NONTARGET EFFECTS OF THE MOSQUITO LARVICIDES, TEMEPHOS AND METHOPRENE, AT BOMBAY HOOK AND PRIME HOOK NATIONAL WILDLIFE REFUGES

Publication No. CBFO-C98-01 Study No. 5N15

Prepared by: Alfred E. Pinkney' Peter C. McGowan 1 Daniel R. Murphy1 Donald W. Sparling2 T. Peter Lowe2 Leonard C. Ferrington3

Under supervision of: Robert J. Pennington, Branch Chief John P. Wolflin, Supervisor

U.S. Fish and Wildlife Service1 Chesapeake Bay Field Office 177 Admiral Cochrane Drive Annapolis, MD 21401

U.S. Geological Survey2 Patuxent Wildlife Reseach Center 11510 American Holly Drive Laurel, MD 20708

University of Kansas3 Department of Entomology 2041 Constant A venue Lawrence, KS 66047-2906 •·· December 1998 EXECUTIVE SUMMARY

The U.S. Fish and Wildlife Service is currently evaluating the environmental hazards associated with mosquito management on National Wildlife Refuges (NWRs). The goal is to ensure that mosquito management procedures are compatible with the objectives and operation of the Refuges.

The objective of the studies described in this report was to examine the nontarget impacts of two larvicides, temephos [O,O'-(thiodi-4,1-phenylene) 0,0,0' ,O'-tetramethyl phosphorothioate)] and methoprene (isopropyl [2E,4E,7S]-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate). These chemicals have been used to control mosquito breeding at Bombay Hook and Prime Hook NWRs in Delaware. The salt marshes of both refuges have been sprayed with temephos and methoprene. The freshwater and slightly saline impoundments at Bombay Hook have been sprayed with methoprene. Temephos was used at the Prime Hook impoundments, but since 1995, only methoprene has been used. Temephos is most commonly applied as Abate 4E, an emulsified concentrate ( 44.6% active ingredient), at an application rate of 1.5 o:z/acre (0.048 lb a.i./acre = 0.054 kg a.i./hectare). Methoprene is applied as Altosid Liquid Larvicide (5% a.i.) at a rate of 3 o:z/acre (0.01 lb a.i./acre = 0.011 kg a.i./ha). Airplanes and, to a lesser extent, helicopters are used for spraying, which is done by the Delaware Department of Natural Resources and Environmental Control (DNREC), Division of Fish and Wildlife, Mosquito Control Section.

Six test plots (approximately 170 x 170 m) were established on the salt marsh at Bombay Hook NWR. In 1994, three were randomly assigned to be sprayed with Abate 4E at 0.054 kg a.i./ha and three were controls. Spraying occurred four times, roughly four weeks apart, beginning in early July. Populations of marsh fiddler crabs (Uca pugnax) were monitored by counting the number of burrow holes in areas along tidal guts. A significant linear association was established between the number of holes and number of crabs. Half of the areas were covered with bird netting so that sub lethal toxic effects, which could increase the crabs' susceptibility to predation, could be monitored. There were no statistically significant differences in the number of holes ( or crabs) in sprayed vs. unsprayed plots or between covered vs. uncovered areas. In situ bioassays were conducted with caged juvenile fiddler crabs on sprayed and unsprayed plots. Survival in the bioassay was significantly reduced (16% lower) in the sprayed vs. control plots. Temephos concentrations decreased rapidly in water whereas degradation was slower in sediments.

The mechanism of action for temephos, an organophosphate pesticide, is through inhibition of cholinesterase enzymes, which are vital for the transmission of nerve impulses. Inhibition of cholinesterase activity is also used as an indicator of exposure and response in nontarget organisms. In 1995, red-jointed fiddler crabs (Uca minax) were collected from areas that were operationally sprayed with Abate 4E before and after spraying and from an unsprayed area. The post-spray median AChE activity in the enlarged claw muscle of the males ( 1.3 7 µmoles per minute per gram tissue) was significantly lower than the median activity of the pooled prespray and control area crabs (1.90 µmoles per minute per gram tissue). A 28% inhibition was calculated against this pooled median.

In 1995, the same plots were reassigned randomly and half were sprayed with Altosid at 0.011 kg a.i./ha. Fiddler crab reproductive potential was analyzed by determining the percentage of females that were gravid in spray and control plots. There was no statistically significant difference in the percentage of gravid females captured at spray and control plots. In situ bioassays with coffee bean snails (Melampus bidentatus) did not result in significant differences in survival at spray and control plots.

Meadow voles (Microtus pennsylvanicus) were collected before and after the temephos spraying in 1994. There was no significant difference in acetylcholinesterase (AChE) activity.

In 1995, due to concerns about possible reduced bird use of a sprayed impoundment at Prime Hook NWR, a monitoring program was established in which refuge biologists harvested emerging insects weekly from June through August. Two ponds were monitored -- pond PMH4a, an oligohaline tidally influenced natural pond, and pond PMH3d, a freshwater, stream fed natural pond. PMH4a was last sprayed with Abate in 1994 by DNREC. During the 1995 monitoring period this pond was treated with Bacillus thuringiensis israeleasis (Bti). Pond PMH3d had been last sprayed in 1993 (with Abate). Dipterans comprised the vast majority of the individuals collected from both ponds. The mean species diversity index (Shannon's H') was higher in pond PMH3d than in pond PMH4a; this difference was of borderline statistical significance (t-test, p=0.058). There was a significantly greater species equitability in pond PMH3d than in pond PMH4a (p=0.04). Factors such as salinity, depth, and organic content of the sediments, as well as spray history may have contributed to the observed differences.

In order to examine the effects of mosquito spraying on freshwater habitats using a statistically rigorous design, three groups of six experimental ponds at Patuxent Wildlife Research Center were randomly assigned to be sprayed with either Abate 4E (at 1.5 oz active ingredient/acre), Altosid (at 3 oz/acre), or distilled water. Spraying was conducted by pumping water through a garden hose attached to a venturi sprayer containing either stock solutions of the pesticides or distilled water. Spraying occurred on May 23, June 14, and July 6, with the three week interval corresponding to an environmentally realistic spraying schedule. The emergence of adult insects was monitored before and after each spray by placing two floating traps in each pond. Traps were allowed to collect insects for 7 days and were harvested on the day before each spray and 13-14 days post-spray. Hester-Dendy artificial substrates were placed in the ponds on May 19 and harvested on July 19. Identification was to the lowest practical taxon. Repeated measures ANOV A was used for the emergence trap data and one way ANOVA for the Hester-Dendy data.

In all of the ponds, there was an initial decrease in numbers of emerging insects followed by a gradual increase and dominance by Collembola (springtails). At several periods during the monitoring, there were statistically significant decreases in the mean numbers of individuals, species, and families in the Abate ponds relative to the control. There were statistically

11 significant decreases in species diversity and equitability in the Abate ponds relative to the control ponds. Certain taxa -- Chaoborus, Chironomidae, Odonata, Ephemeroptera -- appeared to be particularly sensitive to Abate and had significantly lower abundances. Mayflies (Ephemeroptera) were affected most dramatically. Only one emerged from the Abate ponds, compared to 195 ·from the Altosid ponds and 264 from the control ponds. With the artificial substrates, there were significant decreases in species diversity, equitability, Chironomidae, and Ephemeroptera in the Abate ponds relative to the controls.

In 1995, the effects of Abate and Altosid on tadpoles were tested in the experimental ponds and in the laboratory. The pond tests were conducted coincident with the insect monitoring described above. On June 12, 13 very young(< 2 days old) or 8 older (7 - 10 days old) gray treefrog (Hy/a versicolor) tadpoles were placed into each of two plastic containers within each pond. The containers were screened to allow food (algae) and water to enter freely but block predators. On July 13, the containers were removed and the weights of surviving tadpoles was determined. There were no significant differences in mortality or mean weight of the younger tadpoles. The mean weight of the older tadpoles was 0.56 mg in the controls, 0.34 mg in the Altosid ponds, and 0.26 mg in the Abate ponds (a significant decrease).

The median lethal concentration of Abate and its effects on cholinesterase activity in green frog (Rana clamitans) tadpoles were determined. The LC50 was 4.24 parts per million as temephos, which was about two orders of magnitude greater than the concentration measured in the ponds shortly after spray. The activity of butyrlcholinesterase (BChE), expressed as percent of control value, declined precipitously with increasing concentrations of Abate. In contrast, AChE activity increased with increasing concentrations of Abate. Further investigation is needed to resolve these divergent responses.

During the growing season of 1997, Abate 4E andAltosid were sprayed on the experimental ponds in a similar manner as in 1995. In late August and early September, the ponds were sampled for metamorphing southern leopard frogs (Rana utricularia). Preliminary analyses showed that juvenile frogs collected from the Altosid-sprayed_ponds had a higher frequency of deformities than those from control ponds (p=0.025). The most common deformities included missing or partially missing hind limbs, but there were also missing eyes and demelanization resulting in a pale tan coloration. No amphibians captured from Abate-sprayed ponds had deformities but the catch per unit effort was significantly decreased in these ponds relative to Altosid or control ponds. Because of the concern over deformed amphibians in parts of the midwest and northeast U.S., the study will be expanded to confirm these preliminary results.

Based on the studies reported herein, the authors make the following management recommendations:

1. Continued spraying of Abate 4E or Altosid on saltmarsh habitats should be permitted while the U.S. Fish and Wildlife Service, researchers, and mosquito control agencies investigate alternative methods of mosquito control and evaluate the relative risks of various control

lll methods. The study results suggest the need to continue to monitor for non-target impacts of Abate 4E on fiddler crabs and other invertebrates, to evaluate whether effective mosquito control can be achieved at lower application rates of Abate 4E, and to determine whether the observed level of cholinesterase inhibition in fiddler crabs results in short or long-term toxicity.

2. Because nontarget impacts were observed on freshwater insects from the spraying of Abate 4E and because there is preliminary evidence of adverse impacts on frogs from Abate 4E and Altosid, we recommend that refuges discontinue the use of these larvicides on freshwater habitats.

3. Emergence traps are recommended as an effective tool for refuge managers to monitor insect populations in ponds.

IV ACKNOWLEDGMENTS

The cooperation and assistance of William Meredith, Roger Wolfe, and Chet Stahecki of the Delaware Department of Natural Resources and Environmental Control, Division of Fish and Wildlife, Mosquito Control Section are greatly appreciated. The authors are grateful for the contributions of Mary Gustafson, presently, and Woody Hill, formerly, of Patuxent Wildlife Research Center; John Moore and Andrew Belisle of Patuxent Analytical Control Facility; Beth McGee, Robert Foley, Lisa Domico, Amy Derosier, Laurie Hewitt, Keisha Johnson, Patty Mccawley, and Ray Li of the Chesapeake Bay Field Office; Paul Daly, Frank Smith, and Jason Barker of Bombay Hook National Wildlife Refuge; and George O'Shea and Annie Larsen of Prime Hook National Wildlife Refuge. This study was funded by the U.S. Fish and Wildlife Service, Division of Environmental Contaminants under Regional ID number 5Nl5.

V

TABLE OF CONTENTS

Page

1.0 INTRODUCTION ...... 1 1.1 Background ...... I 1.2 Reference ...... 2

2.0 EFFECTS OF TEMEPHOS ON FIDDLER CRABS (Uca minax AND Uca pugnax) AT BOMBAY HOOK NWR ...... 4 2.1 Introduction ...... 4 2.2 Materials and Methods ...... 5 2.3 Results ...... 8 2.4 Discussion ...... IO 2.5 References ...... 13

3.0 EFFECTS OF METHOPRENE ON NON-TARGET INVERTEBRATES AT BOMBAY HOOK NWR ...... 20 3.1 Introduction ...... 20 3.2 Materials and Methods ...... 20 3.3 Results ...... 21 3 .4 Discussion ...... 22 3.5 References ...... 23

4.0 EFFECTS OF TEMEPHOS ON ACETYLCHOLINESTERASE ACTIVITY IN MEADOW VOLES (Microtus pennsylvanicus) AT BOMBAY HOOK NWR ...... 27 4.1 Introduction ...... ·, ...... 27 4.2 Materials and Methods ...... · ...... 27 4.3 Results ...... 27 4.4 Discussion ...... 28 4.5 References ...... 28

5.0 INSECT EMERGENCE MONITORING AT PRIME HOOK NWR ...... 30 5.1 Introduction ...... 30 5.2 Materials and Methods ...... 30 5.3 Results ...... 32 5.4 Discussion ...... 32 5.5 References ...... 34

vi 6.0 EFFECTS OF THE MOSQUITO LARVICIDES, TEMEPHOS AND METHOPRENE, ON INSECT POPULATIONS IN EXPERIMENTAL PONDS ...... 38 6.1 Introduction ...... 38 6.2 Materials and Methods ...... 38 6.3 Results ...... 40 6.4 Discussion ...... 43 6.5 References ...... 46

7.0 EFFECTS OF ABATE AND ALTOSID ON TADPOLES ...... 61 7 .1 Introduction ...... 61 7.2 Chemicals Used to Control Mosquitoes on Refuges Differ to Tadpoles .... 61 7.3 Toxicity of Abate to Green Frog Tadpoles ...... 66 7.4 Preliminary Results ofFollowup Investigation ...... 72

8.0 CONCLUSIONS AND MANAGEMENT RECOMMENDATIONS ..... 73

Appendix A. Water quality data for Chapter 6 Appendix B. Taxonomic data for Chapter 6

e

vii • LIST OF TABLES

Page

Table 2.1. Results of 48-hour in situ bioassays with juvenile fiddler crabs (U pugnax) ...... 15 Table 2.2. Concentrations per unit area oftemephos and Abate on sprayed study plots of Bombay Hook NWR. Per hectare and per acre figures are equivalents of the geometric mean temephos concentrations per square meter. 15 Table 2.3. Concentrations oftemephos in water (µg/L) from sprayed potholes on Bombay Hook NWR before and after spray 3...... 16 Table 2.4. Geometric mean temephos concentrations in sprayed sediments (µgig dry weight) at Bombay Hook NWR before and after spray 3 ...... 16 Table 2.5. Fiddler crab AchE and BchE activities ...... 17 Table 3 .1. Results of in situ bioassays with coffee bean snails (M bidentatus) .. 25 Table 4.1. Meadow vole AChE activities after sprays 2 and 3...... 29 Table 5.1. Insect taxa collected from emergent traps placed on ponds' PMH3d and PMH4a at Prime Hook NWR ...... 35 Table 5 .2. Ecological community indices (Shannon-Wiener H', Equitability J', Percent Similarity, PS) determined for insect taxa collected from emergence traps at ponds PMH3d and PMH4 at Prime Hook NWR ...... 36 Table 5.3. Water quality data collected from ponds PMH3d and PMH4a at Prime Hook NWR on the days that emergence traps were monitored ...... 3 7 Table 6.1. Ranges of water quality parameters...... 49 • Table 6.2. Mean values of ecological indices and mean ±SD number of indiv- iduals per pond of taxonomic groupings ...... 50 Table 6.3. Hester-Dendy substrate data ...... 52 Table 7 .1. Survival and body size (X±SD) of gray treefrog tadpoles exposed to Altosid® and Abate® for 3 wetlands, 1995 ...... 63

viii LIST OF FIGURES

Page

Figure 1.1. Locations of the study sites ...... 3 Figure 2.1. Numbers of burrow holes counted in covered and uncovered areas on study plots at Bombay Hook NWR. Sl, S2, S3, and S4 are the four sprays which occurred on 7/6, 7/27, 8/24, and 9/21 (a) Plot 1 (control) and (b) Plot 2 (sprayed) are shown; other plots are similar ...... 19 Figure 3 .1. Percentages of gravid females in Altosid-sprayed and control plots ...... 26 Figure 6.1. Experimental pond with insect emergence trap ...... 53 Figure 6.2. Mean number of insects emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date)...... 54 Figure 6.3. Mean number of insects (excluding Collembola) emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date) ..... 55 Figure 6.4. Mean number of species emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date)...... 56 Figure 6.5. Mean number of families emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date) ...... 57 Figure 6.6. Total number of Chaoborus sp., Chironomidae, and Diptera • emerging from experimental ponds after the first spray ...... 58 Figure 6.7. Cluster analysis for experimental pond insect data: (a) May 22; .... 59 (b)July19 ...... · ...... 60 Figure 7 .1. 96-hr dose-response curve for green frog tadpoles exposed to Abate in synthetic solft water ...... 64 Figure 7.2. Butyrlcholinesterase activity (as percent of control mean) in green frog tadpoles exposed to Abate for 96-hrs ...... 65

ix 1.0 INTRODUCTION

1.1 Background

The U.S. Fish and Wildlife Service is evaluating the environmental risks associated with mosquito control on National Wildlife Refuges (NWRs). The overall goal is to ensure that mosquito control procedures are compatible with the objectives and operation of the Refuges. Mosquito control methods currently in use include the application of chemical and biological agents, use of fish as natural predators, and the alteration of habitat to encourage predation. One method of habitat alteration, Open Marsh Water Management (OMWM), controls breeding of salt marsh mosquitoes by eliminating the isolated (predator-free) potholes that harbor larvae (Dale and Hulsman 1990). Under OMWM, pothole basins supporting mosquito larvae are hydraulically connected with nearby tidal streams allowing both fish predators (primarily mummichog, Fundulus heteroclitus) and fluctuating water levels to control mosquito larvae. Developing a strategy for mosquito control on NWRs involves a careful evaluation of environmental costs and benefits.

This report summarizes field and mesocosm studies conducted to assess the nontarget impacts of two mosquito control larvicides, temephos and methoprene, used at two refuges in Delaware: Bombay Hook NWR and its satellite refuge, Prime Hook NWR (Figure 1.1 ). The Bombay Hook complex was selected because it (1) has the largest area-of unaltered salt marsh habitat on the East Coast, (2) is a key wintering ground for migratory birds along the Atlantic flyway, and (3) has been sprayed with various larvicides and adulticides for many years. Experimental ponds at Patuxent Wildlife Research Center (Figure 1.1) were used to assess the nontarget impacts of • these chemicals on freshwater habitats. Spraying is initiated on short notice, based on field counts in breeding areas. Larvicides, which are used to a far greater extent than adulticides, are applied by helicopter and fixed wing airplane. The primary larvicides are the organophosphate, temephos (Abate) and the growth hormone, methoprene (Altosid). The bacterial toxin, Bacillus thuringiensis israelensis (Bti), has been used to a much lesser extent. The primary adulticides are the organophosphate, naled (Dibrom), and Scourge (the synthetic pyrethroid resmethrin plus piperonyl butoxide). With the exception of several caged fish and shrimp trials performed about 20 miles south of the Refuge, no recent field tests have been conducted to examine possible nontarget impacts at Bombay Hook (W. Meredith, Delaware Department of Natural Resources and Environmental Control (DNREC), Division of Fish and Wildlife, Mosquito Control Section, personal communication).

