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AMER. ZOOL., 37:354-362 (1997) Alterations in Neurobehavioral Responses in Fishes Exposed to Lead and Lead-chelating Agents1 DANIEL N. WEBER 2 , WENDY M. DINGEL Marine and Freshwater Biomedical Sciences Center, University of Wisconsin, Milwaukee, Wisconsin 53204 JOHN J. PANOS AND RHEA E. STEINPREIS Department of Psychology, University of Wisconsin, Milwaukee, Wisconsin 53201 SYNOPSIS. Lead (Pb) is a neurotoxic element that causes behavioral dysfunction in fishes within days of exposure to sublethal concentrations. To test the hypothesis that internal stores of Pb have long-term behavioral effects, Pb-exposed (0.3 ppm) fish were either treated with the Pb-chelating drug meso-2,3 dimercaptosuccinic acid (DMSA) or returned to control conditions (0.0 ppm Pb). Swimming capacity improved after a 7-day DMSA (1.5 ppm) exposure (ANOVA P < 0.05). Removing fish from conditions of waterborne Pb did not achieve this result; DMSA alone without Pb pretreatment had no significant effect. Blood Pb (BPb) levels in control or DMSA-only fish were not detectable. Treated groups had significantly higher BPb (ANOVA, P < 0.05): Pb-exposed fish—530.5 ± 156.7, 884.6 ± 130.0 ppm (1, 2 wks, respectively); Pb-exposed -> 0.0 ppm water—488.8 ± 67.3 ppm after 2 wks; Pb-exposed —> 1.5 ppm DMSA—202.0 ± 116.0 ppm after 2 wks. Norepinephrine and vanillylmandelic acid levels were altered by Pb exposure (ANOVA, P < 0.05). Whereas removing Pb did not facilitate a return to control values, adding DMSA did (ANOVA, P < 0.05). Temporal-spatial response patterns to a stimulus in Pb-exposed (0.1 ppm) and Pb-exposed —» 0.0 ppm Pb water groups differed from controls (0.0 ppm Pb; 0.0 ppm DMSA) for stimulus response angle, and rate and extent of movement away from stimulus source. While the two control types were similar for stimulus response angle and reaction time, DMSA-only controls, unlike 0.0 ppm Pb controls, did not respond as a tightly-associated group after the stimulus. INTRODUCTION fishes The extensive use and mining of Pb have been major sources of contamination in aquatic systems (Carpenter, 1927; Czarnez' 9?5:JDflhn§er' 1 9 8 6 ;Prosl' 1989)- A s a result, fishes, particularly those in urban areas, have been exposed to high amounts of Pb (Eisler, 1988; D'Antuono and Foye, 1992). Because of the importance of Pb as a measure of overall ecosystem health in urban rivers and lakes (D'Antuono and Foye, 1992) and because behavioral endpoints are sensitive measures of toxicity in (Scherer, 1992), our concerns have been to (1) identify specific short-term effects of p b o n individual a n d social behav. iors ( 2 ) e v a l u a t e u n d e rlying physiological m e c h a n i s m s t h a t a r e a s s o c iated with these behavioral alterations, (3) develop methodologies t h a t u s e s h o rt-term behavioral c h a n g e s t o predict adverse long-term effects a n d ( 4 ) e x a m i n e t h e reversibility of effects a n d t h e r o l e o f i n t e r n a , s t o r e s o f p b i n af_ fecting both a n d individual behavjors This focuses o n locomotor activity 'From the Symposium Behavioral Toxicology: Mechanisms and Outcomes presented at the Annual of Pb behavioral toxicity and the ability of Pb-chelating drugs to reverse those effects. Weber (1991) demonstrated that Pb-exposed fathead minnows ^^S^oZJZ?T9V95anwaCs°hrnPgtoan; D.c. 2 To whom correspondence should be addressed. «>• Pamelas) display lower stamina. Pb decreases heme synthesis (Bolognani Fantin et al., 1989; Tabche et al., 1990) and eryth- tQ m o d d 354 m e chanisms FISH NEUROBEHAVIORAL TOXICOLOGY 355 rocyte concentrations (Dawson, 1930, ber and Spieler, 1995) and Cu (Steele, 1933, 1935) in fishes within a few days. 1989) and may result from changes in unWeber (1991) observed after a 30-d Pb ex- derlying circadian rhythms of brain neuroposure edema, intralamellar hyperplasia and transmitter activity. In adult P. promelas, increased mucus secretion in gill filaments. circadian variations of whole brain norepiCumulatively, lowered oxygen uptake and nephrine and its metabolite vanillylmandeltransport abilities, and ion exchange capac- ic acid levels showed significant changes ities