed before the antenna was turned on, this is a serious problem. Second, often a simple ANCOVA was applied, although a repeated-measures approach is more appropriate. Third, the power of the tests and sampling scheme was low. For example, to sample surface-active arthro- pods, only 10 pitfall traps were used per site, an absurdly low number. In addition, given the large differences between sites and the variability in data, one questions how powerful the BAC! could be in detecting ELF-EMF effects. Fourth, for many of the earthworm analyses, the only data came from the test site, and no serious effort was made statistically to distinguish temporal varia- tion due to antenna activation from temporal variation due to other environ- mental variables. PRESENTATION OF RESULTS Consideration of Alternative Analyses In general, analyses did not include much consideration of alternative approaches. For example, numerous BACI tests were performed, some of which indicated statistically significant effects. No serious effort was made to determine whether those effects were due to ELF EMFs or to some other environmental characteristics associated with the difference between control and treatment sites. Interpretation The interpretations of data seem predisposed to conclude that ELF-EMF

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~ 08 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM exposure had no statistically significant effect; several potentially significant results (as detected via BACI) were consistently dismissed. Biologically, that might be the correct conclusion, but the interpretation gave too little attention to the weakness of the experimental approach and the statistics being used relative to the variance in the data. Some experiments, such as the earthworm-incubation studies, were especially questionable in interpretation. This study cannot distinguish between a test of "natural" versus "unnatural habitat" and treatment versus control, because in the control site the earth- worm species being incubated was very rare. CONCLUSIONS The utility of this research for policy-making is compromised by a failure to ensure that BAC} was properly applied, by the problem of pseudoreplication and the fact that at best only one control site was compared with one treatment site (which differed substantially from the control site before antenna activa- tion), and by a statistical failure to disentangle temporal trends due to antenna activation from other temporally varying environmental factors. Any effects of ELF EMFs were small, compared with the total variation of the processes measured over the Il-year study; but the study included drought years (likely to be important to soil macrofauna), and it might not be consoling to learn that variation due to ELF EMFs is minor in comparison with the variation caused by a severe drought. SOIL AMEBAS PROJECT PROPOSAL This study, carried out over a period of more than 10 years starting in 1983, records data on populations of soil amebas from two treatment sites near the antenna (one next to the ground terminal and one under the antenna) and a control site about 15 km away, where 76-Hz EMFs were at most one-tenth as large. The studies focused on the ameba Acanthamoeba polyphaga and included counts of organisms in the soil (population sizes), growth-rate mea- surements in situ in culture vessels designed to match ELF-EMF exposures in the soil, and species present and determinations of genetic heterogeneity based on isoenzyme analyses.

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EVALUATION OF FINAL REPORTS SPECIES SELECTION 109 Acanthamoeba polyphaga is one of the more common and already well- studied species of soil amebas that occur near the Michigan transmitting facil- ity. Soil amebas are micropredators, and variations in total counts are thought to reflect differences in quality and quantity of food available, especially bacte- ria. They are close to the bottom of the food chain and thus might be highly indicative of effects at that level. Measurements of total bacteria in the soil, a classically indeterminate value, were attempted with a modification of the acridine orange direct-counting technique; numbers were around lO9/g of soil but were highly variable, so attempts to use this technique to explain variance in soil-ameba numbers were abandoned. SELECTION OF RESPONSE VARIABLES The three principal response variables, as noted above, were counts of organisms in the soil, growth-rate measurements, and determinations of spe- cies present and the genetic heterogeneity. These seem well suited to address the overall study from an ecological vantage point, in that they embrace the static situation, the dynamic growth question, and possible genetic effects according to a well-established criterion. EXPERIMENT DESIGN AND IMPLEMENTATION Biologic Sampling Techniques The sampling techniques were well described and appear to have been carried out with professional competence. Similar experimental procedures were used for samples from the control and treatment sites. Physical Measurements and Sites The physical measurements of the fields and currents at both treatment and control sites were made in cooperation with ITTR] personnel and are well described in the report. Extensive measurements were also made of soil chemistry and moisture, the latter having a substantial effect on the biologic

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~ ~ 0 EVALUATION OF ELF ECOLOGICAL MONITORING PROGRAM systems measured. Data on temperature were collected and are presented in full. The sites for sampling the organisms were selected in cooperation with IlTR} personnel. All sites had a similar 60-Hz EMF background, and the control site had 76-Hz EMF intensities no more than one-tenth those at the treatment sites. The ground treatment site was 39 m from the ground termi- nal, whereas the antenna treatment site was about 40 m from the north-south leg of the antenna. The control site was 15 hen south of the ground treatment site. Statistical Methods One-way analysis of variance (ANOVA) was used to detect differences in total-ameba and cyst counts at the three sites. The before-and-after, control-and-impact (BACT) analysis was used for the log maximal ameba counts and the maximal cyst counts and in the measurements of genetic diver- sity. CONCLUSIONS No population-density differences were found between antenna treatment, ground treatment, and control sites. In addition, growth rates of Acanthamoe- bapolyphaga did not differ between sites, and genetic-diversity studies failed to reveal differences between sites. The only statistically significant difference found was in conditions before and after antenna operation; there was a small but statistically significant difference in maximal population densities between the control site and ground treatment site, but by the same method of analysis no differences between the control site and the antenna treatment site or be- tween the antenna and ground treatment sites. The study appears to have been carried out carefully and competently. The inherent variability in the data was great and changes due to temperature and moisture were large, so small effects would not have been detectable. But the study results constitute convincing evidence that large-scale ELF effects did not occur over the time that the study was carried out. It should be noted that the antenna was fully operational only during the last years of the study and that even then there were down periods. Neverthe- less, this study was adequate for the questions being asked.