The remainder of this report is divided into seven chapters. In Chapters 2 and 3, we report the results of field studies which assessed the impacts of controlled spraying of temephos ( 1994) and methoprene (1995) at Bombay Hook NWR on nontarget invertebrates. In Chapter 4, we provide the results of studies conducted in 1994 with meadow voles to determine cholinesterase activity, as a biomarker for exposure and response to temephos. In Chapter 5, we describe monitoring conducted at Prime Hook NWR of insect emergence in two open water areas in the summer of

1 1995. Chapters 6 and 7 are descriptions of studies conducted in 1995 at experimental ponds at Patuxent Wildlife Research Center. Chapter 8 provides the conclusions of the report and e management recommendations.

1.2 Reference

Dale, P.E.R., and K. Hulsman. 1990. A critical review of salt marsh management methods for mosquito control. Rev. Aquat. Sci. 3 :281-311.

•

2 Figure 1.1. Locations of the study sites.

Pennsylvania

New Jersey

Prime Hook NWR

Delaware

Atlantic Ocean Virginia

3 2.0 EFFECTS OF TEMEPHOS ON FIDDLER CRABS (Ucapugnax AND U. minax) AT BOMBAY HOOK NWR

2.1 Introduction •

The insecticide, temephos [O,O'-(thiodi-4,1-phenylene) 0,0,0',0'-tetramethyl phosphorothioate)] has been regularly applied to control mosquito production on the salt marsh at Bombay Hook National Wildlife Refuge (NWR), 13 km from Dover, Delaware (Figure 1.1 ). Temephos, an organophosphorus insecticide, acts through inhibition of cholinesterase enzymes which play a critical role in the nervous system. On the refuge, the emulsified liquid larvicide, Abate® 4E (44.6% active ingredient; American Cyanamid, Princeton, NJ), is sprayed at a rate of 1.5 ozlacre (0.048 lb a.i./acre = 0.054 kg a.i./hectare). Airplanes and, to a lesser extent, helicopters are used for spraying, which is done by the Delaware Department of Natural Resources and Environmental Control (DNREC), Division of Fish and Wildlife, Mosquito Control Section.

Fiddler crabs are the most abundant and conspicuous macroinvertebrates on many salt marshes . (Montague 1980). Their extensive burrowing activities aerate the marsh, enhancing decomposition through increasing surface area and stimulating aerobic bacteria. They are a vital component of the diet of birds such as clapper rail, herons, and willets (Burger et al. 1991). At Bombay Hook NWR, two species of fiddler crabs, the red-jointed fiddler crab (Uca minax) and the marsh fiddler crab (U. pugnax), are found on the salt marsh. Large populations of fiddler crabs (primarily U. pugnax), which live in the intertidal zone along small tidal guts, are exposed • to insecticides when the marsh is sprayed. ·

In a field test on a New Jersey salt marsh, Ward et al. (1976) demonstrated that application of granular Abate (2% temephos), at the 0.1 lb a.i./acre (0.112 kg a.i./ha) rate used for operational control, reduced the population of marsh fiddler crabs by about 30%. These investigators tested both open (uncovered) field plots as well as plots in which bird predators were excluded ( covered). Because the reduction only occurred in uncovered plots, the authors concluded that temephos increased the crabs' susceptibility to bird predation. Laboratory experiments indicated that temephos interfered with the crabs' escape behavior (Ward and Busch 1976). In the field study, although crabs were observed eating Abate granules, temephos tissue concentrations did not increase through the growing season presumably because the compound is metabolized rapidly.

The primary objective of the present study was to examine the non-target impacts of Abate 4E on fiddler crabs. Effects on U. pugnax were analyzed using the caged and uncaged plot design of Ward et al. (1976) and through in situ bioassays. In addition, we investigated possible effects on cholinesterase activity in U. minax, and evaluated the persistence of temephos in pothole water and marsh sediments.

4 2.2 Materials and Methods I e

Study Plots: In the spring of 1994, six approximately 170 x 170 m study plots were delineated along Duck Creek and Shearness Gut on Bombay Hook NWR. Each plot was traversed by a small tidal creek or gut ~ith an intertidal zone populated with fiddler crabs. The corners of the plots were marked with two-m tall metal stakes topped with two-liter orange-painted bottles to increase visibility. The plots were numbered and randomly assigned to be spray (2, 3, and 5) or control (1, 4, and 6) plots. Adjacent plots were separated by at least 500 m.

Spray Application and Chemical Monitoring: In the summer of 1994, Abate 4E was applied by helicopter to three spray plots at 0.054 kg a.i./ha on four occasions: July 6, July 27, August 24, and September 21. The spraying rate and 3-4 week interval between spraying corresponds to typical operational spraying procedures (W. Meredith, DNREC, pers. comm.). Spray was applied early in the morning (before 0800 hours) when the air was still. Temephos concentrations in water and sediments were measured in samples collected before and after the third spraying.

Spraying efficacy was determined for the second, third, and fourth sprays by collecting the sprayed larvicide from nine equal-sized subdivisions within each sprayed plot and four subdivisions in each control plot. Sprayed larvicide was collected on 10 x 20 cm pieces of glass fiber filter paper clipped to aluminum foil covered horizontal wooden platforms attached to vertical wooden stakes. Approximately 18 hours before the scheduled spray, the stakes were pushed into the ground as close as practicable to the center of each of nine equal-sized subdivisions until each stake's platform was IO cm above the surface of the vegetation. Filter • papers in treated plots were removed from the platforms within one hour after spraying and placed immediately in 125-mL glass containers filled with methylene chloride (CH2Cl2) to begin the extraction. Filter papers in the control plots were removed within one hour after spraying was completed at the nearest sprayed plot.

Five potholes within each sprayed plot were sampled 24 h before and 6 h, 24 h, and 48 h after the third spraying to determine the rate oftemephos degradation. Approximately 0.8 L water was collected, using a dipper made with an 0.8-L beaker attached at the end of a 2-m piece of 2.54- cm diameter dowel. The sampling depth was a few centimeters below the water surface. The average water depth of the potholes was difficult to measure due to the soft mud bottoms, but can be estimated as 15-30 cm. Water was poured into 1-L amber-colored glass bottles containing

125 mL of CH2Cl2• Immediately after each sample was collected, the bottle was shaken vigorously for about two minutes to mix the CH2CI 2 and extract the temephos, then put in iced coolers. One duplicate and one spiked sample were collected each time water was sampled. Spiked samples were injected with 160 µg oftemephos (U.S. Environmental Protection Agency standard, 98.5% purity, EPA, Beltsville, MD).

Sediment was collected from upstream and downstream intertidal locations of the tidal guts passing though the sprayed study plots on the same schedule that water was collected. Each

e 5 composite sediment sample (100 g) was composed of 5 - 10 individual samples scraped from the top 1 cm of sediment, using a 50-mL glass beaker attached perpendicularly to the end of 1.3-m piece of dowel. The composite samples were placed immediately into chemically clean glass jars, packed into iced coolers, and stored in a freezer at -20°C.

The samples were analyzed for temephos by the U.S. Fish and Wildlife Service, Patuxent Analytical Control Facility (PACF), Laurel, MD, based on the methods of Belisle and Swineford (1988). Water samples were extracted three times with methylene chloride, extracts were pooled, cleaned by florisil column chromatography, and analyzed by gas chromatography on a DB-1 capillary column using a flame photometric detector. Detection limits were 0.3 µg/L. Sediment samples were mixed with sand and sodium sulfate, placed in a chromatography column and eluted with 1: 1 acetone/methylene chloride, then concentrated and analyzed by gas chromatography using the same column and detector. Detection limits were 0.10-0.16 mg/kg dry weight. Quality control procedures included the analysis of blanks, field and laboratory duplicates, and matrix spikes.

Chemical data were analyzed using analysis of variance (ANOVA) within the framework of general linear models (Proc GLM) because the data did not fit into a balanced design (SAS 1989). Duncan's multiple range test was used to separate means of main effects that were significantly different in the ANOVA. Data from samples in which temephos concentrations were below detection limits were represented as half of the limit of detection. All statistical analyses were performed on log-transformed values to avoid heteroscedasticity.

Fiddler Crab Monitoring: The numbers ofmarsh fiddler crabs were estimated by counting the number of burrow holes in locations that were visited repeatedly. A linear relationship between the number of holes and number of marsh fiddler crabs (~0.5 cm carapace diameter) was established on a Massachusetts salt marsh by Krebs and Valiela ( 1978) as number of crabs = 2.233 + 0.9712 (holes), (r=0.95, p<0.01, n=13). This relationship was determined by sinking a 0.5 x 0.5 square m wooden frame into the mud, counting the holes, and then digging to a depth of 0.5 m, sieving through a 4 mm mesh, and counting the crabs. Using this same technique, the correlation between holes and crabs at Bombay Hook NWR was established over the course of the study.

Covered and uncovered areas were established so that both lethal and sublethal effects (increased susceptibility to bird predation) could be examined. On each of the six study plots in the intertidal zone along a tidal gut, two 30 m x 1 m areas were marked out. These areas were covered by approximately 10 cm water at high tide and were out of the water at low tide. One half of each area was covered with plastic bird netting (1.9 x 1.6 cm mesh) to exclude avian predators. The netting was dug into the mud and fixed with horseshoe-shaped metal stakes. Within each area two 1 m x 1 m square counting areas were marked with fluorescent pink cord. Within the counting area, all vegetation was kept to a height of less than 2 cm by frequent clipping with hand shears. The number of burrow holes in each counting area was determined on the day before each spray (day -1), and on days +1, +2, and +7. Only holes at least one cm in

6 diameter were counted, since this is the minimum size for U. pugnax burrow holes (Montague 1980).

A repeated measures ANOV A (SAS 1989) was used to test for the effects of spraying on the number of holes. The following model variables were identified and included in the analysis: treatment (spray or control), plot number, cover (covered or uncovered), and spray (whether the counting was performed pre-spray or post-spray). Interactions between these variables were also evaluated. Correlation analysis was used to relate the numbers of holes with the numbers of crabs.

In situ Bioassays: Juvenile U. pugnax (approximately 2-4 mm carapace width) were collected along Petersfield Ditch, an area of Prime Hook NWR (Figure 1.1) that had not been sprayed with mosquito-control chemicals in 1994 (W. Meredith, pers. comm.). The crabs were kept in plastic trays containing collection site mud and held overnight in the laboratory. The next day crabs were transported to the study plots, where 10 crabs each were placed in 1-L polyethylene jars having 5 x 7.5 cm windows on two sides covered with I-mm mesh polyethylene screen. Each jar was tied to a galvanized minnow trap such that one window was facing upwards. One tablespoon of study plot mud was placed in each jar to provide moisture for the crabs. At each study plot on the day before the third and fourth sprays, two traps were placed in an unvegetated area just above the high tide line The crabs were collected two days after spraying and the numbers of living and dead crabs were counted. Data were analyzed using two way ANOVA on arc sin square root-transformed survival data to test for effects of the spray, the spray event (3rd • or 4th spray), and the interaction (SAS 1989).

U. minax Cholinesterase and Residue Analysis: In 1995, the investigators were given several days notice by DNREC of an upcoming operational spray with Abate 4E at 0.054 kg a.i./ha. Red­ jointed fiddler crabs (U. minax) were collected for cholinesterase assay and residue analysis because they are larger than U. pugnax. The crabs were collected from an area of Bombay Hook NWR along the Leipsig River that was to be operationally sprayed with Abate 4E and from a control area (Marshall Island) that has never been sprayed (W. Meredith, Delaware DNREC, pers. comm.). The spray occurred on July 18. Crabs were collected from Marshall Island on July 14 and July 20 and from the spray area on July 17 and July 20. On each date, male crabs were collected for residue analysis (25 per site per collection date) and cholinesterase assays (10 per site per date). Crabs were kept on ice after collection and stored at -20 °C until analysis.

U. minax samples were analyzed for temephos by the P ACF, based on Belisle and Swineford ( 1988). Carapaces were removed and composites of five crabs were analyzed as a single sample. The weight of the samples ranged from 14.9 g to 26.4 g. The soft tissues were homogenized with acetone and methylene chloride. The organic extract was filtered and adjusted to volume for gas chromatography on a megabore capillary column using a flame photometric detector. The detection limit was 0.019 to 0.023 mg/kg wet weight.

7 For the cholinesterase assays, the large claw muscle tissues from individual male crabs were analyzed. Acetylcholinesterase (AChE) assays were conducted according to the methods of Hill and Fleming (1982), at a buffer:tissue (volume:weight) ratio of20:1 and a homogenate volume of • 0.1 mL. Butyrlcholinesterase (BChE) assays were conducted according to the same method, using the substrate butyrlthiocholine iodide instead of aceytlthiocholine iodide. The buffer:tissue ratio was 20: 1 and the homogenate volume was 0.3 mL.

Statistical differences in cholinesterase activity before and after spraying were analyzed by Mann-Whitney U tests because parametric assumptions were not met even after log transformation. Data from the Marshall Island control plot were used to analyze for differences in time unrelated to the spray and to evaluate variability between the sprayed and control plots.

2.3 Results

Fiddler Crab Population Monitoring: There was a significant linear relationship between the number of holes and the number of crabs with a carapace diameter ~0.5 cm: (number of crabs= 13.945+0.535(holes); r=0.50, p<0.001; n=46). A similar result was obtained when the relationship was calculated for crabs with a carapace diameter ~ 1.0 cm: ( crabs = 13 .365 + 0.521(holes); r=0.49, p<0.001, n=46).

The number of holes fluctuated considerably through the course of the monitoring ( examples given in Figure 2.1 ). Based on the repeated measures ANOV A, there was no significant effect of spraying on the mean number of holes (p=0.51). The effect of cover (p=0.28), the interactive a effects of spraying and cover (p=0.91 ), and the interaction of spraying, cover, and pre vs. post • spray (p=0.55) were also not significant. The mean number of holes was similar in the sprayed (53.4 holes/m2) vs. control (49.1 holes/m2) areas, and in the covered (46.6 holes/m2) vs. uncovered (55.9 holes/m2) sections. Using the linear equation for crabs with a carapace width~ 0.5 cm, the mean numbers of crabs were: 42.5 (sprayed), 40.2 (control), 38.9 (covered), and 43.9 (uncovered).

In situ Bioassays: The mean survival of juvenile fiddler crabs in the sprayed jars (77.5%) was significantly less than that in the control jars (93 .3%; two way ANOVA on arc sin square root transformed data; p =0.01, Table 2.1). This represents a 15.8% decrease in survival ofjuvenile crabs exposed to a temephos application.

Chemical Monitoring: The application of temephos was uneven among sprays, and highly variable within sprays (e.g., spray 4 range was 75 µg/m2 to 11,500 µg/m2; Table 2.2). Mean temephos concentrations were significantly different among sprays (ANOVA; p<0.001) with the second spray significantly lower than the third and fourth sprays. The average per hectare application oftemephos during sprays 2, 3, and 4 were 0.0105, 0.0633, and 0.1047 kg a.i./ha, respectively (Table 2), compared to the desired 0.054 kg a.i./ha application rate. Thus, sprays 3 and 4 appear to be within a factor of two of the desired rate, whereas spray 2 appears to be only

8 about 20% of the desired rate. Concentrations on filter paper collected from unsprayed plots were consistently below the limit of detection(< 0.50 µglm2).

Temephos concentrations in pothole water at 6 h after spray 3 ranged from 7.69 to 132 µg/L with an average of 22.6 µg/L. Mean temephos concentrations in pothole water were significantly different for all collection times, with the lowest concentrations occurring before spraying and the highest occurring 6 h after spraying (Table 2.3). Concentrations decreased rapidly over the first 24 h. Mean concentrations in water collected 24 hand 48 h after spraying was 7.7% and 3 .2%, respectively of the mean concentration in water collected 6 h after spraying. For the sediment concentrations after spray 3, the results of the ANOVA indicated that study plot and collection times were both significant (p = 0.013 for both variables; Table 2.4). Changes in temephos concentrations after the spray were not consistent among plots. Concentrations declined slightly at plot 2, decreased and then increased at plot 3, and increased and then decreased at plot 5. Mean values for the three plots indicate that approximately 63% of the temephos measured in sediments 6 h post spray was still present 48 h post spray.

Analysis of the blank samples did not indicate detectable temephos. Mean spike recovery from Bombay Hook NWR water (82. 7%) was nearly equal to that of reverse osmosis water (81.6%; range 70-94%); however, the variability among samples was greater in the Bombay Hook water. In the five spiked samples with Bombay Hook water, recovery in three was 78-81 %, however one sample had 41 % recovery and one had 134% recovery. Recoveries of spiked sediment samples were higher (91 % and 114% ) and more consistent than those for water samples . Duplicate sediment samples were more consistent (relative percent differences of 6% and 12%) • than duplicate water samples (relative percent differences of 0%, 19%, 49%, and 171 %). U minax Cholinesterase and Residue Analysis: There was a significant decrease in the AChE activity of the crabs collected after spraying compared to those collected before spraying (Table 2.5). Median activity was 2.18 µmoles per min per g tissue at the spray site on the day before the operational spray (July 17) vs. 1.37 µmoles per min per g tissue two days post-spray (July 20). At the control site, median activity was nearly identical on July 14 (1.83 µmoles per min per g tissue) and July 20 (1.85 µmoles per min per g tissue). The activity in the crabs collected post­ spray on the spray site were also compared with the pooled data from the pre-spray collection at the spray site and the two collections from the control site. These data were pooled after an analysis of variance indicated no significant differences between the three collections (p=0.058). The post-spray median activity (1.37 µmoles per minute per gram tissue) was significantly lower than the median of the pooled data (1.90 µmoles per minute per gram tissue). A 28% inhibition of AChE activity was calculated against this pooled median.

BChE activity was not significantly inhibited at the spray site (Table 2.5). Median activity was 0.245 µmoles per min per g tissue on July 17 and 0.191 µmoles per min per g tissue on July 20 (Mann-Whitney U test, p=0.10). At the control site, activities were nearly identical -- 0.200 µmoles per min per g tissue on July 14 and 0.198 µmoles per min per g tissue on July 20.

9 There were no detectable residues oftemephos in any of the U minax samples. Detection limits were O.019 to O. 023 mg/kg wet weight.