at gill surfaces (Tabche et al., 1990) between control and Pb-exposed groups could result in decreased swimming capac- (Spieler et al., 1995). ity. Yet, another explanation for decreased Pb-exposed male Medaka (Oryzias latiswimming capacity is Pb-induced CNS and pes) maintain hyperactivity after being PNS dysfunction. The medulla, a critical placed into 0.0 ppm Pb water for 17 days site for respiratory control, contains both (Weber, unpublished). It is possible that Pb cholinergic and catecholaminergic nuclei damaged pathways critical to controlling that control such functions as mucus secre- this behavior. Alternatively, due to internal tions on the gill surface, opercular stroke stores being mobilized, these neural pathfrequency, gill and systemic blood vessel ways may still be exposed to toxic levels vasoconstriction, and heart stroke (Lagler et of Pb, thus maintaining altered states of beal, 1962; Randall, 1970; Butler and Met- havior. Previous data (Weber, 1991) indicalfe, 1983; Smith, 1984). Pb interferes cate that Pb reaches very high bioconcenwith selected muscarinic receptors in the tration factors (internal: ambient concentrabrain and may be an important mechanism tions), remains in various tissues, especially for some Pb-induced behavioral alterations bone, even after Pb is no longer present in (Costa and Fox, 1983; Schulte et al, 1994; the water, and may decrease from specific tissues at a very slow rate. A similar pattern Cory-Slechta and Pokora, 1995). Group responses to directional stimuli of bone-Pb mobilization has been observed are directly controlled by two interconnect- in humans (Todd et al, 1996). Little research has been done with vered, neural pathways, the Mauthner cells (M-cell) and lateral line system, that induce tebrates on the reversibility of lead effects highly coordinated and uniform group on CNS function. In children exposed to Pb movements away from the perturbation and then treated with various Pb-chelating (Partridge, 1982; Eaton et al, 1991). This drugs, blood Pb does decrease. When chilhighly polarized, stereotypical startle reflex dren cease taking the drug, blood Pb inis controlled by the M-cell located on the creases, suggesting that internal stores are medulla surface. Sensitive to transitory dis- being released (Graziano et al, 1988), alplacements of water, the lateral line gives though the implications for long-term beinformation to individuals regarding posi- havioral change are not clear (Eileen Helztion and velocity within a group (Partridge, ner, MacNeil Consumer Products Company, 1982). During exposure to cadmium (Cd) personal communication), During Pb-cheor copper (Cu), positions relative to others lation therapy, Frumkin and Gerr (1993) within the group, velocity and angular dis- noted that Pb-induced depression was repersion are altered (Sullivan et al., 1978; versed. Ruff et al. (1993) observed imKoltes, 1985). To date, there have been no proved cognitive abilities in Pb-poisoned reports on Pb effects on either neural sys- children treated with the Pb-chelating drug tem. meso 2,3-dimercaptosuccinic acid (DMSA). While some neural pathways control in- Mechanisms of action were not investigated stantaneous reactions to specific stimuli in either study. Friedheim et al. (1978) de(Eaton et al, 1991), others influence loco- scribed the efficacy of treating Pb poisoning motor activity patterns that control specific with DMSA. Several workers investigating circadian rhythms, e.g., anticipatory behav- Pb redistribution during DMSA chelation ior associated with feeding schedules (We- therapy found that DMSA significantly reber and Spieler, 1995). Prolonged states of duced brain and bone Pb levels (Coryhyperactivity are induced by both Pb (We- Slechta, 1988; Jones etal, 1994; Tandon et 356 D. N. WEBER ET AL. al., 1994), although the mode of action at the cellular level is not clear (Goyer et al., 1995). By decreasing levels of Pb in the primary storage site (bone) and in those tissues critical for controlling behavior (brain), DMSA has the potential to affect changes in Pb-induced behavioral dysfunction. Our goal in using this drug, as a part of a multifaceted approach to study Pb neurobehavioral