2.4 Discussion •

Effects on Fiddler Crab Populations

The lack of effect of Abate 4E on fiddler crab populations, as evidenced by the burrow hole monitoring, contrasts with the studies of Ward et al. (1976) with granular Abate. This may be a result of the much lower exposure in the present study. First, the application of Abate 4E (0.054 kg temephos/ha) was less than half the 0.1125 kg temephos/ha rate of application used by Ward et al. (1976). Second, since Ward et al. (1976) observed crabs eating the granules; it is likely that some crabs obtained much higher concentrations of the compound than would be expected by broadcast of an emulsified insecticide. Finally, the Abate 4E would be expected to be rapidly diluted by the incoming and outgoing tides in the tidal gut, whereas the granular Abate might adhere to mud or vegetation and remain available for consumption for longer periods. In a study with the coffee bean snail (Melampus bidentatus), Fitzpatrick and Sutherland (1976) reported substantially higher uptake of granular vs. emulsified temephos, and attributed the increase to the snails' ingestion of whole granules.

The 0.50 correlation coefficient for the association between the number of holes and number of crabs was considerably weaker than the 0.95 coefficient reported by Krebs and Valiela (1978) on a Massachusetts salt marsh. The present study was based on 46 counts, however, while Krebs and Valiela based their relation on only 13 counts. Further investigation of the relationship between the number of burrow holes and the number of crabs is recommended. Alternate methods of censusing fiddler crabs, such as enclosing the crabs within frames at high tide and • capturing and counting at low tide (Cammen et al. 1984), while more time-consuming, might be more accurate.

Pierce (1993) reported that application of Abate 4E to the south Florida saltmarsh and fringing mangrove forest communities raised concerns for lethality to the saltmarsh fiddler crab, U rapax. His laboratory test reported an estimated toxic threshold to U rapax larvae ( concentration at which there was no significant increase in mortality through two days past the first molt) of 5 µg/L. In the field, spraying Abate 4E at 0.5 ounce temephos/acre (0.018 kg/ha) on the mid- and lower-marsh killed fiddler crab larvae, whereas spraying only the upper marsh area did not kill fiddler crab larvae.

Non-target effects of mosquito larvicides on larval fiddler crabs may not be as great a concern in the Delaware salt marsh as in the mangrove marsh. In the mangrove, the larval fiddler crabs may be present in the mid-marsh habitats where mosquito larvae are breeding and where spraying may occur (Pierce 1993). In the Atlantic coast salt marshes, U pugnax larvae are released on a semi-lunar cycle coinciding with the high tides associated with new and full moons, which results in maximum dispersal from the marsh to the estuary (Wheeler 1978). Thus, it is unlikely

10 that the fiddler crab larvae would experience substantial exposure to larvicides sprayed on the Delaware marsh.

Juvenile fiddler crabs may be the life stage most likely to be affected by Abate 4E. As shown in the in situ bioassays, exposure can result in a 16% decrease in survival. Juveniles may be more sensitive than adults to the pesticide because they are receiving a higher exposure per unit body weight. The caged exposure which occurred just above the high tide line, however, may overestimate exposure in the intertidal zone, where there would be dilution with each tidal cycle.

Chemical Monitoring

Temephos concentrations on the filter paper ranged over several orders of magnitude within sprays 2, 3, and 4 (Table 2.2). The concentration at the high end of the range might reflect a temephos buildup in the overlap zones of adjacent spraying swaths. The concentration at the low end of the range may reflect an area that received low temephos exposure, as might occur between spraying swaths.

The range in temephos concentrations was greater than the results that have been reported in another study using a similar technique. Temephos concentrations measured during two sprayings of a Florida mangrove swamp varied from 1,315 to 3,130 µg/m2 (Pierce, et al. 1989). The lower concentrations measured on the filter papers from the second spray probably reflect the severe weather conditions that preceded spraying, rather than real differences in coverage. Heavy rains the night before spraying left the filter paper saturated with water, which displaced

large amounts of the CH2Cl2 in the storage vessels. Because the storage vessels were just large • enough to accommodate the filter papers, much of the CH2Cl2 with the dissolved temephos could not be salvaged.

The mean temephos concentration in pothole water at 6 h after spraying (22.6 µg/L) is in reasonable agreement with expected concentrations. For the 0.054 kg/ha application rate, the theoretical concentration immediately after spraying would be 63 µg/L for an average depth of 15 cm and 31.5 µg/L for an average depth of 30 cm (based on Lores et al. 1985). A lower measured concentration can be expected due to the degradation that would have occurred over the 6 hours after the spray and due to binding to suspended particles or sediments.

Degradation rates over the first 24 h in this study appear to have been similar to the rates reported in some other studies. Geometric mean temephos concentrations in water collected 24 h post spray were 7.7% of those measured at 6 h post spray (Table 2.3). Hughes et al. (1980) reported that 90% oftemephos had disappeared in 1.05 d post spray in a study of two freshwater ponds that were sprayed at a per square meter area rate resulting in an initial concentration of 10.0 µg/L. The 24 h post spray concentrations reported by Lores et al. (1985) were about 4% of the concentrations reported 1 h post spray. Degradation rates in some studies appear to have been slower than those seen in this study or given above. Helgen et al. (1988) reported mean 24 h post spray concentrations for five experimental sites that were 21 % of the mean 1 h post spray

11 concentrations, and Pierce et al. (1989) reported 24 h post spray concentrations that ranged from 19% to 46% of 1 h post spray concentrations. In a later study, however, Pierce et al. (1996) reported 24 h post spray concentrations more in line with those reported by the earlier mentioned investigators, which were 12.5% and 4.6% of the 1 h post spray concentrations. •

Four out of 13 pothole water samples collected 24 h before spray 3 contained detectable concentrations oftemephos (mean of 0.23µg/L or about 1% of the 6 h post spray concentration). Thus, some traces oftemephos from spray 2, which occurred 27 days earlier, were still evident in pothole water.

Mean temephos concentrations did not decline as rapidly in sediments as in water. Overall mean temephos concentrations in sediments 24 hand 48 h post spray had declined to 62% and 63%, respectively, of 6 h post spray concentrations (Table 2.4), whereas water concentrations during the same period had declined to 7.7% and 3.2%, respectively {Table 2.3). Pierce et al. (1989) also indicated that temephos degradation in sediment samples proceeded more slowly than in water. Concentrations 24 h post spray were 44.4%, 35.8% and 92.5% of 1 h post spray concentrations at three sprayed sampling sites. Concentrations 48 h post spray were 68% of 1 h post spray concentrations at one sprayed sampling site. The high variability in sediment concentrations among sites and at different sampling times observed in the present study was also observed by Pierce et al. (1989).

No studies have been conducted to determine the persistence and fate oftemephos applied to sediment; however, an investigation may be advisable because of the implications that slower degradation might pose when managing lands that experience regular tidal flooding. For example, if temephos were applied to recently exposed intertidal sediments, very little degradation of temephos would take place before the sediments were flooded again several hours • later by the next high tide. If temephos were released from the sediments during flooding, it could be carried into aquatic areas where non-target organisms could be exposed.

Cholinesterase inhibition

The 28% inhibition observed in the present study is only slightly below the 30-50% inhibition range suggested by Edwards and Fisher ( 1991) as an indicator of exposure in terrestrial and aquatic invertebrates, and the 30% threshold for freshwater mussels suggested by Moulton et al. (1996). No values for "normal" fiddler crab muscle tissues were identified in the literature, nor has there been an inhibition threshold proposed. It is reasonable to conclude that the 28% level of inhibition is indicative of exposure, and that laboratory studies would be needed to establish the relationship between this level of inhibition and lethal or sublethal effects.

Conclusions

Although spraying with Abate 4E at an operational rate of 0.054 kg a.i/ha did not affect populations of adult fiddler crabs (U pugnax), it caused a significant increase in mortality in

12 juveniles in in situ bioassays. Acetylcholinesterase activity was significantly inhibited in U minax exposed to Abate 4E at the same rate. In view of the toxicity to juvenile crabs and the cholinesterase inhibition, we recommend continued monitoring for non-target impacts of Abate 4E on fiddler crabs, especially population-level effects, and research to establish whether the reported level of cholinesterase inhibition results in acute or chronic toxicity.

2.5 References

Belisle, A.A., and D.M. Swineford. 1988. Simple, specific analysis of organophosphate and carbamate pesticides in sediments using column extraction and gas chromatography. Environ. Toxicol. Chem. 7: 749-752.

Burger, J., J. Brzorad, and M. Gochfeld. 1991. Immediate effects of an oil spill on behavior of fiddler crabs (Uca pugnax). Arch. Environ. Contam. Toxicol. 20:404-409.

Cammen, L.M, E.D. Seneca, and L.M. Stroud. 1984. Long-term variation of fiddler crab populations in North Carolina salt marshes. Estuaries 7: 171-175.

Edwards, C.A. and S.W. Fisher. 1991. The use of cholinesterase measurements in assessing the impacts of pesticides on terrestrial and aquatic invertebrates. In: Cholinesterase-inhibiting Insecticides. Their Impact on Wildlife and the environment. P. Mineau, ed., Elsevier Science Publishers, Amsterdam, pp. 255-275 .

Fitzpatrick, G. and D.J. Sutherland. 1976. · Uptake of the mosquito larvicide temefos by the salt • marsh snail, New Jersey -- 1973-74. Pesticides Monit. J 10:4-6. Helgen J.C., N.J. Larson, and R.L. Anderson. 1988. Responses ofzooplankton and Chaoborus to temephos in a natural pond and in the laboratory. Arch. Environ. Contam. Toxicol. 17:459- 471.

Hill, E.F. and W.J. Fleming. 1982. Acetylcholinesterase poisoning of birds: field monitoring and diagnosis of acute poisoning. Environ. Toxicol. Chem. 1:27-38.

Hughes, D.N., M.G. Boyer, M.H. Papst, C.D. Fowle, G.A.V. Rees, and P. Baulu. 1980. Persistence of three organophosphorus insecticides in artificial ponds and some biological implications. Arch. Environ. Contam. Toxicol. 9: 269-279.

Krebs, C.T., and I. Valiela. 1978. Effect of experimentally applied chlorinated hydrocarbons on the biomass of the fiddler crab, Uca pugnax (Smith). Estuar. Coastal Mar. Sci. 6:375-386.

Lores, E.M., J.C. Moore, P. Moody, J. Clark, J. Forester, and J. Knight. 1985. Temephos residues in stagnant ponds after mosquito larvicide applications by helicopter. Bull. Environ. Contam. Toxicol. 35:308-313.

13 Montague, C.L. 1980. A natural history of temperate western Atlantic fiddler crabs (genus Uca) with reference to their impact on the salt marsh. Contrib. Mar. Sci. 23:25-55.

Moulton, C.A., W.J. Fleming, and C.E. Purnell. 1996. Effects of two cholinesterase-inhibiting pesticides on freshwater mussels. Environ. Toxicol. Chem. 15:131-137.

Pierce, R.H. 1993. Temephos distribution and toxicity in a South Florida saltmarsh community. Mote Marine Laboratory Technical Report No. 333. Sarasota, FL. 24 p.

Pierce, R.H., R.C. Brown, K.R Hardman, M.S. Henry, C.L.P. Palmer, T.W. Miller, and G. Wichterman. 1989. Fate and toxicity oftemephos applied to a intertidal mangrove community. J Am. Mosquito Control Assoc. 5:569-578.

Pierce, R, M. Henry, D. Kelly, P. Sherblom, W. Kozlowsky, G. Wichterman, and T.W. Miller. 1996. Temephos distribution and persistence in a southwest Florida salt marsh community. J Am. Mosq. Control Assoc. 12:637-646.

SAS Institute Inc, 1989. SAS/STAT User's Guide Version 6. 4th edition. SAS Institute Inc., Cary, NC. 846 p.

Ward, D.V., B.L. Howes, and D.F. Ludwig. 1976. Interactive effects of predation pressure and insecticide (Temefos) on populations of the marsh fiddler crab Uca pugnax. Mar. Biol. 35:119-126.

Ward, D.V. and D.A. Busch. 1976. Effects oftemefos, an organophosphate insecticide, on survival and escape behavior of the marsh fiddler crab Uca pugnax. Oikos 27:331-335.

Wheeler, D.E. 1978. Semilunar hatching periodicity in the mud fiddler crab Uca pugnax (Smith). Estuaries 1:268-269.

14 e Tba le 2.1. Results o f 48-hour in situ bioassays with juvenile fiddler crabs (U. pugnax % Survivala

Control plots Spray plots Spray 3 68.3 (80 , 65, 60) 93.3 (90, 100 b, 90) Spray 4 86.7 (80, 85, 95) 93.3 (95, 90, 95)

OveraUC 77.5 93.3

a Mean survival followed by survival per plot (based on two Jars per plot with 10 crabs per jar) b Survival for one jar, in the other jar the mud was dried and all crabs were dead c Results of two way ANOVA on arc sin square root transformed data: treatment (spray vs. control) - p=0.01; spray event (3 vs. 4)- p=0.24; treatment x spray - p=0.10.

Table 2.2. Concentrations per unit area of temephos and Abate on sprayed study plots of Bombay Hook NWR. Per hectare and per acre figures are equivalents of the geometric mean temep h os concentraf ions per square met er. • Spray Number Temephos (µglm 2) Temephos Abatec of samples mean±SDa range (Kg/ha, fl oz/acb) (Kg/ha, fl oz/ac)

2 24 1083±1000 A 2.5-4250 0.0047, 0.06 0.0105, 0.13

3 27 4911±3471 B 2.5-11500 0.0283, 0.36 0.0633, 0.81 4 23 6023±2801 B 75-11000 0.0468, 0.59 0.1047, 1.33 a Mean values with different letters are significantly different using the Duncan's multiple range test (p<0.05) b Assumes that the specific gravity ofTemephos and Abate (1.0756 g/ml) are equal c Based on temephos composing 44.6% of Abate

15 Table 2.3. Concentrations oftemephos in water (µg/L) from sprayed potholes on Bombay Hook NWR b e tiore and a ft er spray 3 . Collection time Frequency of Geometric Minimum Maximum detection mean

24h before spray 4 /13 0.23 Aa 0.15b 0.74 6h post spray 15/15 22.6B 7.69 132.3 24h post spray 15/15 1.74 C 0.44 28.0

48h post spray 11/15 0.72D 0.15 b 59.6 a Geometric mean values with different letters are significantly different using ANOVA (p=0.013) and Duncan's multiple range test (p<0.05) b Equal to one half of the limit of detection for all samples

Table 2.4. Geometric mean temephos concentrations in sprayed sediments (µgig dry weight) at B om b ay H 00 k NWR b e tiore and a ft er spray 3 Collection Study plot Number of Overall time samples geometric 2 3 5 mean 24h before 0.067a nab 0.075a 4 0.071 Ac spray

6h post spray nab 0.570 1.392 4 0.890 B 24h post 0.216 0.485 1.613 6 0.553 B spray

48h post 0.169 0.726 1.452 6 0.563 B spray a Mean of half of the limits of detection for all samples in a group b Not analyzed cMean values with different letters are significantly different using ANOVA (p=0.013) and Duncan's multiple range test (p<0.05)

16 Table 2.5. Fiddler crab AChE and BChE activities.a Enzyme Area Pre-sprayb Median Post-spray (day +2) Statisticsc (Min, Max) (n=lO) Median (Min, Max) (n=lO)

AchE Spray 2.18 (1.66, 3.11) 1.37 (1.27, 2.46) S: U test, p=0.009

Control 1.83 (1.28, 3.33) 1.85 (1.58, 2.03) NS: U test, p=0.70

BChE Spray 0.245 (0.199, 0.290) 0.191 (0.141, 1.125) NS: U test, p=0.10 Control 0.200 (0.139, 0.256) 0.198 (0.179, 0.273) NS: U test, p=0.90 a Units: µmoles per minute per g tissue b Pre-spray sampling on 7-14-95 (control) and 7-17-95 (spray) c Mann-Whitney U tests because data did not meet assumptions for parametric statistics; S - significant (p<0.05); NS - not significant (p>0.05)

•

17 Figure 2.la. Numbers of burrow holes counted in covered and uncovered areas on study plots at Bombay Hook NWR. Sl, S2, S3, and S4 are the four sprays which occurred on 7/6, 7/27, 8/24, and 9/21. Site 1 (control)

Fiddler Crab Burrow Data -uncov Site 1 (control) -a-uncov 120 --e--COV

-9-COV ... 100 ..,Cl) Cl) E Cl) 80 ...ca :::s er "' 60 -"'Cl) -0 .c.... 40 0. 0 z 20

0

It) ~ co M N CD N co a, M M M It) CD 0 "11:t' N M co ""'" N N N N N N N ""'"M N N N N ~ en ~ ~ ""'" en co en en - - - -~ -~ -~ --~ ~ - -co -co -co C0 a,- --a, a, -a, Date • Figure 2. lb. Numbers of burrow holes counted in covered and uncovered areas on study plots at Bombay Hook NWR. SI, S2, S3, and S4 are the four sprays which occurred on 7 /6, 7/27, 8/24, and 9/21. Site 2 (sprayed)

Fiddler Crab Burrow Data Site 2 (sprayed)

120 -uncov ... 100 -a-uncov ...,Cl) G) ...... ,_cov E G) 80 -&-cov ...ns :::J O"',,, 60 - ,,, -G) -0 .c 40 It- 0. z0 20

0 N C") en en Date 3.0 EFFECTS OF METHOPRENE ON NON-TARGET INVERTEBRATES AT BOMBAY HOOKNWR

3 .1 Introduction

The mosquito larvicide, methoprene (isopropyl [2E,4E,7S]-11-methoxy-3,7,11-trimethyl-2,4- dodecadienoate) is regularly applied to the salt marsh at Bombay Hook National Wildlife Refuge (NWR), located approximately 13 km from Dover, Delaware, USA. The most commonly used formulation is Altosid® Liquid Larvicide, (5% (S)-methoprene; Zoecon Corp., Dallas, TX), which is applied by helicopter and fixed wing aircraft at 3 fluid ounces/acre(= 0.011 kg active ingredient (a.i.)/ha). Methoprene is applied when larvae are in the second, third or fourth instar stages and acts as a juvenile growth hormone mimic, preventing mosquitoes from emerging from the pupa stage as viable adults (Zoecon Corp. 1993).

Two of the most abundant invertebrates on the salt marsh are the marsh fiddler crab, Uca pugnax, and coffee bean snail, Melampus bidentatus. Few data are available on the effects of methoprene on these taxa. Barber et al. (1978) reported that continuous laboratory exposure of adult U pugilator to Altosid SR-10 at 0.02 ppm methoprene had no effect on molting (either the time to molt or the percentage molting) or mortality. Molting occurred in an average of 37 days in both the control and exposed crabs; the percentage of crabs that molted was 53% in the controls and 51 % in the exposed. According to the authors, 0.02 ppm methoprene was approximately equal to the recommended field dosage. Although no studies on reproductive effects on fiddler crabs were identified, Payen and Costlow (1977) reported that laboratory exposure of mud crabs (Rhithropanopeus harrisii) to 1.30 ppm methoprene inhibited reproduction. No studies with M bidentatus were identified. Miura and Takahashi (1974) reported no detectable population impacts on snails (Physa and Lymnaea sp.) in ponds treated with Altosid SR-10 at 0.02 lb and 0.025 lb a.i/acre (0.022 and 0.028 kg a.i./ha). The authors, however, did not census the populations and these observations should be viewed as qualitative.

The objective of the present study was to examine non-target effects of methoprene on coffee bean snails and fiddler crabs exposed under a realistic scenario. This was achieved by conducting controlled sprays on study plots established on the salt marsh.

3 .2 Materials and Methods

Study Plots: The six Bombay Hook NWR study plots used in 1994 (Chapter 2) were randomly reassigned to be either spray (1, 2, and 4) or control (3, 5, and 6) plots. Adjacent plots were separated by at least 500 m.

Spray Application: Altosid Liquid Larvicide (5% methoprene) was applied to the three spray plots by helicopter at 0.011 kg a.i./ha on three occasions: June 14, July 12, and August 9. The four-week interval between spraying corresponds to the typical pattern for operational spraying

20 (W. Meredith, Delaware Department of Natural Resources and Environmental Control, pers. comm.). Spray was applied early in the morning (before 0800 hours) when the air was still.

Snail Bioassays: Two days before the first and second sprays, coffee bean snails (10-12 mm width) were collected from the Marshall Island area of Bombay Hook NWR, an area where no spraying had been reported (W. Meredith, pers. comm.). The snails were kept in plastic trays containing collection site mud and held overnight in the laboratory. The next day snails were transported to the study plots, where 10 snails each were placed in 1-L polyethylene jars with 5 x 7.5 cm windows on two sides covered with 1-mm mesh polyethylene screen. Each jar was tied to a galvanized minnow trap such that one window was facing upwards. One tablespoon of study plot mud was placed in each jar to provide moisture for the snails. At each study plot on the day before the sprays, two traps were placed in an unvegetated area just above the high tide line. The traps were retrieved two days after spraying and the numbers of living and dead snails were counted. Survival data were analyzed using arc sin square root transformed data to meet the assumptions for parametric statistics.

Fiddler Crab Monitoring: The percentage of female fiddler crabs observed to be gravid was monitored at the control and spray plots as an indicator of possible reproductive impacts of methoprene spraying. The carrying of eggs by the female occurs after copulation and, therefore, signifies successful courtship, copulation, and fertilization. Grimes et al. (1989) and Montague ( 1980) summarized life history data for U pugnax. Males signal their interest in mating by making a series of courtship displays. Fiddler crabs are believed to copulate in their burrows, after which the females carry the developing eggs in a mass or sponge for 11-13 days (Montague 1980). Ovigerous females are found during the warmer months of the year, beginning in April in Florida and July in Massachusetts (Grimes et al. 1989). Females are believed to bear several clutches through the season.

Female fiddler crabs were collected weekly from spray and control plots. To test the hypothesis that spraying of Altosid affected the reproductive capacity of the crabs, the proportion of the females that were gravid was recorded. Size (carapace width) was also recorded to determine whether crabs were of similar size at spray and control plots. The collections began one week before the first of three sprays and ended three weeks after the last spray. Crabs were collected by hand in areas within 10 meters of tidal guts. After collection, crabs were placed in plastic bags, frozen, and transported to the laboratory to determine whether they were gravid and to be measured. Crabs were collected on the following dates: June 8, June 15 ( one day after spray 1), June 22, June 29, July 6, July 14 (two days after spray 2), July 20, July 27, August 3, August 10 (one day after spray 3), August 18, August 23, and August 31. By collecting through the month of August, the possibility of cumulative impacts of multiple exposures on later clutches could be evaluated.

Data were analyzed by covariate analysis of variance (SAS 1989) using treatment (spray or control) as the class variable. The percent gravid data were transformed using the arc sin square root transformation. The model tested for differences in slopes between the percent gravid at the

21 spray and control plots over the course of the study. It also tested for the effects of time ( collection date) on the percent gravid.

3.3 Results

Snail Bioassays: There were no significant differences in mortality in the snails exposed at the spray and control plots in both bioassays (Table 3 .1 ). The mean percent survival in the spray .1 test was 85% for the control and 87% for the spray plots (p=0.45). The mean percent survival for the spray 2 test was 90% for the control and 87% for the spray plots (p=0.66).

Fiddler Crab Monitoring: Results of the covariate ANOV A indicated that there was no significant treatment effect (p=0.38) or interaction between time and treatment (p=0.93). There was a significant decrease in the percentage of females that were gravid through time (p=0.0001). Analysis of the graph of the percentage of gravid females over the course of the study (Fig. 3 .1) indicated that considerably fewer females were gravid in August, compared with June or July. In August, the percent gravid never exceeded 20%, whereas in June as many as 57% were gravid and in July as many as 33% were gravid. Over the 13 collections, the percentage of gravid females was 16.4 ± 3.7% (mean± one standard deviation) in the control plots and 21.8 ± 6.0% at the spray plots. A Mann-Whitney U-test indicated no significant differences in the sizes of the crabs captured at the control and spray plots (p=0.93), which had identical median carapace widths of 15.2 cm.

3 .4 Discussion

The results of the snail bioassay indicate that the sprays had no effect on mortality of coffee bean snails. It is difficult to determine whether the amount of spray that reached the snails was an overestimate or underestimate of exposure to uncaged animals. Snails in the intertidal zone may receive less of an exposure than the caged snails (placed above the high tide line) because the methoprene would be diluted with the tidal cycle. However, some of the spray was probably intercepted by the mesh of the minnow traps and polyethylene.

In the Payen and Costlow (1977) study with mud crabs, seven adult males and seven adult females were exposed to 20 ppt seawater containing 1.30 ppm Altosid (90% a.i.). Equal numbers of crabs were tested as seawater and acetone controls. The authors reported that three of the control females laid eggs whereas none of the experimental animals did. Crabs sacrificed after 12-15 days of Altosid exposure had inhibition ofvitellogenesis (females) and stimulation of spermatogenesis (males). In crabs exposed for 30-45 days, there was inhibition of vitellogenesis in the females and spermatogenesis in the males. The authors stated, however, that the 1.30 ppm concentration approached the solubility limit of methoprene and greatly exceeded an environmental dose. In the present study, application of 0.011 kg a.i./ha corresponds to an initial concentration of 7 .5 ppb in a water body 15 cm deep (Ross et al. 1994); this concentration is only 0.6% of the 1.30 ppm concentration used by Payen and Costlow (1977).

22 The lack of effect from methoprene spraying on the percentage of gravid female fiddler crabs may be attributable to several possibilities. One hypothesis is that methoprene may not affect mating behavior or the development of eggs at realistic environmental concentrations and that the effect observed by Payen and Costlow (1977) on mud crabs may only occur at extremely high concentrations. A second hypothesis is that exposure to fiddler crabs in the present study may have been minimal since the compound would have been diluted and dispersed with tidal inundation of the mud flat habitats. It also degrades rapidly in water. For example, Ross et al. (1994) reported that methoprene concentrations in microcosm tanks decreased from 2.3 ppb on the day of treatment to 0.51 ppb on day 4 and to 0.23 ppb on day 14.

There appeared to be three peaks in the percentage of gravid females through the course of the monitoring: the first on June 8, then on June 22-29, and the third on July 27. Based on the tide charts, the highest tides (associated with new and full moons) would have occurred on June 13, June 27-28, July 11-12, and July 26-28. Thus, three of the four periods with high tides seem to be associated with peaks in the percent of females that were gravid. Since females are gravid for 11-13 days (Montague 1980), it is to be expected that weekly monitoring will not coincide precisely with peaks in the percent that are gravid. It is apparent, however, that the percent gravid is cyclical and is most likely related to the release of larvae at the periods of greatest tides (Wheeler 1978).

The study results suggest that application of methoprene does not affect the measured endpoints for these two taxa. To rule out alternative explanations, a study which includes repeated analyses of methoprene concentrations in sediments, water, and biota would be required.

3. 5 References

Barber, J.T., E.G. Ellgaard, and R.J. Castagno. 1978. Crustacean molting in the presence of Altosid SR-10. Mosquito News 38:417-418.

Grimes, B.H., M.T. Huish, J.H. Kerby, and D. Moran. 1989. Species profiles: Life histories and environmental requirements of coastal fishes and invertebrates (Mid-Atlantic). Atlantic marsh fiddler. U.S. Fish and Wildlife Serv. Biol. Rep.82(11.114). Washington, DC.

Miura, T. and R.M. Takahashi. 1974. Insect developmental inhibitors. Effects of candidate mosquito control agents on nontarget aquatic organisms. Environ. Entomol. 3 :631-636.

Montague, C.L. 1980. A natural history of temperate western Atlantic fiddler crabs (genus Uca) with reference to their impact on the salt marsh. Contrib. Mar. Sci. 23:25-55.

Payen, G.G. and J.D. Costlow. 1977. Effects of a juvenile hormone mimic on male and female gametogenesis of the mud crab, Rhithropanopeus harrisii (Gould) (Brachyura: Xanthidae). Biol. Bull. 152: 199-208.

23 Ross, D.H., D. Judy, B. Jacobson, and R. Howell. 1994. Methoprene concentrations in freshwater microcosms treated with sustained-release Altosid formulations. J Am. Mosq. Control Assoc. 10:202-210.

SAS Institute. Inc, 1989. SAS/STAT User's Guide Version 6. 4th edition. SAS Institute Inc., Cary, NC. 846 p.

Wheeler, D.E. 1978. Semilunar hatching periodicity in the mud fiddler crab Uca pugnax (Smith). Estuaries 1 :268-269.

Zoecon Corp. 1993. Altosid product literature. Zoecon Corp., Dallas, Texas.

24 T a bl e 3 ..1 R esu 1ts o f m. situ. b.10assavs wit"h co ffiee bean snai·1 s (M b"d1 entatus ) . % Survivala

Control plots Spray plots Statistics

Spray 1 85 (82\ 90, 85) 87 (96c, 90, 75) NSd; p=0.45

Spray 2 90 (70\ 100, 100) 87 (80, 80, 100) NS\ o=0.66 a Mean survival followed by survival per plot (based on two cages per plot with 10 snails per cage) b Based on 11/13 surviving in one jar and 8/10 in the other c Based on 11/12 surviving in one jar and 10/10 in the other d t-Test on arc sin square root transformed data e Based on one jar; the mud in the other jar had dried and all snails were dead r Mann Whitney U test because parametric assumptions were violated

25 Figure 3 .1 Percentages of gravid females in Altosid-sprayed and control plots.

Percent Gravid Female Fiddler Crabs in Control and Spray Plots

70 -,------,

60

-a-Control 50

"'C ---spray ·-> a..ca 40 C).... s::: Cl) CJ 30 a.. Q) CL 20

10

0 "lit' It) N 0, (0 N "lit' 0 0 CIC) C") ... N N t? ! ...... ~ ... """ CIC) I ...... t? U) (0 U) (0 -""" ~ ~ r::: CIC) co ~ CIC) rn - -rn""" rn - Date • 4.0 EFFECTS OF TEMEPHOS ON ACETYLCHOLINESTERASE ACTIVITY IN MEADOW VOLES (Microtus pennsylvanicus) AT BOMBAY HOOK NWR

4.1 Introduction

Temephos, an organophosphate insecticide, acts on target organisms through inhibition of a class of enzymes known as cholinesterases which play a critical role in the transmission of signals in the nervous system. Measurement of cholinesterase activity in nontarget organisms has been widely used as a biomarker for pesticide exposure and response (Mayer et al. 1992). In mammals, the primary cholinesterases are acetylcholinesterase (AChE) and butyrlcholinesterase (BChE). In birds and mammals, diagnosis of pesticide-induced mortality involves a depression in brain cholinesterase equal to or greater than 50% (Rattner and Fairbrother 1991 ). Hill and Fleming (1982) suggested that a 20% depression in brain cholinesterase in fish and birds can be used as an indicator of exposure to cholinesterase-inhibiting chemicals.

The objective of the present study was to determine if exposure to Abate 4E (44.7% temephos) altered cholinesterase activity in meadow voles, Microtus pennsylvanicus.

4.2 Materials and Methods

Sampling: The meadow vole cholinesterase work was conducted as part of the monitoring program described in Chapter 2. In the summer of 1994, meadow voles were captured from spray plots 1 and 4 and spray plots 2 and 5 using museum special traps. Animals were collected on day+ 1 and day +2 following the second and third spraying. Traps were collected the morning after being set and animals were kept on ice while being transported to the laboratory. The brains were excised and stored at -20°C until analysis.

Cholinesterase Assay Procedures: Meadow vole assays for AChE were conducted according to the methods of Hill and Fleming (1982), as stated in Patuxent Wildlife Research Center Technical Operating Procedure: Brain Cholinesterase as a Diagnostic Tool. AChE, data from the two sprayed or the two control plots were pooled if there were· no significant differences determined by t-tests or Mann-Whitney tests. Then, differences between control and sprayed plots were analyzed by t-tests or Mann-Whitney tests. Separate analyses were performed for animals collected on day+ 1 and day +2.

4.3 Results

Cholinesterase: In meadow voles collected after the second spray, AChE activity was not significantly different among the control and spray plots (Table 4.1). Mean activities were 7.45 µmoles per minute per g wet wt. tissue for the animals collected from control plot 1 and 7 .20 µmoles per minute per g tissue in the pooled animals collected from spray plots 2 and 5. For the second day after spray, the mean activity in voles from control plots 1 and 4 was 8.25 µmoles per minute per g tissue and the mean activity in voles from spray plots 2 and 5 was 7 .91 µmoles per

27 minute per g tissue. For the third spray, there were no significant differences in voles collected from the control and sprayed plots on the day after the spray. Only a single animal was collected from the control plot. Its AChE activity (7.83 µmoles per minute per g tissue) was not significantly different than the pooled median activity in the 32 animals collected from spray plots 2 and 5 (6.83 µmoles per minute per g tissue). For spray 3, day 2, the da:ta from the two spray plots could not be pooled because they were significantly different. The Kruskal-Wallis test was used to test for differences between the voles collected from control plot 4, spray plot 2 and spray plot 5. There were significant differences between the two spray plots (median activities: 4.98 and 7.27 µmoles per minute per g tissue) but not between the control plot (5.03 µmoles per minute per g tissue) and either spray plot (Table 4.1).

4.4 Discussion

The median AChE activity in meadow voles was in the 5 to 8 µmoles per minute per g tissue range. These levels of activity are similar to those reported in the literature for unexposed animals. Jett (1986) reported mean brain AChE activity of 6.05-6.56 µmoles per minute per g tissue. Mean activity of 5.6 µmoles per minute per g tissue was reported by Rattner and Hoffman (1986).

4. 5 References

Hill, E.F. and W.J. Fleming. 1982. Acetylcholinesterase poisoning of birds: field monitoring and diagnosis of acute poisoning. Environ. Toxicol. Chem. 1:27-38.

Jett, D.A. 1986. Cholinesterase inhibition in meadow voles (Microtus pennsylvanicus) following field applications of Orthene. Environ. Toxicol. Chem. 5:255-259.

Mayer, F.L., D.J. Versteeg, M.J. McKee, L.C. Folmar, R.L. Graney, D.C. McCume, and B.A. Rattner. 1992. Physiological and nonspecific biomarkers. In: Biomarkers: Biochemical, Physiological and Histological Markers ofAnthropogenic Stress, R.J. Huggett, R.A. Kimerle, P.M. Mehrle, Jr., and H.L. Bergman, eds. Lewis Publishers, Boca Raton, FL., pp. 5-85.

Rattner, B.A. and D.J. Hoffman. 1984. Comparative toxicity of acephate in laboratory mice, white-footed mice, and meadow voles. Arch. Environ. Contam. Toxicol. 13:483-491.

Rattner, B.A. and A. Fairbrother. 1991. Biological variability and the influence of stress on holinesterase activity. In: Cholinesterase-inhibiting Insecticides. Their Impact on Wildlife and the Environment. P. Mineau, ed., Elsevier Science Publishers, Amsterdam, pp. 89-107.

28 Tabl e 4 1. Mead owvo1 e AChE act1v1t1es a ft er sprays 2 and3 a

Spray Day Control Spray Statistics b

2 +1 Median: 7.45 Median: 7.20 NS; U test, p=0.66 Min:5.96, Max: Min: 4.87, Max: 10.98 (n=3) 10.25 (n=25)

+2 Mean: 8.25 Mean: 7.91 NS; t-test, p=0.56 SD: 1.79 (n=14) SD:1.32 (n=l5)

3 +1 7.83 (n=l) Median: 6.83 NS; U test, p=0.32 Min:2.32, Max: 9.61 (n=32)

+2 Median: 5.03 Plot 2 Median: 4.98 S; Kruskal-Wallis; p<0.001; Min: 4.54, Max: Min: 4.65, Max: Dunn's method: Plot 5 > Plot 2; 9.79 (n=5) 5.77 (n=9); Plot 5 no other comparisons significant Median: 7 .27 Min: 4.70, Max: 13.88 (n=21)

.a Units: µmoles per minute per g tissue b t-Test if parametric assumptions were met, otherwise Mann-Whitney or Kruskal-Wallis tests; NS: Not significant; S: significant

e 29 5.0 INSECT EMERGENCE MONITORING AT PRIME HOOK NWR

5 .1 Introduction

Prime Hook National Wildlife Refuge (NWR), a satellite refuge of Bombay Hook NWR, is located near Milford, Delaware, bordering the shores of Delaware Bay (Figure 1.1). Approximately 3,612 hectares (ha) in size, this refuge provides ideal breeding habitats for dipteran pests such as mosquitoes. Mosquitoes are an important food source, especially the larvae, for a variety of aquatic and terrestrial organisms. However, as with most coastal areas, human development has encroached into the surroundings areas around Prime Hook NWR. As can be expected, human tolerance of mosquitoes is severely limited, primarily due to the irritating bites caused by the females of the species. In an effort to reduce the mosquito populations and their contact with humans, the Delaware Department of Natural Resources and Environmental Control (DNREC) implemented a mosquito control program that includes spraying with larvicides such as temephos (Abate), methoprene (Altosid), and Bacillus thuringiensis israelensis israelensis (Bti).