toxicity in fishes, is to determine the longevity and reversibility of Pb-induced behavioral effects. These investigations are unique in that we use fish as a biomedical model for evaluating drug efficacy and physiological mechanisms of behavioral dysfunction. METHODS Blood Pb levels As with clinical samples, whole blood from rainbow trout {Oncorhynchus mykiss) (approximately 250 g) was used to evaluate Pb levels using graphite furnace atomic absorption spectrophotometry with deuterium background correction (GBC Scientific Equipment, Model 904AA). Blood (1.0 mL) from control (0.0 ppm Pb), Pb-exposed (0.3 ppm for 2 wks), Pb-exposed for 2 wks followed by 0.0 ppm Pb for 2 wks, Pb-exposed for 2 wks followed by 1.5 ppm DMSA for 2 wks, and 1.5 ppm DMSA alone for 2 wks to control for any druginduced effects (5 fish per group) was collected weekly from the caudal vein with a heparinized hypodermic syringe. Samples were diluted 1:10 in a matrix modifier of 0.2% NaH3PO4, 0.2% HNO3 and 0.5% Triton X-100. Additional 1:10 dilutions of these samples were prepared as needed. Samples were read at 283.3 nm to minimize background interference normally observed at 217 nm. Data were analyzed using ANOVA (two-way for Pb and DMSA concentrations, with replicates) with significance defined as P < 0.05. Duncan's Multiple Range Test was used to evaluate significance of intergroup differences. Neurotransmitter analyses P. promelas (approx. 25 mm; 5/treatment) were exposed to either 0.0, 0.05 or 0.10 ppm Pb for 1 wk, or Pb for 1 wk followed by 0.0 ppm Pb for 1 wk, or Pb for 1 wk followed by 0.25 or 0.50 ppm DMSA for 1 wk, or DMSA alone for 1 wk. Fish were anesthetized in an ice-water bath. Brains (approx. 2-10 mg wet weight) were removed and snap-frozen in 0.1 M perchloric acid (150 uX/100 mg tissue) until homogenized. Homogenate was centrifuged at 14,800 g for 1 hr at 4°C. Supernatant was recentrifuged with Z-spin filtering centrifuging tubes at 4,000 g for 20 min. Supernatant was injected into an HPLC with electrochemical detection. Dihydrobenzoic acid was used as an internal standard. Mobile phase consisted of phosphate and citrate buffers with o-sulfonic acid as an ion-pairing agent. Data were analyzed using ANOVA (two-way for Pb and DMSA concentrations, with replicates) with significance defined as P < 0.05. Duncan's Multiple Range Test was used to evaluate significance of intergroup differences. Group response to a stimulus If removing Pb from the water should yield results similar to those seen in Pb-exposed fish, it is possible that internal stores of Pb are an important consideration in Pb toxicity studies. If short-term exposure to DMSA is able to return the fish to control behaviors, then the drug may have an important role to play in how internal stores of Pb interact with other tissues. These experiments represent initial tests to evaluate the ability of group responses to detect Pb toxicity and the degree of permanent damage due to Pb exposure. Group startle responses, were recorded on professional quality video tape (Maxell) by a remote camera (Sony Model XC711RR) connected to a 13" color video monitor (Sony, Trinitron model) and a Panasonic Super VHS video recorder with a jog shuttle for frame-by-frame analyses. P. promelas (15 fish/group; 3-4 cm length) were placed in each of three aerated aquaria (static-renewal, 100% water changes every second day) and allowed to acclimate for 1 wk. The applied stimulus involved using a nylon stopper at the end of a glass rod to hit the side of the aquarium at which the group was congregated. At the end of each 1 wk exposure period the area the group occupied, direction of swimming (180° = exact 357 FISH NEUROBEHAVIORAL TOXICOLOGY opposite direction of the stimulus), and mean distance of each fish (at snout) in the group from the source of the stimulus were recorded before the stimulus was applied (to) and every 0.05 sec for 0.5 sec and then at 1.0, 2.0, 5.0 and 10.0 sec thereafter. With each group being its own control, this procedure was repeated after 1 wk exposure to 0.1 ppm Pb, then followed by 0.0 ppm Pb, and finally followed by 1.0 ppm DMSA only. To control for any drug-induced effects, an additional set was exposed to DMSA only. Data were transformed to mean angle and angular distribution using formulae for circular distributions (Zar, 1974). During protocol development, we noticed that control groups responded similarly to a stimulus regardless of time spent in the aquarium. Therefore, separate sets to control for time were not included. Swimming behavior Testing protocol for O. mykiss (approx. 