Several ponded areas on the refuge are used by a variety of waterfowl and other water birds that feed heavily on benthic organisms such as chironomids. However, it has been observed, by refuge staff, that bird use in some of the ponds is significantly reduced after spray applications (G. O'Shea, pers. comm.). Because of these observations, insect emergence monitoring was conducted on two ponds during the spring and summer of 1995. The objective of the monitoring was to establish quantitative data on insect taxa on these ponds and provide refuge managers information for decision making on mosquito management.

5 .2 Materials and Methods

Site Description: Pond PMH3d is a freshwater, stream fed (Prime Hook Creek) natural pond with an area of approximately 0.81 ha. Water depths during June and July 1995 ranged from 0.25 to 0.46 m; PMH3d, was dry during the month of August. Sediments within PMH3d are composed of unconsolidated silts and clays, forming an organic rich substrate. Dominant vegetation within the pond consists of cattail (Typha sp.), wild millet (Echinochloa walteri), and duckweed (Lemna sp.). Fall and winter are seasons in which waterfowl utilization of the pond is the greatest. Refuge biologists estimate that bird use within the pond is moderate to heavy during these two seasons. PMH3d was last sprayed with Abate in 1993 by DNREC.

Pond PMH4a is an oligohaline, tidally influenced (Deep Hole Creek), natural pond that is approximately 40.5 ha in size. Freshwater inputs into the pond are derived from an unnamed tributary, and from ground water. Water depths during June and July 1995 ranged from 0.05 - 0.15 m; PMH4a was dry during the month of August. Sediments within PMH4a are composed of consolidated clays. Dominant vegetation within the pond consists of sprangletop (Leptochloa fasicularis), wild millet (Echinochloa walteri), and sea purslane (Sesuvium maritimum). The fall .and winter are seasons in which waterfowl utilization of the pond is the greatest. During spring 30 - and summer, shorebirds and waterbirds utilize the pond. Refuge biologists estimate that bird use within the pond is moderate to heavy during the fall and winter seasons. PMH4a was last sprayed with Abate in 1994 by DNREC. During the 1995 monitoring period the pond was treated with Bti.

Monitoring: One floating emergent insect trap (English 1987) was placed in each of the two ponds. The emergent insect traps sampled a pond surface area of approximately 0.3 meters. Weekly monitoring of the traps was conducted by Prime Hook NWR personnel. The monitoring period began in mid-June and ended in late August, 1995, and consisted of retrieving the traps from the ponds and removing the entrapped insects from the collecting bottles. The contents of the collection bottles were transferred to specimen vials containing 70% ethanol. The collecting bottles were refilled with 200 mL of 95% ethanol, reattached to the traps, and re-deployed to their former position on the pond. Taxonomic identifications were performed by Dr. Leonard Ferrington of the University of Kansas.

Water quality monitoring was also conducted on the same day as trap monitoring. Water temperature and salinity were measured with a YSI model S-C-T 33 meter (YSI Inc., Yellow Springs, OH). Dissolved oxygen was measured with a YSI model 58 D.O meter, and pH with an Orion Model 201 pH meter (Orion Research, Cambridge, MA).

Data Analysis: Species abundance was calculated for each of the ponds and dominant taxa were determined. From the species abundance data, several ecological indices were also calculated and used to compare the sites statistically. Ecological indices used to describe the two pond communities were species diversity (an expression of community structure), equitability (an expression of how evenly distributed species are in a given community), and community similarity (a measure of how similar two communities are with respect to the number of species found in both communities; Brower et al. 1990).

Species diversity was calculated using Shannon-Wiener Index H', where H' = pi logpi (Zar 1984, Brower et al. 1990). Equitability was calculated using H' /H' max where H' = Shannon­ Wiener Index and H' max = log s, where s = number of species present. Community similarity was calculated using Percent Similarity PS, where PS= minimum Pa or qa, where Pa= proportion of species a from community 1 (PMH3d), and qa = the same species a from community 2 (PMH4a) (Baker and Wolfe 1987; Brower et al. 1990).

Statistical analysis of insect data from each pond was conducted using the Statistica package (StatSoft, Inc., Tulsa, OK). Analysis consisted of conducting a t-test for independent samples on the following variables: Shannon-Wiener Index H', and equitability J'. Before each t-test was conducted, data from each pond were tested for normality using the Shapiro-Wilk test (Zar 1984). If the data were not normally distributed then the data were normalized using log transformation. After ensuring that the data were normally distributed, the Levene's test for homogeneity of variances was conducted. The Levene's test was run because the standard t-test for independent samples assumes that variances in the two groups being compared are the same

31 (homogenous). If the Levene test was significant (p = <0.05), non-parametric statistics were used. If the result approached significance (p between 0.05 and 0.3), then at-test with separate variance estimates was used. All statistical analyses were conducted at a 95 percent confidence level (p = 0.05).

5.3 Results

Emergent Insects: Four insect orders were represented in each of the ponds, with Diptera being the dominant order (Table 5 .1 ). Pond PMH3d had 15 taxa represented, 11 of which were dipterans. Pond PMH4a had 11 taxa, 8 of which were dipterans. A total of 112 and 206 individuals were collected from PMH3d and PMH4a, respectively. Chironomids (midges) were the dominant group of dipterans, representing 95% of the total species collected from each pond. Two unidentified species of chironomids, Chironomus sp. 2 and 3, were the most dominant taxa found in each of the ponds (Table 5.1). Only one species of mosquito (Aedes sp.) was collected (PMH3d), and represented < 1 percent of the total individuals collected.

Mean Shannon-Wiener diversity indices were 1.14 for PMH3d and 0.82 for PMH4a. There was a borderline significant difference (t-test, p=0.058) between the two ponds in terms of species diversity. There was a significant difference in equitability between the two ponds (t-test with separate variance estimates, p=0.04). Mean equitability scores for PMH3d were 0.73, and 0.48 for PMH4a. Percent similarity calculations for the entire sampling period resulted in a score of 0.42 (Table 5.2).

Water Quality: For most of the monitoring period, dissolved oxygen concentrations were high enough to support a healthy aquatic community. However, in the initial monitoring period, there were periods when dissolved oxygen concentrations fell below levels needed to support a healthy aquatic community (Table 5.3). Dissolved oxygen levels among ponds were comparable during each sampling period. Salinity was higher in PMH4a, ranging from 1.0 - 3.0 ppt, typical of oligohaline environments. PMH3d was considerably less saline, ranging from 0.0 - 1.0 ppt, characteristic of freshwater sytems. Temperature and pH were similar in the two ponds (Table 5.3).

5.4. Discussion

There were significant differences in the insect communities within the two ponds, with PMH3d having consistently higher ecological community indices scores than PMH4a. Several physical, chemical, and biological factors may be responsible for the differences found between the two ponds. Salinity, water depth, spray history, organic matter, and predation may all contribute to the decreased numbers of species found in PMH4a.

Freshwater habitats, such as those found in PMH3d, are usually more diverse than their brackish water counterparts, such as those found in PMH4a (Gasner 1971, Wiley 1976). The presence and dominance of dipteran species found in both ponds, especially PMH4a, is not suprising. 32 - Similar studies found that dipterans are the dominant insect taxa found in oligohaline environments (LaSalle and Bishop 1987). The consistently higher salinities found in the oligohaline environment of PMH4a (Table 5.3) could be a limiting factor responsible for the reduced numbers of species. The added stress and expenditure of energy needed to facilitate osmoregulation is a major factor in the reduced number of species found in brackish waters (LaSalle and Bishop 1987, Wiley 1976).

The shallower waters of PMH4a may also be affecting the insect assemblages found in this pond. The decreased water depths in PMH4a may make the insect community more vunerable to predation by wading waterbirds. This particular pond is subject to use by shorebirds and waterbirds, especially during the spring and summer months, the period in which this study was conducted. The deeper waters of PMH3d may afford greater protection from these predatory birds.

The spray history of the ponds and the effects of chemical and physical parameters on spray bioavailability may contribute to the differences in the communities. Pond PMH4a was sprayed during the 1995 monitoring period with Bti, whereas pond PMH3d was not sprayed. Pond PMH4a was also sprayed in 1994 with Abate. Pond PMH3d was last sprayed in 1993 with Abate. Chironomids are known to be especially sensitive to Bti applications, even more so than mosquito larvae (Hershey et al. 1995). The shallower water depth of pond PMH4a may increase the toxicity of Bti to chironomids, since this effect was observed in a pond study conducted in Minnesota (Charbonneau et al. 1994).

The lack of organic matter in pond PMH4a sediments may also affect the insect populations adversely in two manners. First, sediments enriched with organic matter tend to adsorb Bt, decreasing its availability and toxicity to benthic organisms. Thus, the sediments in pond PMH4a would be unlikely to adsorb much of the Bt. Second, most benthic organisms, including insects such as chironomids, are primarily detritivores that feed on organic particles found on and within the sediments (Pennak 1978, Gaston et al. 1988). Pond PMH4a sediments are composed of consolidated clay particles, with little associated organic matter, limiting the food supply to only a few species adapted to these type of enviromental conditions, and therefore reducing species diversity. The rich organic composition of sediments found in PMH3d and its higher species diversity scores support this possibility.

In summary, differences between the two ponds appear to exist. However, at present we cannot determine the relative importance each of the five factors discussed may have in regulating the insect communities. Salinity is a known factor determing the distribution of certain species, however, the remaining four factors: water depth, spray history, sediment organic matter, and predation are variables that need to be investigated further before drawing conclusions about the differences between the two ponds. The observation of only a single mosquito during the summer monitoring period suggests that the decision to spray these ponds should be based on dip survey data.

33 5.5 References

Baker, J.M., and W.J. Wolff. 1987. Biological Surveys ofEstuaries and Coasts. Cambridge University Press, London.

Brower, J.E., J.H. Zar., and C.N. von Ende. 1990. Field and Laboratory Methods for General Ecology. Third Edition. William C. Brown, Dubuque, Iowa.

Charbonneau, C.S., R.D. Drobney, and C.F. Rabeni. 1994. Effects of Bacillus thuringiensis var. israelensis on nontarget benthic organisms in a lentic habitat and factors affecting the efficacy of the larvicide. Environ. Toxicol. Chem. 13:267-279.

English, W.R. 1987. Three inexpensive aquatic invertebrate samplers for the benthos, drift, and emergent fauna. Entomological News 98: 171-179.

Gaston, G.R., D.L. Lee, and J.C. Nasci. 1988. Estuarine macrobenthos in Calcasieu Lake, Louisiana. Estuaries 11: 192-200.

Gesner, K.L. 1971. Guide to Identification ofMarine and Estuarine Invertebrates. John Wiley and Sons, New York.

Hershey, A., L. Shannon, R. Axler, C. Ernst, and P. Mickelson. 1995. Effects of methoprene and Bti (Bacillus thuringiensis var. israelensis) on non-target insects. Hydrobiologia 308:219-227.

LaSalle. M. W., and T.D. Bishop. 1987. Seasonal abundance of aquatic diptera in two oligohaline tidal marshes. Estuaries 10:303-314.

Pennak, R.W. 1978. Freshwater Invertebrates ofNorth America. Second Edition. John Wiley and Sons, New York.

Wiley, M. 1976. Estuarine Processes: Uses, Stresses, and Adaptations to the Estuary. Academic Press, New York.

Zar, J.H. 1984. Biostatistical Analysis. Second Edition. Prentice-Hall, Englewood Cliffs, New Jersey.

34 Table 5.1. Insect* taxa collected from emergent traps placed on ponds' PMH3d and PMH4a at Prime Hook NWR.

Abundance Rel. Abundance TAXA PMH3d PMH4a PMH3d PMH4a Diptera

Chironomus sp~ 2 2 12 0.02 0.06

Chironomus sp. 3 60 175 0.54 0.84

Polypedilum sp. 1 0 0.01 0.00

Glyptotendiptes sp. 29 4 0.26 0.02

Parachironomus sp. 4 0 0.04 0.00

Chironomini sp. 2 2 0.02 0.01

Zavreliella sp. 1 0 0.01 0.00

Tanytarsini sp. 3 0 0.03 0.00

Cricotopus sp. 0 1 0.00 0.004

Tanypus neopunctatus 0 2 0.00 0.01

Dolichopodidae I 0 0.02 0.00

Ccratopogonidae 0 2 0.00 0.01

Aedes sp. 1 0 0.01 0.00

Ephydridae 3 1

Odonata

Libelludidae 2 0- 0.02 0.00 - Coenagrionidae 1 0 0.01 0.00

Coleoptera

Hydrophiladae 1 0 0.01 0.00

Berosus sp. 0 2 0.00 O.oI

Troposternus lateral/is 0 1 0.00 0.004

Hemiptera

Salididae 1 0 0.01 0.00

Corixidae 0 4 0.00 0.02 I Total I 112 206 I 1.00 1.00 I * Data consists only of aquatic or semi-aquatic insects listed on data sheets

35 Table 5.2. Ecological community indices (Shannon-Wiener H', Equitability J', Percent Similarity, PS) determined for insect taxa collected from emergence traps at ponds PMH3d and PMH4 at Prime Hook NWR. - Ecological Community Indices

Shannon's Equitability %Similarity

Date Site H' J' psa 06-19-95 PMH3d 2.46 0.82 0.36 06-26-95 PMH3d 1.72 0.75 0.58 07-03-95 PMH3d 0.34 N/Ab 0.00

07-10-95 PMH3d 1.52 0.66 0.60

07-17-95 PMH3d 1.27 0.63 0.72

07-31-95 PMH3d 2.85 N/Ab 0.68

08-08-95 PMH3d 1.30 0.82 0.00

08-28-95 PMH3d 1.06 0.67 N/AC meanct 1.56 0.74 0.42 el

06-19-95 PMH4a 1.46 0.73

06-26-95 PMH4a 0.53 0.50 07-03-95 PMH4a 0.362 .. -0.36

07-10-95 PMH4a 0.615 0.31 07-17-95 PMH4a 0.361 0.23 07-31-95 PMH4a 0.497 0.31 08-08-95 PMH4a 1.84 0.92

mean 0.81 0.48 a PS values are based on a comparison of data from each pond for each sampling date b Insufficient data collected to calculate J'; only 1 species collected c PS value unavailable because only PMH3d had data collected for this date ct Mean values do not include data for 08-28-95 when PMH4a was dry e 36 Table 5.3. Water quality data collected from ponds PMH3d and PMH4a at Prime Hook NWR on t h e d ays t h at emergence t raps were mom·t ore d . D.O (mglL) Salinity (ppt) Temp. (°C) pH

PMH3d PMH4a PMH3d PMH4a PMH3d PMH4d PMH3d PMH4a 06-12-95 0.4 0.4 NIA 2.0 NIA 26.0 NIA NIA

06-19-95 0.7 1.0 0.0 2.0 27.0 25.0 8.0 8.4

06-26-95 11.7 10.5 0.0 2.0 31.0 30.0 7.9 8.1

07-03-95 14.1 10.8 0.5 1.0 30.0 29.5 8.5 9.1

07-10-95 2.5 5.0 0.5 2.0 28.0 22.0 ?:6 7.1

07-17-95 7.3 6.4 1.0 3.0 28.5 28.0 6.5 7.4

07-24-95 6.5 10.7 0.0 2.0 35.0 36.0 6.9 7.7

07-31-95 7.6 3.3 0.5 2.5 26.0 27.0 7.5 8.3 .NIA data not available for these parameters or for 8-8-95 and 8-28-95 e

37 6.0 EFFECTS OF THE MOSQUITO LARVICIDES, TEMEPHOS AND METHOPRENE, ON INSECT POPULATIONS IN EXPERIMENTAL PONDS

6.1 Introduction

The U.S. Fish and Wildlife Service (FWS) is investigating the hazards of mosquito control pesticides that are used on National Wildlife Refuges (NWRs). At issue is the compatibility between mosquito spraying and the production of waterfowl, which is often one of the key management objectives of the refuges. FWS is concerned that non-target effects of these pesticides may reduce the invertebrate populations that are food for waterfowl. Previous efforts have focused on the non-target impacts of Bacillus thuringiensis israelensis (Bti) (Charbonneau et al. 1994).

Temephos and methoprene are the mosquito control larvicides comonly used at Bombay Hook NWR, which is 13 km from Dover, DE. Temephos is most frequently applied as Abate® 4E (hereafter referred to as Abate; American Cyanamid, Princeton, NJ), an emulsified formulation containing 44.6% temephos and 55.4% inert ingredients listed as petroleum distillates. Methoprene is applied as Altosid® Liquid Larvicide (5% methoprene, 95% inerts; hereafter referred to as Altosid; Zoecon Corp., Dallas, TX). These chemicals have been sprayed by helicopter and fixed wing aircraft to control mosquito breeding in isolated potholes on the salt marsh (temephos and methoprene) and over impoundments that are fresh or slightly saline (methoprene).

Non-target insects may be affected by the larvicides in both habitats. In the salt marsh, the physical and chemical conditions in the isolated potholes are extremely harsh and a depauperate insect community exists (Campbell and Denno 1978). Because a more diverse community exists in the impoundments and ponds (Chapter 5), this study was focused on non-target impacts on these habitats. The objective was to determine the effects oftemephos and methoprene on non­ target invertebrates in freshwater ponds. To reduce the natural variability of ponds with respect to factors such as water quality, size, depth, and organic content of the sediments, experimental ponds constructed for scientific studies were used.

6.2 Materials and Methods

Spray application

Test sprays with the mosquito larvicides, Abate 4E and Altosid Liquid Larvicide (supplied by the Delaware Department of Natural Resources, Division of Fish and Wildlife, Mosquito Control Section) were conducted in May-July 1995 in experimental ponds at the Patuxent Wildlife Research Center, Laurel, Maryland, USA. A total of 18 ponds (average volume: 105 m3; average area: 202 m2; maximum depth: 0.7 m) were tested (Fig. 6.1). The ponds contain considerable stands of submerged aquatic vegetation, primarily cattails (Typha latifolia). Spraying was conducted by pumping water from a nearby natural pond through a garden hose

38 attached to a one quart venturi sprayer containing either stock solutions of the pesticides or distilled water for the controls. Six ponds each were randomly assigned for Abate 4E, Altosid, or distilled water spray. Stock solutions for each pond were calculated based on pond area.

Spraying occurred on May 23, June 14, and July 6. The sprays were at the rates prescribed on the pesticide application label and used by the Mosquito Control Section: Abate -- 1.5 ounces active ingredient/acre (0.054 kg a.i./ha); Altosid -- 3 ounces a.i./acre (0.011 kg a.i./ha). The three week spray interval corresponds to an environmentally realistic schedule for application of methoprene to freshwater wetlands (Metropolitan Mosquito Control District 1993). Certified pesticide applicators sprayed at approximately 0700 hour when the air was usually still. The applicator sprayed while walking slowly around each pond to achieve uniform coverage.

Chemical and Water Quality Monitoring

Temperature, dissolved oxygen, conductivity, and pH were monitored weekly beginning on May 10 and continuing through July 19, with a Hydrolab Surveyor 2 instrument (Hydrolab Corp., Austin, TX). Alkalinity and hardness were measured weekly on the same days according to American Public Health Association (1992) methods. Samples collected on the spray dates were analyzed for dissolved organic carbon (Menzel and Vaccaro 1964), and total dissolved phosphorus and nitrogen (D'Elia et al. 1977; Valderrama 1981) at the University of Maryland, Chesapeake Biological Laboratory (Solomons, MD).

Samples were collected for analysis of temephos in the six Abate-sprayed ponds within 30 minutes post spray. Each sample was composed of four 200-mL aliquots of water collected from 10-20 cm depth at different locations in the ponds. Samples were collected with a beaker attached to a 5 m pole. The water was poured into a previously labeled 1-L amber colored glass bottle containing 125 mL of methylene chloride. After all four aliquots were collected, the bottle was closed and shaken vigorously for approximately one minute to disperse the methylene chloride through the water. This constituted the first of three extractions with methylene chloride used in the sample preparation. Following shaking, the bottles were kept on ice. Approximately 1.6 L of water was collected from one pond and splif info two ·800-mL aliquots to provide material for spiking. Abate 4E (0.224 µL) was added to one aliquot before shaking to give a 100 percent spike recovery level of 0.125 mg/L of active ingredient. A second 800-mL composite sample was collected from a pond for duplicate analysis. The ponds from which spike and duplicate samples were taken were randomly selected before each spray application. Deionized water for one procedural blank was prepared and treated identically to a routine water sample.

The samples were analyzed for temephos by the U.S. Fish and Wildlife Service, Patuxent Analytical Control Facility (PACF; Laurel, MD), based on Belisle and Swineford (1988). The water samples were extracted twice more with methylene chloride, extracts were pooled, cleaned by florisil column chromatography, and analyzed by gas chromatography on a DB-1 capillary column with a flame photometric detector. The detection limit was 0.9 µgL. No chemical analysis was conducted for methoprene because the methodology was not available at P ACF.

39 Invertebrate Monitoring

Two insect emergence traps (English 1987; Fig. 6.1) were placed in each pond and allowed to collect insects for seven days. The traps were harvested on the day before each of the three sprays and again, after a seven day collection period, on day 13-14 post spray. The timing of the post-spray sampling period was based on the time course of the mechanism of action for methoprene, a juvenile growth hormone mimic which acts on insect larvae and prevents them from emerging from the pupa stage as viable adults (Zoecon Corp. 1993). Chironomids in the pupa stage, which would not be affected by methoprene, would emerge during the first six days post-spray (L. Ferrington, pers. comm.). Thus, any change in emergence occurring during days 6-13 or 7-14 would be from impacts on larvae (not pupae). Insects were collected in one quart mason jars containing 95% ethanol. Two Hester-Dendy artificial substrates were assembled and placed in each pond on May 19 and harvested on July 19, according to American Society for Testing and Materials (1992) methods. During the course of the study, Hester-Dendy substrates for one Altosid pond and one Abate pond accidentally became buried in the mud, resulting in loss of these data. The harvested invertebrates from both types of samplers were transferred to specimen vials containing 70% ethanol and identified to the lowest practical taxon.

From the species abundance data, several ecological indices were calculated to compare the sites statistically. Ecological indices used were species diversity (using Shannon's H', an expression of community structure which takes into account both the number of species and distribution of individuals among the species) and equitability (using J', an expression of how evenly distributed species are in a given community) (Brower et al. 1990). Emergence data were analyzed by repeated measures analysis of variance on square root transformed data using General Linear Models and the Tukey' s Studentized Range multiple comparison test (SAS Institute 1989). If parametric assumptions were not met, the data set was analyzed using ANOVA on ranks. Hester-Dendy data were analyzed by one way ANOVA followed by Tukey's test. If the assumptions of parametric statistics were not met, the Kruskal-Wallis test was used with Dunn's method for multiple comparisons.

Emergence data were also evaluated with Bray-Curtis cluster analysis (Clifford and Stephenson 1975), using an unweighted pair-group linkage scheme on the number of individuals per taxa per pond. Collembola were excluded because high numbers of a particular taxon can bias the analysis. Clustering was performed on the first (May 22) and last (July 19) sampling dates.

6.3 Results

Water Quality

Ranges of values for the various water quality parameters were generally similar for the control, Altosid, and Abate® ponds (Table 6.1; Appendix A). A critical parameter for the survival of invertebrates is the level of dissolved oxygen in the ponds, which range from 0.4 mg/L to 12.2

40 mg/Lover the course of the study. A two-way ANOVA, indicated no significant differences in dissolved oxygen among the three treatments (p=0.65). There were significant differences in dissolved oxygen among the different weeks of sampling (p<0.001 ); these largely reflect declines that began in mid-June and continued through the end of the study. Median dissolved oxygen levels in the control ponds ranged from 0.73 to 4.5 mg/L during this period; in the Altosid and Abate ponds the ranges were 1.7 to 4.4 mg/Land 2.2 to 5.7 mg/L, respectively.

Temephos Concentrations

The nominal temephos concentrations, based on the application rate and the average depths of the ponds ranged from 20 µg/L to 24 µg/L. Mean (± SD) measured temephos concentrations in the six ponds treated with Abate 4E were 30 ± 16 µg/L for spray 1, 32 ± 7 µg/L for spray 2, and 27 ± 16 µg/L for spray 3. The second spray had the most consistent concentrations, with a minimum of 25 µg/L and a maximum of 40 µg/L, whereas the first and third sprays had somewhat wider concentration ranges (spray 1: 10-54 µg/L; spray 3: 7.3-43 µg/L).

Emergent Insect Data

The mean number of individuals in ponds sprayed with Altosid, Abate, or distilled water all showed the same pattern. There was an initial decrease from the number of insects recorded on May 22 (spray 1, day -1 = SID-I) to the number recorded on June 6 (SID14). Thereafter the number increased steadily (Fig. 6.2). Based on the repeated measures ANOV A, the number of individuals was significantly lower in the Abate ponds relative to the control on SID14, but was not significantly lower on succeeding days: June 13 (S2D-1), June 28 (S2D14), July 5 (S3D-1), or July 19 (S3D13). A complete listing of taxonomic data is given in Apprendix B.

One order, Collembola (springtails) became dominant in all of the ponds over the course of the monitoring. The mean number of Collembola per pond, which ranged from 0.5 to 3 .8 on S 1D-1, increased to 262.3-362.7 on S3D13. (There were no significant differences in the number of Collembola among the three treatments.) Because of the dominance of this order, the data were analyzed in two ways, including and excluding Collembola. The mean number of individuals excluding Collembola was significantly lower in the Abate ponds relative to control ponds on the final monitoring period of the study (Fig. 6.3). When Collembola was excluded from the analysis, there was an initial decrease in the number of insects in all ponds, then a steady level with a slight increase at the end of the study in the control and Altosid ponds. In the Abate ponds, there was a steady level until S2D 14 when a slow decrease began and continued through the end of the study. There were statistically significant decreases in the mean number of species and families in the Abate ponds relative to the control (Figs. 6.4 and 6.5). The significant decreases began on S 1D 14 and continued through the remainder of the monitoring period. Except for a significant decrease in the number of species on S 1D 14, there were no significant differences between the Altosid and control ponds in mean numbers of species or families.

41 Species diversity excluding Collembola was significantly reduced in the Abate ponds relative to the control ponds (Table 6.2) beginning on SID14 and continuing through the duration of the study. Species diversity including Collembola was significantly reduced on SlD14 and once again on S3D13. Equitability (J') including Collembola was significantly reduced in the Abate ponds only on the final day of monitoring (S3D13); equitability excluding Collembola was significantly reduced in the Abate ponds on days S2D-1 and S2D 14 relative to the control ponds (Table 6.2). Although there were significant reductions in the Altosid ponds for these indices in the early part of the study (S 1D 14 and S2D-1 ), there were not significant reductions from the control ponds on later dates.

The results were analyzed to determine differences in the emergence of various taxa. In all cases, there were no statistically significant differences between the Altosid and control ponds. With the exception of Collembola, dipterans were the dominant order. The total number of dipterans was significantly reduced in the Abate-treated ponds only on the final day of monitoring (Table 6.2). The family, Chironomidae, was similar to the order Diptera in the pattern in emergence. A significant reduction in Chironomidae occurred in the Abate ponds only on the last day of monitoring. The genus Chaoborus (Family: Culicidae), however, was affected in a different manner. There was a significant decrease in the numbers of this genus on SID14, S2D-1, S2D14, and S3D13. Later in the monitoring period, there were few Chaoborus emerging from any of the ponds (Table 6.2). The total number of Chaoborus emerging from the Abate ponds after the first spray was 26 compared with 183 from the control ponds and 150 from the Altosid ponds (Figure 6.6).

Certain non-Dipteran taxa appear to extremely sensitive to the Abate treatment. A total of one Ephemeroptera (mayfly) emerged from all of the Abate ponds after the first spray, compared with 196 from the Altosid ponds and 264 from the control ponds. In the control and Altosid ponds, beginning on S2D-1 and continuing throughout the study, small numbers of mayflies were consistently collected. The number of mayflies was significantly reduced in the Abate-treated ponds beginning on S2D-1 and continuing through the end of the study (Table 6.2).

Odonates also appear to be affected adversely by exposure to Abate, although few odonates emerged from any of the ponds (Table 6.2). The median number of odonates emerging from the Abate-treated ponds was less than 0.5/pond; significantly less than the 1-3 emerging after day S2Dl4 in the control and Altosid ponds (since data did not meet parametric assumptions, tests of individual dates could not be performed). After the first spray, a total of 7 odonates emerged from the Abate ponds compared with 34 from the control ponds and 36 from the Altosid ponds.

The differences in taxa between the Abate ponds and the control or Altosid ponds is shown in the cluster analysis conducted on SID-I and S3Dl3, using the common distance criterion of0.7 units for similarity. The SlD-1 samples, collected before the first spray, clustered randomly with no obvious groupings according to treatment (Fig. 6.7a). In contrast, at the end of the study, the S3D13 samples clustered into two groups: one containing the Altosid and control ponds, the other containing the Abate ponds (Fig. 6. 7b ).

42 Hester-Dendy Data

There were no significant differences in the number of individuals collected from substrates from the Abate, Altosid, or control ponds. The mean number of species obtained in the substrates exposed in the Abate-treated ponds (13.2) was less than the number obtained in the control (18.0) or Altosid (18.2) ponds. This difference was statistically significant at the 90% confidence level (p=0.09) but not at the 95% level. Species diversity (H') was significantly reduced (p=O.O 1) in the Abate ponds (mean H'=0.74) relative to the control (H'=l.08) or Altosid ponds (H'=0.99). Equitability was also significantly reduced (Table 6.3).

The majority of the individuals in all of the ponds were dipterans. Chironomidae were most affected by the Abate spraying. The mean number of chironomids was 88.5 in the control ponds, 75.2 in the Altosid ponds, and only 23.0 in the Abate ponds (p=0.02; Table 6.3). Chironomus sp. was also significantly reduced (p=0.054), with means of 12.2 (control), 10.0 (Altosid), and 0.8 (Abate). The results suggest that Ceratopogonidae may be resistant to Abate. The mean number in the Abate ponds was 47.6 compared with 29.2 (control) and 20.6 (Altosid) (p=0.33). In contrast, Ephemeroptera appeared to be sensitive to the Abate spraying. The median numbers of Ephemeroptera were 14.5 (control), 6.0 (Altosid), and 2.0 (Abate) (p=0.052). Odonata did not appear to be affected by the spraying.

6.4 Discussion

In the Abate ponds, after the first spray, the numbers of emerging insect species and families, and • the species diversity were reduced and did not recover over the course of the study. The most dramatic effects of Abate on insect emergence were on Ephemeroptera, Odonata, and the genus Chaoborus.

With the Hester-Dendy substrates, there was a statistically significant decline in species diversity and a borderline reduction in the number of species in the Abate ponds relative to the controls. The average number of Chironomidae was significantly reduced to about 26% of the average in the control ponds (Table 6.3). This effect on Chironomidae was far more dramatic than that observed in the emergence traps, where the total number emerging in the Abate ponds after the first spray was 73% of the total emerging during this period in the control ponds (Fig. 6.6). On the Hester-Dendy substrates, there was a slight increase (relative to controls) in the number of non-Chironomid dipterans, especially Ceratopogonidae, which may be more resistant to Abate.

In addition to mosquito control, Abate has been used to control Simulium (Mohsen and Mulla 1992) and chironomids (Tsai 1978, Ali et al. 1992, Stevens and Warren 1992). Thus, the observed effects on chironomids were not unexpected. In laboratory tests in Taiwan, where Abate was evaluated for control of Chironomus /ongilobus larvae, Tsai ( 1978) reported that LC50s for a 50% a.i. emulsified formulation were 2.5 and 5.1 µg/L Abate (1.25 and 2.55 µg/L temephos). In field tests, Sanders et al. (1981) applied emulsified Abate (43% a.i.) at 0.18 kg

43 a.i./ha to ponds once a month for three months. Mean water concentrations one day after spraying ranged from 4.9-8.0 µg/L Abate or 2.1-3.4 µg/L temephos. They observed an initial decrease in benthic dipteran biomass followed by an increase in non-dipteran biomass (attributed to decreased competition). They reported that the emergence of two Chironomid species, Tanytarsus and Dicrotendipes, was inhibited and concluded that the treatment eliminated sensitive species, reduced competition, and enabled a greater number of less sensitive dipterans to emerge. Both of these studies reported lethal temephos concentrations that are considerably lower than the 27 to 32 µg/L measured in the experimental ponds within one hour of spraying. A similar response may be occurring on the Hester-Dendy substrates where chironomids were drastically reduced and Ceratopogonidae somewhat increased.

The genus, Chaoborus, appears to be particularly sensitive to temephos. In a laboratory test, Helgen et al. (1988) reported a 24 hour LC50 of 1.2 µg/L for Chaoborus. They sprayed a pond with emulsified Abate at 0.005 kg a.i./ha on two occasions (22 May and 5 June). In an in situ bioassay, Helgen et al. (1988) reported that 90% of the Chaoborus were dead within 24 hours. Temephos concentrations were 4.2 µg/L within 1 hour of treatment and decreased to 0.9 µg/L 24 h post-treatment. There was an 84% decrease in Chaoborus americanus population within 24 h of the first treatment and a 89% decrease within 24 h of the second treatment. By the end of June, densities had recovered to pretreatment levels. The initial concentrations reported in the present study are likely to have been lethal to Chaoborus. The duration of the lethal conditions in the ponds is not known.

Laboratory studies with mayflies suggest that lethal concentrations of temephos may have • occurred in the Abate ponds. Mohsen and Mulla (1981) reported a 24 hour laboratory LC50 of 9.7 µg/L for the mayfly, Baetis parvus, using an Abate emulsifiable concentrate formulation containing 50% active ingredient. Thus, the LC50 for temephos would be 4.85 µg/L, which is also well below the initial concentration reported in the experimental ponds. In a continuous- flow laboratory test, Muirhead-Thomson (1978) reported that exposure of larval mayflies, Baetis rhodani, to 1 to 2 µg/L temephos for one hour resulted in 90-95% mortality over a 24 hour observation period. In a stream study, Helson and West (1"978) reported that treatment with a particulate formulation of Abate (50% a.i.) at a nominal concentration of 0.1 ppm for 15 minutes resulted in increased drift and mortality of certain mayflies of the family, Ephemerellidae.

The lack of recovery of certain taxa may be attributable to the persistence of temephos in sediments. Hanazato et al. (1989) added Abate (5% temephos) to two enclosures within a lake. The enclosed area was 5 m x 5 mat a site that was 4.2 m deep to produce an initial water column concentration of 500 µg/L. Ekman and core samples revealed that concentrations greater than 2 mg/kg could still be measured in surface sediments collected 4 7 days after application.

Methoprene effects on insects other than mosquitoes have been reported, although application rates were often higher than the 0.011 kg a.i./ha sprayed on the experimental ponds. Ali (1991), in a study aimed at determining if methoprene could be used to control chironomids, reported that Altosid Liquid Larvicide (5% a.i.) was effective against Tanytarsini and Chironomini when

44 applied to ponds at 0.28 kg a.i./ha, which is 25 times the 0.011 kg a.i./ha rate applied to the experimental ponds. He reported that this formulation did not control chironomids when applied at 0.015 kg a.i./ha. Norland and Mulla (1975) applied an emulsified concentrate formulation of Altosid to experimental ponds at 0.27 lb a.i./acre (=0.03 kg a.i./ha) and placed caged mayfly nymphs (Callibaetis pacificus) in the ponds at 4 hours and at 4 days after treatment and tracked their emergence. They reported a substantial decrease in the percentage emerging in both exposure groups relative to unexposed controls. Their application rate, however, was nearly three times the 0.011 kg a.i./ha used in the present study.

The 0.011 kg a.i./ha rate corresponds to a nominal concentration of l. lµg/L, based on experimental pond dimensions and depth. This concentration is similar to the median concentration (0.5 µg/L) reported by Hershey et al. (1995) who applied slow release (150 day) methoprene briquets to three temporary woodland ponds in Minnesota. The ponds were divided during a dry period and one third of each pond received methoprene, one third received Bti, and one third was untreated. Repeated sampling of benthic invertebrates using cores revealed no significant differences among the treatments with respect to the density or biomass of any invertebrate group or on species richness.

To date, the most comprehensive studies on the non-target effects of methoprene on freshwater habitats have been conducted in Minnesota by the Metropolitan Mosquito Control District ( 1996; Hershey et al. 1998). Wetlands in Wright County were sampled for three years (1988-90) to examine "natural" variability in insect populations. Beginning in 1991, and continuing in 1992 and 1993, eight of the wetlands were treated six times during the spring and summer at three week intervals with methoprene at 1.1-13.2 lb/acre (0.05-0.58 kg a.i./ha, based on 4% a.i. formulation). The methoprene was applied as a 20-day release granule. Nine sites were • untreated and nine were treated with Bti. Benthic invertebrates were sampled with a coring device. The investigators reported that there were no significant differences in benthic invertebrate density during the first year of treatment. In years 2 and 3, however, there were significant decreases in the biomass and density of chironomids, Tipulidae, Ceratopogonidae, and Stratiomyidae in the wetlands treated with either pesticide relative to the reference wetlands. Examination of the reproductive success ofred-winged blackbirds (Agelaius phoeniceus) did not indicate that either larvicide had an adverse impact.