7 cm standard length) followed Peterson et al. (1987) with exposure regimes as with tests for BPb. Data were transformed to critical swimming velocity (Beamish, 1978) and analyzed using ANOVA (two-way for Pb and DMSA concentrations, with replicates) with significance defined as P < 0.05. Duncan's Multiple Range Test was used to evaluate significance of intergroup differences. RESULTS Blood levels The effect of DMSA on blood Pb (BPb) was greater than that of simply removing environmental Pb (Fig. 1; ANOVA, P < 0.05). However, DMSA-treated fish still had a large concentration of Pb in their blood after 2 weeks. Because fish erythrocytes are nucleated, it is possible that these cells have nuclear Pb inclusions similar to that found in catfish oocytes (Katti and Sathyanesan, 1987). It is not clear at present whether these Pb inclusions are a protective mechanism for the cell or a source of internal Pb mobilization. If there are nuclear Pb inclusions, it is not clear from these data if DMSA interacts with them. Pb-DMSA 2 Pb-DMSA 1 I Pb-OPb 2 5> Pb-OPb 1 I Pb2 5 Pb1 I DMSA 2 5 DMSA1 control 2 control 1 200 400 600 800 Blood Pb (ppb) 1000; FIG. 1. Whole blood Pb analysis from rainbow trout (Oncorhynchus mykiss). Fish were exposed to either control (0.0 ppm Pb or 1.5 ppm DMSA) for two weeks, 0.3 ppm Pb for two weeks (Pb), 0.3 ppm Pb for one week followed by 0.0 ppm Pb for two weeks (Pb-0), 0.3 ppm Pb for one week followed by 1.5 ppm DMSA for two weeks (Pb-DMSA). Histogram bars = mean values of 5 fish ± S.E. All Pb treatments are significantly different from control and DMSA-only values; Pb-DMSA at two weeks is significantly different from both Pb and Pb-0 (ANOVA, P < 0.05). Note: No S.E. shown on Pb 2 because 4 of 5 fish in that group died after 1 wk. Whole brain neurotransmitter levels The neurotransmitter dynamics of norepinephrine (NE) and its metabolite vanillylmandelic acid (VMA) in whole fish brains after exposure to each of the experimental regimes is described in Figure 2. Pb exposure results in significant decreases in NE levels at either concentration; a significant drop in VMA occurred only at 1.0 ppm Pb (ANOVA, P < 0.05). Removal of Pb did not ameliorate the condition (ANOVA, P > 0.05 compared to Pb-exposure groups and P < 0.05 compared to control groups). In the presence of DMSA, a return to control values was observed for VMA and a significant increase over the Pb removal group was observed for NE (ANOVA, P < 0.05 for NE and P > 0.05 for VMA). Swimming capacity Individual fish tested without exposure to either Pb or DMSA displayed no significant change in swimming capacity; DMSA alone also did not cause any significant change in swimming capacity (ANOVA, P > 0.05; Fig. 3). Fish exposed for 1 wk to 358 D. N. WEBER ET AL. A. Pb-DMSA .1 Pb-DMSA Pb-0 „, .05 Pb-DMSA £ o> or a .1 Pb-OPb .05 Pb-OPb DMSA control .05 Pb control 40 20000 pg NE/mg brain 40000 FIG. 3. Mean critical swimming velocities in Oncorhynchus mykiss as per cent of control values ± SE Treatments: 1 wk exposure to 0.3 ppm Pb (Pb), 1 wk exposure to 0.3 ppm Pb followed by 1 wk exposure to 0.0 ppm Pb (Pb-0), 1 wk exposure to 0.3 ppm Pb followed by 1 wk exposure to 1.5 ppm DMSA (PbDMSA), or 1 wk exposure to 0.0 ppm DMSA followed by 1 wk exposure to 1.5 ppm DMSA (DMSA). Each individual fish was tested before the exposure regime was begun and again after the exposure regime was completed. Values shown are for the second set of tests, i.e. increases in control values are due to exercise-induced improvements. Controls, DMSA-only, and Pb-DMSA are significantly different from Pb and Pb-0 (ANOVA, P < 0.05) but not from each other. SE for the DMSA-only group = ±27.5. .1 Pb-DMSA o .05 Pb-DMSA E oi .1 Pb-OPb 60 80 100 120 140 180 % Control 0 20000 pg VMA/mg brain 40000 Only by the addition of DMSA for 1 