The results of the long-term Minnesota experiment cannot be compared directly with those of the present study. Due to their different degradation rates, granular and liquid formulations of methoprene can be expected to vary considerably in their long-term impacts on non-target insects. The more stable granular formulations would be likely to have greater impacts than the shorter-lived liquid formulations (Ali 1991). The multi-year nature of the Minnesota study resulted in findings that could not be detected from the single season study in the experimental ponds. The pre-treatment monitoring provided the investigators with valuable data on the variability in insect populations due to factors other than pesticide exposure. The multi-year nature of the study also provided investigators an opportunity to detect subtle changes that may affect invertebrate recruitment.

45 Further non-target impact studies with temephos and methoprene should focus on short- and long-term effects on a variety of invertebrate taxa, including benthos, emerging insects, and zooplankton. In view of the dramatic effects of temephos on insect taxa, studies should also e investigate possible impacts on animals such as birds and amphibians which rely on the pond invertebrates as a food source.

6.5 References

Ali, A. 1991. Activity of new formulations ofmethoprene against midges (Diptera: Chironomidae) in experimental ponds. J Am. Mosq. Control Assoc. 7:616-620.

Ali, A., L.C. Barbato, G. Ceretti, S. Della Sala, R. Riso, G. Marchese, and F. D'Andrea. 1992. Efficacy of two temephos formulations against Chironomus salinarius (Diptera:Chironomidae) in the saltwater lagoon of Venice, Italy. J Am. Mosq. Control. Assoc. 8:353-356.

American Public Health Association (APHA). 1992. Standard Methods for the Examination of Water and Wastewater. 18th Edition. APHA, Washington, DC.

American Society for Testing and Materials (ASTM) 1992. Standard practice for collecting benthic macroinvertebrates with multiple-plate samples. E 1469-92. ASTM, Philadelphia, PA.

Belisle, A.A. and D.M. Swineford. 1988. Simple, specific analysis of organophosphate and • carbamate pesticides in sediments using column extraction and gas chromatography. Environ. Toxicol. Chem. 7:749-752.

Brower, J.E., H.Z. Jerrold, and C.N. von Ende. 1990. Field and Laboratory Methods for General Ecology. Third Edition. William C. Brown, Dubuque, Iowa.

Campbell, B.C. and R.F. Denno. 1978. The effect of temephos and chlorpyrifos on the aquatic insect community of a New Jersey salt marsh. Environ. Entomol. 5:477-483.

Charbonneau, C.S, R.D. Drobney, and C.F. Rabeni. 1994. Effects of Bacillus thuringiensis var. israelensis on nontarget benthic organisms in a lentic habitat and factors affecting the efficacy of the larvicide. Environ. Toxicol. Chem. 13:267-279.

Clifford, H.T. and W. Stephenson. 1975. An Introduction to Numerical Classification. Academic Press, New York.

D'Elia, C., P.A. Syteudler, and N. Corwin. 1977. Determination of total nitrogen in aqueous samples using persulfate digestion. Limnol. Oceanogr. 22:760-764.

46 English, W.R. 1987. Three inexpensive aquatic invertebrate samplers for the benthos, drift, and emergent fauna. Entomological News 98: 171-179.

Hanazato, T., T. Iwakuma, M. Yasuno, and M. Sakamoto. 1989. Effects oftemephos on zooplankton communities in enclosures in a shallow eutrophic lake. Environ. Pollut. 59:305- 314.

Helgen, J.C., M.K. Larson, and R.L. Anderson. 1988. Responses of zooplankton and Chaoborus to temephos in a natural pond and in the laboratory. Arch. Environ. Contam. Toxicol. 17:459- 471.

Helson, B.V, and A.S. West. 1978. Particulate formulations of Abate and methoxychlor as black fly larvicides: their selective effects on stream fauna. Canad Entomol. 110:591-602.

Hershey, A, L. Shannon, R. Axler, C. Ernst, and P. Mickelson. 1995. Effects ofmethoprene and Bti (Bacillus thuringiensis var. israelensis) on non-target insects. Hydrobiologia 308:219- 227.

Hershey, A., A.R. Lima, G.J. Niemi, and R.R. Regal. 1998. Effects of Bacillus thuringiensis israelensis (Bti) and methoprene on nontarget macroinvertebrates in Minnesota wetlands. Ecol. Applic. 8:41-60.

Menzel, D.W. and R.F. Vaccaro. 1964. The measurement of dissolved organic and particulate carbon in seawater. Limnol. Oceanogr. 9:138-142.

Metropolitan Mosquito Control District. 1993. Interim report of the Scientific Peer Review panel of the Metropolitan Mosquito Control District to the Minnesota Environmental Quality Board. St. Paul, MN.

Metropolitan Mosquito Control District. 1996. An assessment of non-target effects of the mosquito larvicides, Bti and methoprene, in methropolitan area wetlands. A report from the Scientific Peer Review Panel to the Metropolitan Mosquito Control District, St. Paul, MN.

Mohsen, Z.H. and M.S. Mulla. 1981. Toxicity ofblackfly larvicidal formulations to some aquatic insects in the laboratory. Bull. Environ. Contam. Toxicol. 26:696-703.

Mohsen, Z.H. and M.S. Mulla. 1982. Field evaluation of Simulium larvicides: effects on target and nontarget insects. Environ. Entomol. 11 :390-398.

Muirhead-Thomson, R.C. 1978. Relative susceptibility of stream macroinvertebrates to temephos and chlorpyrifos, determined in laboratory continuous-flow systems. Arch. Environ. Contam. Toxicol. 7:129-137.

47 Norland, R.L. and M.S. Mulla. 1975. Impact of Altosid on selected members of an aquatic ecosystem. Environ. Entomol. 4:145-152. e Sanders, H.O., D.F. Walsh, and R.S. Campbell. 1981. Abate: effects of the organophosphate insecticide on bluegills and invertebrates in ponds. U.S. Fish and Wildlife Service Tech. Pap. 104, Washington, DC.

SAS Institute Inc. 1989. SAS/STAT User's Guide Version 6. 4th edition. SAS Institute Inc., Cary, NC.

Stevens, M.M. and G.N. Warren. 1992. Insecticide treatments used against a rice bloodworm, Chironomus tepperi (Diptera: Chironomidae): suppresion of larval populations. J Econ. Entomol. 85: 1606-1613.

Tsai, S-C. 1978. Control of chironomids in milkfish ( Chanos chanos) ponds with Abate (temephos) insecticide. Trans. Am. Fish. Soc. 107:493-499.

Valderrama, J.C. 1981. The simultaneous analysis of total nitrogen and total phosphorus in natural waters. Mar. Chem. 10:109-122.

Zoecon Corp. 1993. Altosid product literature. Zoecon Corp., Dallas, TX.

48 T a bl e 6 ..1 R anges o f water quar 1ty parameters. Control Altosid Abate Temperature 20.4-28.6 20.3-30.2 20.0-28.2 pH 6.5-8.3 6.5-8.0 6.6-7.7 Dissolved oxygen 0.4-11.9 0.26-11.1 0.61-12.2 Conductivity 0.14-0.22 0.13-0.23 0.13-0.24 Alkalinity 58.4-99.0 49.8-103.2 56.9-120.8 Hardness 52.0-104.0 52.-116.0 52.0-124.0 DOC 12.1-24.9 11.3-22.4 11.0-20.4 TDN 0.86-2.0 0.86-1.6 0.90-1.6 TDP 0.022-0.2 0.025-0.21 0.024-1.05

Units: Temperature - (°C); dissolved oxygen - mg/L; conductivity - mmhos/cm; alkalinity, hardness - mg/L CaC03; dissolved organic carbon (DOC) - mg C/L; total dissolved nitrogen (TDN) - mg NIL; total dissolved phosphorus (TDP) - mg P/L

49 T a bl e 6 2 M ean va ues o f eco ogica1. m d"ices an d mean (± SD) numb er o f m d"ivi "d ua1 s per pond o f taxonomic groupmgs. Parameter 22-May 6-Jun 13-Jun 28-Jun 5-Jul 19-Jul

Shannon diversity (H') • Control 0.64 (0.62) 0.82 (0.78) 0.63 (0.79) 0.61 (0.86) 0.44 (0.86) 0.40 (1.01)

Altosid 0.74 (0.73) 0.65b (0.64) 0.32b (0.66) 0.40 (0.82) 0.32 (0.81) 0.26 (0.85)

Abate 0.59 (0.58) 0.32b (0.27b) 0.35b (0.21 b) 0.43 (0.50b) 0.27 (0.54b) 0.09b (0.54b)

Equitability (J') • Control 0.63 (0.62) 0.81 (0.81) 0.61 (0.80) 0.56 (0.80) 0.39 (0.81) 0.32 (0.85)

Altosid 0.70 (0.71) 0.72 (0.76) 0.35b (0.80) 0.39 (0.88) 0.28 (0.83) 0.23 (0.78)

Abate 0.65 (0.65) 0.55 (0.54b) 0.49 (0.44b) 0.57 (0.73) 0.36 (0.86) 0.12b (0.87)

Diptera Control 86.5 ± 40.5 33.0 ± 20.4 35.8 ± 29.8 24.8±9.6 30.2 ± 18.6 36.0± 22.0

Altosid 120.8 ± 172.0 14.7 ± 6.8 17.7±15.2 19.8± 12.3 29.17 ± 22.9 21.2 ± 9.4

Abate 58.7 ±36.3 21.0±20.9 24.8 ± 20.0 23.2 ± 25.4 15.2 ±13.1 6.5b ± 2.2

Chironomidae Control 63.0±42.7 20.3 ±20.8 26.3 ± 29.7 19.2 ± 9.4 21.5 ± 16.3 28.8 ± 13.6

Altosid 88.3 ± 151.0 5.0 ±1.6 9.2±7.9 13.5 ± 13.2 21.2 ± 20.6 16.7 ± 7.3

Abate 318.7 ± 31.0 20.3 ±21.3 22.5 ±18.3 21.8 ± 25.2 13.2 ± 13.1 4.8b ± 2.5 ' Chaoborus Control 20.0± 12.9 11.7 ± 5.8 8.5±2.9 2.6 ± 1.5 5.2 ± 5.0 3.0± 3.2

Altosid 3,0.2 ± 24.2 · 8.7 ± 7.5 7.0 ± 9.4 2.5 ± 1.8 6.5 ± 6.4 0.3 ± 0.5

Abate 17.8 ± 8.8 0.17b± 0.41 2.0b± 3.0 O. l 7b±0.41 1.7 ± 1.6 0.3b ± 0.5

Odonata< Control 0.0 (0.0) 1.0 (1.3) 0.67 (1.2) 0.80 (0.80) 1.2 (1.6) 2.2 (2.1)

Altosid 0.2 (0.4) 0.7 (0.5) 0.0 (0.0) 1.0 (1.6) 1.2 (1.3) 3.2(2.l)

Abate 0.3 (0.5) 0.2 (0.4) 0.2 (0.41) 0.3 (0.5) 0.0 (0.0) 0.5 (0.8)

Ephemeroptera Control 0 1.3 ± 3.3 5.2 ± 7.2 10.4± 8.6 7.5 ± 6.4 21.3 ±15.2

Altosid 0 0.3 ± 0.5 1.3 ± 1.9 7.2 ± 6.3 5.0 ± 4.1 18.7 ±11.3

Abate 0 0 ob ob ob 0.17b ± 0.40

Collembola Control 1.2 ± 1.2 4.3 ± 3.1 53.3 ± 36.7 61.4 ± 26.7 126.7 ± 57.8 262.3 ± 112.5 Altosid Abate 3.8 ± 6.4 6.8 ± 4.8 88.2 ± 37.6 118.0 ± 72.8 209.5 ± 99.2 362.7 ± 136.7

0.5 ± 0.5 2.5 ± 2.4 21.5 ± 15.3 35.2 ± 23.2 89.0 ± 48.8 304.2 ± 205.0 e • Values in parentheses are for the index excluding Collembola b Significantly different from control (repeated measures ANOVA on square root transformed data, p<0.05; Tukey's Studentized Range Test, p<0.05) codonata data were non-parametric; median and range are shown. The numbers emerging from the Abate ponds over the entire course of the monitoring was significantly different from control (Kruskal-Wallis test, p<0.05) Table 6.3. Hester-Dendy substrate data. Parameter Control Ponds Altosid Ponds Abate Ponds Statistics e (n=6} (n=S) {n=S)

Individuals 154.7 ± 96.6 126.2 ± 36.6 112.0 ± 28.7 p=0.72a,b Species 18.0 ± 3.4 18.2 ± 2.6 13.2 ± 5.0 p=o.09a,b Families 9.8 ± 2.1 9.4 ± 2.6 8.0 ± 1.9 p=0.4oa,b Diversity (H') 1.08 (0.87-1.10) A 0.99 (0.95-1.13) A, B 0.74 (0.33-0.97) B p=O.Olc

Equitability (J') 0.82 ± 0.09 A 0.81 ± 0.04 A 0.61 ± 0.13 B p=0.005a

Diptera 117.8 ± 81.8 96.4 ± 33.8 70.6 ± 14.7 p=0.39a

Chironomidae 88.5 ± 48.6 A 75.2 ± 36.5 A, B 23.0 ± 20.5 B p=0.02a,b

Chironomus 12.2 ± 9.8 10.0 ± 6.9 0.8 ± 0.4 p=o.054a

Ceratopogonidae 29.2 ± 41.5 20.6 ± 10.0 47.6 ± 17.2 p=0.33a

Odonata 8.0 (5-17) 8.0 (1-14) 8.0 (1-50) p=0.92c Ephemeroptera 14.5 (4-33) 6.0(4-11) 2.0 (0-32) p=0.052c a AN OVA; if p<0.05, Tukey's test used to compare treatments; groups with different capital letters are significantly different b square-root transformed data • c Kruskal-Wallis test; if p<0.05, Dunn's method used to compare treatments; those with different capital letters are significantly different