wk did the critical swimming velocity return to FIG. 2. Effects of Pb and DMSA on catecholamine near-control values (89% of control; ANOneurotransmitter dynamics in juvenile fathead minnow VA, P > 0.05). The DMSA group was also (Pimephales promelas). Exposure regimes correspond significantly different from the two to: control (0.0 ppm Pb or DMSA-only); 0.05 ppm Pb Pb-treated groups (ANOVA, P < 0.05). for 2 wk; 0.1 ppm Pb for 2 wk; 0.05 ppm Pb for 1 wk Therefore, just as Pb did not alter hemoglothen 0.0 ppm Pb for 1 wk; 0.1 ppm Pb for 1 wk then 0.0 ppm Pb for 1 wk; 0.05 ppm Pb for 1 wk then 0.25 bin levels, DMSA-induced reversals of beppm DMSA for 1 wk; 0.1 ppm Pb for 1 wk then 0.5 havior may not have been due to physioppm DMSA for 1 wk. A. norepinephrine, B. vanillyl- logical changes in the blood. mandelic acid. * = Significant difference from control; t = Significant difference from Pb-exposed group; + = Significant difference from Pb and no Pb group. Significance: ANOVA, P < 0.05. Pb displayed a 38% loss in critical swimming velocity; this value did not improve by simply removing environmental Pb (-36%; ANOVA, P < 0.05). This is of interest in that even a 2 wk exposure to Pb may not significantly affect hemoglobin concentrations in fish blood (Christensen et al., 1977). Thus, over this time period, changes in swimming capacity may not be due to changes on O2 carrying capacity. Group response to stimulus Under conditions of 0.0 ppm Pb and 0.0 ppm DMSA fish respond to a stimulus by swimming in a closely-knit group in the opposite direction (Fig. 4). After one week of Pb exposure, pre-stimulus group size was initially larger than control groups and the post stimulus flash expansion occupied a larger area and lasted longer, time to react to the stimulus appeared to be longer (0.05 sec in control groups vs. 0.15 sec in Pb-exposed groups), the group took longer settling to a final position (10 sec vs. 2 sec for control group) FISH NEUROBEHAVIORAL TOXICOLOGY which was not at the opposite side of the aquarium, i.e., there was less movement away from the stimulus source (Fig. 5). The response after one week of 0.0 ppm Pb was similar to the reaction of Pb-exposed groups. With only one week of exposure to DMSA, the group again displayed a pattern similar to groups exposed to control conditions. Because approximately 33% of the DMSA-treated fish remained near the stimulus source and the rest moved to the other side of the aquarium, the mean distance traveled appears to be similar to Pb-exposed groups. However, most fish within the group did react similarly to groups under control conditions. Groups treated only with DMSA displayed reaction times and mean stimulus response angle similar to control groups. However, like the Pb-DMSA group, about one-third of the fish in the DMSA-only group did not move very far from the stimulus source. Reaction times of workers exposed to occupational sources of Pb were, as with fish, increased (Stollery, 1996). Interestingly, -y-amino butyric acid (GABA) located in the medulla, where the M-cell is located and where the anterior and posterior lateral lines connect with the M-cell, is involved with the startle response (Smith, 1984); GABA levels in both mammalian and fish brains are decreased by Pb (Silbergeld et al., 1980; Katti and Sathyanesan, 1986). CONCLUSIONS In the experiments reported here, Pb-exposed fishes still maintained their disrupted behavioral patterns 1 wk after they were transferred to aquaria in which all environmental sources of Pb were removed, perhaps due to high internal stores of Pb still being mobilized (Weber, 1991). This behavioral pattern is reflected by whole brain neurotransmitter dynamics remaining altered even after fish were placed in aquaria with 0.0 ppm Pb. Whether individual behaviors, e.g., swimming capacity, or group interactions, e.g., startle responses, were examined, the short-term removal of environmental Pb did little to facilitate a return to the activity patterns observed under control con- 359 ditions. If these observations were primarily due to tissue damage, the addition of a Pb-chelating drug would not be expected to be effective over such short exposure times. However, it was observed that DMSA could reverse some neurobehavioral changes, specifically attenuation of altered startle responses and reversals of norpeinephrine levels and metabolism even when declining BPb levels were still high. Thus, this Pb-chelating agent may be decreasing the internal, labile stores of Pb that are important in affecting behavior. Although several reversals of behavioral dysfunction were affected by DMSA, not all Pb-induced behavioral alterations fall into this category. Previous studies (Weber et al., 1991) demonstrated that psychomotor coordination in P. promelas was adversely affected by Pb. Yet, preliminary data (unpublished) suggest that there is no significant difference between control groups and Pb-exposed fish placed into water with either no Pb or no DMSA. This may be due to the complex nature of neural networks involved in feeding, i.e., olfactory, visual, neuromuscular (jaw muscles for capturing, trunk muscles for swimming), appetitive/motivation (hunger status), and mechanosensory (anterior lateral line). Amelioration in only a few of these systems may not be sufficient to counteract long-term effects of Pb in other neural networks. The length of response, i.e., the fear factor, depends upon processing centers in the medulla. That Pb-exposed groups neither moved very far from the stimulus source nor oriented 180° from the stimulus source, yet did return to the stimulus source, unlike either control group, would indicate that the fear response was quickly attenuated by Pb; the delayed response may be indicative of altered neurotransmitter dynamics in the M-cell and/or slower nerve conduction velocities in the M-cell-interneuron-muscle pathway. These data resemble the changes in vibrational sensitivity seen in some Pb-exposed monkeys (Rice and Gilbert, 1995), i.e., Pb changed the threshold level and response time for mechanosensory neural net- 360 D. N. WEBER ET AL. A. B. A. B. Distance Travelled Area ot Group Distance Travelled Area ot Group 200- 250 0 .1 .2 .3 .4 .5 1 2 510 Time (sec) 0 .1 .2 .3 .4 .512510 Time (sec) 0 .1 .2 .3 .4 .5 1 2 510 Time (sec) .1 2 .3 .4 .51 a 510 Time (sec) .1 .2 .3 .4 . 5 1 2 5 1 0 Time (sec) IV. III. Distance Travelled 0 Area of Group .2 .3 .4 .51 2 510 Time (sec) Distance Travelled Area of Group .2 .3 .4 . 5 1 2 5 1 0 Time (sec) FIG. 4. Group response of fathead minnows (Pimephales promelas) to a stimulus. A. mean group direction from stimulus (arrow) ± angular dispersion (rays surrounding arrow), and mean group distance (cm; intersection point of rays and arrow) from stimulus source as a function of time; B. area of a group (cm2) as a function of time after a stimulus applied. I. 1 wk exposure to 0.0 ppm Pb (control); II. 1 wk exposure to 0.1 ppm Pb; III. 1 wk exposure to 0.1 ppm Pb followed by 1 wk exposure to 0.0 ppm Pb; IV. 1 wk exposure to 0.1 ppm Pb followed by 1 wk exposure to 1.0 ppm DMSA; V. 1 wk exposure to 0.0 ppm DMSA followed by 1 wk exposure to 1.0 ppm DMSA. works. Short-term treatment with DMSA apparently modified, but did not totally reverse, those changes. There exists, however, an alternate hypothesis that requires additional investigation. Because DMSA by itself, affects some parameters of the startle response, specifically group size, it may interact with the neurons and, therefore, directly induce reversals of Pb-induced neurobehavioral toxicity. If true, FISH NEUROBEHAVIORAL TOXICOLOGY V. A. Area of Group Distance Travelled 0 810- 20- 25 0 .1 2 3 A .5 1 2 510 Time (sec) 0 .1 .2 .3 .4 .5 1 2 510 Time (sec) this hypothesis might explain the higher, albeit statistically insignificant, swimming capacity of fish treated only with DMSA. ACKNOWLEDGMENTS These studies were supported by the Marine and Freshwater Biomedical Sciences Center (University of Wisconsin-Milwaukee; NIH grant ES04184 to D. Petering), Medical College of Wisconsin (NIH grant ES07292 to J. Lech), EPA block grant to Wisconsin Department of Natural Resources, and the Conservation and Research Foundation. Special thanks to T. Egan, K. Kosteretz, B. Wimpee and M. Zogg for technical assistance, and S. McKay for figure preparation. REFERENCES Beamish, F W. 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