52 e

Figure 6.1. Experimental pond with insect emergence trap.

~~~~-Eme~e~ vegetation -4-~~1--1--Water

11------Plexiglass ---- Plastic funnel Mason jar Ethanol

Window screen (four sides) ~iiillllS------~!liii.-iiiiiiT PVC pipe (float) Emergence trap (English 1987) Figure 6.2. Mean number of insects emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date.).

Spray 1 Spray 2 Spray 3 500

m, I"'#JMt I!)ill .....Cl) 400 ,i --•-- Abate (.) Altosid Q) • Cl) I<4 w - Control C 300 I - • ,~,I I 0 <]:',>'. - t ' I.. }Jt i~ Q) 200 w; .0 ,,\1W Abate= 24.0* E :::J z 100 ..",,,:.---- ... ------~ 0 22 May 6 June 13 June 28 June 5July 19 July •

54 Figure 6.3. Mean number of insects (excluding Collembola) emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date).

Spray 1 Spray 2 Spray 3 -ro 150 \¼', 0 fj .0 I~.;;,, fl ,w E 120 I (I.) --•-- Abate Altosid • 0 I () '4 • - Control I \0 .. 90 •- -en t5 (I.) en 60 I C -0 I... (I.) 30 .0 ~ E ::J ------·~--- z 0 22 May 6June 13 June 28 June 5July 19 July •

55 Figure 6.4. Mean number of species emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date).

Spray 1 Spray 2 Spray 3 20 ------~ Ab I •. "'0 --•-- ate II i; I • Altosid I.. ;q Cl) ~ _ .,. _ Control r2 I+ JI Q) ;

©---

0 22 May 6 June 13 June 28 June 5July 19 July •

56 Figure 6.5. Mean number of families emerging in experimental ponds sprayed with Abate, Altosid, or distilled water ( circled data points are significantly different than the control for the same date).

Spray 1 Spray 2 Spray 3 8 i1X I F:i U') *t --•-- Abate I -~ 7 • Altosid E - .. - Control 11 ctS 6 u. ' 0 - 5 I.. Q) .0 E 4 z::::, 3

2 22 May 6June 13 June 28 June 5 July 19 July •

57 Figure 6.6 Total number of Chaoborus sp., Chironomidae, and Diptera emerging from experimental ponds after the first spray.

Total emerging from experimental ponds after the first spray.

1000 -,------,

tn 900 0 control -cu !El Altos id :::s 800 - ---~------·--·------· ------·-·------~- -·- "C •Abate ·-.2: 700 -t--~- ~~------"C C: 600 -+------~---~- ·····-··--· ·---~------~------11- ...0 500 -1------~------··· Cl) .0 400 __,______------... ------E :::s 300 ------~---·· ... ------·-··· C: -....cu 200 ...0 100 -

0 -t-----'--­ C ha o bo rus Chironomidae Diptera Figure 6.7a. Cluster analysis for experimental pond insect data: May 22.

Site

0 C B C C C T T T B T T B B C B B C T

.1

.2 17

15 16 .3 -- 14 12 13 G) CJ ' 11 ~ .4 10 I .ii 7 9 8 C .5 5 I 6

.6 4 3

.7 2 C = Control T = Altosid B =Abate 1 .8

.9 L------Figure 6. 7b. Cluster analysis for experimental pond insect data: July 19.

Site

0 C C C T T T T T T C C CB BBB BB

.1

.2 12 17 .3 -- 15 Cl) 16 CJ C a:, .4 13 14 I 1;; I I 11 10 ·-Cl 9 .5 ' 8 6 7 5 .I .6 4 I 3 .7

.8 C =Control 2 T =Altosid B =Abate .9 1

1.0 7.0 EFFECTS OF ABATE AND ALTOSID ON TADPOLES

7 .1 Introduction

This chapter describes studies conducted in the laboratory and experimental ponds at Patuxent Wildlife Research Center to assess the effects of Abate and Altosid on green frog (Rana clamitans) and gray treefrog (Hy/a versicolor) tadpoles. The pond studies were conducted simultaneously with the insect emergence study described in Chapter 6. This chapter is a compilation of two reports, prepared by D.W. Sparling, T. P. Lowe, and A.E. Pinkney. The first report is entitled, Chemicals Used to Control Mosquitoes on Refuges Differ in Toxicity to Tadpoles and was broadcast on PWRC's site on the World Wide Web. The second report, Toxicity of Abate to Green Frog Tadpoles has been published in the Bulletin of Environmental Contamination and Toxicology, volume 58, pages 475-481, 1997. In addition, there is a brief description of a preliminary study examining the effects of Abate 4E and Altosid on the prevalence of deformities in southern leopard frogs (Rana utricularia).

7.2 Chemicals Used to Control Mosquitoes on Refuges Differ in Toxicity to Tadpoles

Two chemicals widely used by National Wildlife Refuges to control mosquito populations, Altosid and Abate differed in their toxicity to amphibian larvae in field and laboratory trials. National Wildlife Refuges are instrumental in protecting a large number of fresh, brackish, and saline wetlands for migrating and wintering waterfowl and for other functions inherent to wetlands. Unfortunately, when these refuges are in proximity to areas populated by humans, they can be potential sources of nusiance or disease to humans and livestock due to breeding populations of mosquitos. This concern is especially high along the western and eastern seaboards. The potential conflict between maintaining the natural functions of refuge wetlands and the concern for public health has led to a search for integrated pest control methods that are both effective on mosquitos and relatively safe for nontarget organisms. One method involves application of chemicals to either directly kill larvae or inhibit their emergence as adults.

We tested Abate (44.6% temephos), an organophosphate insecticide, and Altosid (5% methoprene ), a juvenile growth inhibitor on tadpoles. Abate is the most widely applied mosquito abatement chemical in National Wildlife Refuges whereas Altosid and the bacterium, Bacillus thuringensis, are used less frequently.

Abate® Depressed Growth of Gray Treefrog Tadpoles

In one experiment we determined that Abate reduced growth of gray treefrog (Hy/a versicolor) larvae in constructed ponds located at Patuxent Environmental Science Center, MD. The ponds averaged 202 m2 in area and 105 m3 in volume. Each chemical was applied to six ponds with a 1 hand sprayer following manufacturers' label instructions - 1.5 ounces/acre (21.6 µL'L- ) for Abate 1 and 3 ounces/acre (42.8 µL'L- ) for Altosid. Six control ponds were sprayed with water but no chemical. Spray dates were 24 May, 13 June, and 6 July 1995 which were selected to simulate actual application times at Prime Hook National Wildlife Refuge, DE. On 12 June, 13 very

61 young ( < 2 days old) or 8 older (7 - 10 days old) tadpoles were placed into plastic containers within each pond. The containers were screened to allow food (algae) and water to enter freely but block predators. On 13 July, the containers were removed and the length of each tadpole from snout to vent, their collective displacement volume, and their collective weight were measured.

The mortality rate of tadpoles is reportedly very high under natural conditions and there was no difference in mortality among treatments for either age class. However, the very young tadpoles experienced a higher mortality rate than the older tadpoles (Table 7.1). By the end of the experiment, older tadpoles showed a significant difference in size due to treatment with those in Abate®-treated ponds smaller than those in Altosid®-treated or control ponds. Body size of very young tadpoles was marginally different among treatments.

Abate was More Toxic than Altosid in Laboratory Tests

In a second set of experiments we attempted to determine the 96-hr median lethal concentration (LC50) of both Altosid and Abate for green frog (Rana clamitans) tadpoles using a static renewal protocol in synthetic soft water. The assays were conducted in an environmental chamber at 21 °ࣧ
C and 16:8 light:dark cycle. Concentrations ranged from 1.86 to 10 µL'L" 1 for Abate and 18.6 to 100 µL'L" 1 for Altosid. Four tadpoles were used in each 2-L bell jar and each concentration was run in duplicate. At the end of the study, survivors were euthanized and frozen. Butyrlcholinesterase (BChE) and acetylcholinesterase (AChE) assays were run on whole bodies of control and Abate-treated tadpoles.

The dose-response curve for Abate was: 1 % Mortality= -4.408 + l.046*Concentration, LC50 = 4.21 µL'L" (95% confidence interval= 3.61 - 5.10 µL'L-1), standard error of slope= 0.295, p = 0.0004 (Fig. 7.1). After 5.5 hr of treatment on the first day, three of the eight tadpoles at 10 µL'L" 1 and one at 5.1 µL'L" 1 were unable to right themselves. By 24 hr, half of the tadpoles at 10 µL'L" 1 were dead. The estimated 1 48 hr LC50 was 7.70 µL'L" . BChE activity (mmol/cm/sec) declined sharply with the lowest dose of Abate and remained low in all treated tadpoles (Fig. 7.2}. However, AChE activity did not decrease with dosage.

Two tadpoles died at each of the middle concentrations of Altosid but none at the highest level. Because this mortality was not correlated with concentration, we were unable to determine a dose-response curve for Altosid.

Management Considerations

Of the two mosquito abatement chemicals, Abate clearly presents the greater risk to amphibian larvae. Abate appeared to inhibit the growth of young gray treefrog larvae in the pond experiments. Whether this was a direct physiological response or due to a secondary effect such as chemically-induced decreased food availability is unknown. In addition, the median lethal

62 dose for Altosid, although undetermined, must be at least 10 times greater than that for Abate and Altosid is applied at only twice the concentrations as Abate®.

One of the problems in relating the LC50 value to actual conditions is the calculation of field concentrations. Label directions on Abate and Altosid prescribe application rates on an ounce per surface acre basis. Although this is practical for surface-dwelling mosquito larvae, interpretation of concentration is difficult for organisms such as tadpoles which live at indeterminate depths. Based on our pond study, the calculated LC50 is well within expected field concentrations. However, care must be taken in extrapolating the laboratory study to field conditions because of the absence of ligands such as dissolved organic carbon (DOC), the inability of the tadpoles to escape exposure, and the length of the study. Under field conditions temephos is known to bind with DOC and to degrade within a day or two. In a related experiment, we found that full strength pond water decreased the toxicity of Abate compared to synthetic soft water. Thus our laboratory experiment could be interpreted as a worst case situation for either compound. Further work on additional species and formulations of Abate are required.

Table 7.1. Survival and body size (X±SC) of gray treefrog tadpoles exposed to Altosid®. and Abate®. for three wetlands, 1995. Treatment and Initial Mortality Average Weight (mg) Average Displacement Age of Tadpolesa Volume (ml) Control 2d 83.4 + 18.0 0.29 ± 0.21 0.28+0.l 7 8d 51.2 + 28.1 0.56 + 0.37 0.50+0.35 Abate 2d 75.4 + 19.8 0.19 + 0.17 0.16+0.14 8d 47.4 ± 26.4 0.26 ± 0.15 0.24±0.17 Altosid 2d 80.3 + 13.2 0.25 ± 0.23 0.19±0.18 .. ~

63 Figure 7 .1. 96-hr dose-response curve for green frog tadpoles exposed to Abate in synthetic solft water. e 100 90 80 -+-% Mortality 70 ~ 60 ·-ii t: 0 50 :E ';I. 40 30 20 10 0 0 2 4 6 8 10 Concentration (I.IUL)

64 Figure 7.2. Butyrlcholinesterase activity (as percent of control mean) in green frog tadpoles exposed to Abate for 96-hrs.

120

100 --+-BChE ~ 80 > =u ca 60 w s:::. 0 m 40

20

0 0 2 4 6 8 10 Abate (mgn)

65 7.3 EFFECTS OF ABATE AND ALTOSID ON TADPOLES Toxicity of Abate® to Green Frog Tadpoles. e Donald W. Sparling1, T. Peter Lowe 1, and Alfred E. Pinkney2.

1National Biological Service, Patuxent Wildlife Research Center, 11510 American Holly Dr., Laurel, MD 20708

2U.S. Fish and Wildlife Service, Chesapeake Bay Field Office, 177 Admiral Cochrane Dr. Annapolis, MD 21401

Wetlands are essential habitats for many beneficial species of plants and animals. However, when wetlands are in proximity to urbanized areas, they may be breeding areas for mosquitoes which may become nusiance species and vectors for numerous zoonoses and epizootics. Public health officials, therefore, sometimes call for measures to control these populations of mosquitoes. Because many control measures affect non-target organisms, mosquito control may result in a conflict between maintaining the benefits of wetlands and reducing the negative affects of pests. This is particularly true for many U.S. Fish and Wildlife Service National Wildlife Refuges along the Atlantic and Pacific coasts that were established years ago to provide habitat but now are surrounded by urbanization.

In response to this concern, the U.S. Fish and Wildlife Service has sought ways of controlling mosquitoes that are both efficacious and comparatively safe to non-target species. Potential control methods include various biological control agents ( e.g. Gambusia affinis, Bacillus thuringiensis, Lagendium giganteum ), juvenile growth inhibitors (e.g. methoprene) and several pyrethroid and organophosphorus pesticides (e.g. e pyretrhin, temephos, fenthion, malathion) . Temephos [O,o'-(thiodi-4, 1-phenylene) 0,0,0',0'-tetramethyl phosphorothioate)] is a widely used organophosphorus pesticide to control mosquitoes (Mu Lee and Scott 1989), but it is lethal to fish and non-target invertebrates. Field and laboratory studies also indicate that temephos may affect the behavior and survival of fiddler crabs (Ucapugnax) (Ward and Busch 1976, Ward et al. 1976). Little, if anything, has been published about the toxicity of temephos to amphibians.

The purpose of this paper is to determine the median lethal concentration of an emulsifiable formulation oftemephos, Abate® 4-E (American Cyanamid Co., hereafter referred to as Abate) and to study its effects on the behavior and cholinesterase activity in green frog (Rana clamitans) tadpoles. Abate contains 44.6% temephos and 55.4% inert ingredients listed as petroleum distillates on the label.

66 4

Cl) +,,) 3 c-s ~ 2 ~ ·~> ·~+,,) 1 <:.> < 0

1 2 3 4 5 6 7 8 9 Sample Period

Dose (µL/L) - Control !J-l!Jo[) 2.60 !!!!-l>-1!1 7.14 Figure 1. Activity (number of swimming bouts/tadpole/2 min) of green frog tadpoles exposed to Abate for 96-hr; two sample periods per day.

METHODS AND MATERIALS

The bioassay generally followed ASTM (1988) guidelines for 96-hr static renewal tests. Green frog tadpoles (stage 25-27, Gosner 1960) were collected from unsprayed wetlands located at Patuxent Wildlife Research Center, Laurel, MD and kept in aerated pond water within an environmental chamber (temperature 20-21 ° C, 16:8 light:dark photoperiod) for I wk prior to testing. Tadpoles were then acclimated to reconstituted soft water (RSW, 1 hardness 40-48 mg CaC03r , ASTM 1988) by successively diluting pond water with RSW over an 8 d period. Four tadpoles were placed in each of 4-L glass bell jars containing 2 L of soft water and one of six geometrically arranged concentrations of Abate ranging from 1.86-10 µL'L-1. Each concentration was run in duplicate. Water was replaced with fresh solutions of Abate and RSW every morning. Tadpoles were fed a half pellet (ca 0.5 g) of rabbit chow (alfalfa) every other day.

Twice each day starting approximately 15 min after water was renewed, we sampled the activity level of tadpoles by counting the number of swimming events (active propulsion for 2: 1 cm distance) during 2 min divided by the number of living tadpoles. Movement caused by another tadpole was not counted as an activity. Death was determined by lack of movement including signs of respiration when tadpoles were prodded with a glass rod. All dead tadpoles were collected as soon as possible, placed in glass jars for each treatment, and frozen at -20 °C. Survivors were anesthetized by chilling and then frozen.

To confirm nominal Temephos concentrations in water, we extracted 10 ml samples of water from a 10 µL'L" 1 and a 1.86 µL'L" 1 solution immediately after mixing. Temephos in water was extracted in triplicate with methylene chloride and the extracts were pooled, concentrated, and cleaned through a florisil column and analyzed via gas

67 100 :>-. .,> •,-I 80 c;S -.,> 1-1 0 60 ::E .,> ~ 40 Q) C) 1-1 Q) 20 ~ 0 0 1 2 3 4 5 6 7 8 9 10 Concentration (µL/L))

Figure 2. Percent mortality of green frog tadpoles exposed to Abate for 96-hr. Eight tadpoles per concentration.

chromatography on a DB-1 capillary column. Detection limits were 0.08 µLL- 1• Analyzed values were± 12% of nominal concentrations.

Within 2 wk, the intestinal coils of the tadpoles were removed and the remaining whole bodies were homogenized using a tissue grinder and analyzed for butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) activities using the method of Ellman et al. (1961 ); activity rates of control tadpoles were used in lieu of specific activities. Acetylthiocholine iodide and y-butyrylthiocholine iodide were used as substrates for AChE and BChE, respectively. To obtain repeatable activity rates with slopes> zero we had to wait 5 min before taking the first spectrophotometric reading and used 200 and 400 µL of tissue homogenate for AChE and BChE. Each sample was run at least in duplicate or until two readings were within 10% of each other and then averaged.

Differences in activity levels among concentrations of Abate were compared with analysis of variance after data were log-transformed; a posteriori comparisons were made with Tukey's test. The LC50 and associated statistics were calculated using logit analysis (Proc CA TMOD, SAS 1987) which is more appropriate than probit analysis when multiple organisms are used per test chamber. The responses of BChE and AChE to dose were tested with regression analysis.

RESULTS AND DISCUSSION

Activity levels varied with concentration of Abate and time (Fig. l ). Tadpoles in control chambers tended to be more active in the morning (odd numbered sample periods) than in the afternoon. However, there was no general trend through time (r2=0.019, p=0.582).

68 140 120 ni1 ;...-, 11 ...... ,; 100 ...,....;.... C) 80· ~ i:.::l 60 .c: u 40 i::r::. 20 0 0 1 2 3 4 5 5 7 8 9 10 Concentration (µ.L/L)

Figure 3. Mean(± S.E.M.) activity of BChE expressed as percent of control activity in green frog tadpoles exposed to Abate for 96-hr.

Differences between morning and afternoon activity levels increased for tadpoles at intermediate concentrations of Abate (e.g. 2.60 µLL-1).

The parent compound of temephos is less toxic to mammals and target invertebrates than its sulfoxide or sulfone forms. These forms can be generated either through photocatalytic degradation within hours or metabolically (Eto 1974, Lores et al. 1985). The enhanced bimodality in activity at the intermediate concentrations compared to controls suggests that tadpoles have to be exposed to or metabolize temephos to these breakdown products in order to display symptoms but can recover with further degradation of temephos. Tadpoles at high dose levels ( e.g. 7 .14 µLL- 1) nearly ceased moving by the afternoon of the second day of treatment. These tadpoles lay on the bottom of the jars and respired laboriously. Differences in activity rates were depende_nt on dose (p = 0.0001 ), sample period (p = 0.0001) and the interaction between dose and sample period (p = 0.026).

Specifically, tadpoles at 5.10, 7.14, and 10.0 µLr 1 were less active than controls (p < 0.05). By 24 hr of exposure, half of the tadpoles at 10 µLL- 1 had died. At the end of 96 hr, the percent mortality for each concentration was: control - 0%; 1.86 µLr 1 - 0%; 2.60 and 3.64 µLL- 1 - 12.5%; 5.10 µLr 1 - 87.5%; and 7.14 and 10 µLr 1 - 100% (Fig. 2). The jar (p=0.805) and jar by dose interaction (p=0.903) effects were not significantly different from zero, indicating that these effects were trivial. The dose-response curve for Abate could be described as: logit(mortality) = -8.1329 - 1.917* Concentration (µLL- 1 ), 1 1 LC50=4.24 µLL- , S.E. on slope=0.594 µLr , df=6, p=0.0017. This median lethal concentration is slightly higher than that found for salmonids (Mayer and Ellersieck 1986) and about the same as found by Tsai (1978) for other species of fish.BChE and AChE responded very differently to these levels of Abate. The activity rate (± SD) for BChE in control tadpoles was 0.979 ± 0.415 mol min- 1 g- 1 of tissue which is consistent with values published for some fish species (Magnotti et al. 1994 ).

69 105

~, 155 ....l ';""'-1 ....•' ,..,) 145 ·,:C) ' ~ 125 ' .c:: I; u ii ~ 105

85 0 1 2 3 4 5 6 7 8 g 10 Concentration (µ,L/L)

Figure 4. Mean (± S.E.M.) AchE activity as percent of control in green frog tadpoles exposed to Abate for 96-hr.

The activity of this enzyme, expressed as percent of control value, declined precipitously with concentration of Abate, even at 1.86 µL'L- 1 of Abate (r2 = 0.577, p = 0.0001) (Fig. 3). In contrast, AChE (mean activity rate in controls= 18.28 ± 2.96 mol min- 1 g- 1) increased with concentration of Abate (r2 = 0.348, p=0.0001) (Fig. 4).

BchE is produced by muscle and liver tissue in fish but its function is uncertain. The amount of enzyme present, or its activity rate, varies considerably among fish species and even tissues within a species (Kozlovskaya et al. 1993). AChE is the principal neurotransmitter in animals and tends to be concentrated in brain tissue. BChE is substantially more sensitive to cholinesterase-inhibiting pesticides than AChE in fish (Magnotti et al. 1994), birds (Thompson et al. 1991) and mammals (Ecobichon and Comeau 1973). Thus greater depression in the activity ofBChE compared to AChE was expected. However, the increase in AChE with concentration of Abate demands further interpretation.

Typically, organophosphorus (OP) pesticides depress AChE activity (Hill and Fleming 1982, de Llamas et al. 1985). In fact, AChE depression compared to controls is often used as a diagnostic of OP poisoning (Ludke et al. 1975) . Rarely, animals may respond to low, sublethal, levels of OP exposure by increasing cholinesterase activity. Thompson et al. ( 1991) observed increased carboxylesterase activity in house sparrows (Parus domesticus ) treated with demeton-S-methyl. Thompson and Walker (1994) suggested that this elevated cholinesterase could have come from the liver. Although the majority of AChE is in nervous tissue, Kozlovskaya et al. (1993) reported AChE activity in muscle and liver of fish. The parent compound oftemephos has relatively low toxicity and must be

70 metabolized to sulfone or sulfoxide forms to be lethal. Therefore, one possible answer to the diverging activities of AChE and BChE upon exposure to Abate is that green frog tadpoles are inefficient in metabolizing the pesticide. The more sensitive BChE was probably inhibited by the amount oftemephos that was metabolized, but the AChE levels were unaffected. Tadpoles at the higher dose levels were stressed for long periods of time - sometimes for more than a day - and this stress could have stimulated the nervous system to produce more acetylcholine and AChE. Alternatively, tadpoles that died during the study laid for variable periods of time before they were frozen and these delays may have affected AChE in some way that is presently unclear. Further studies are planned to examine this situation.

Label instructions on Abate prescribe application rates of 0.5 to 1.5 ounces per surface acre (ca. 244 mJ·ha-'). This complicates hazard assessments based on concentration because water depths vary among wetlands. However, in an associated study, we determined that, following label instructions, application to a series of constructed wetlands with a mean depth of 0.5 m would result in a concentration of 0.02-0.05 µLL- 1 which is two orders of magnitude less than the LC 50 for this species. Because we renewed the Abate concentration each day, whereas temephos degrades rapidly under field conditions (Lores et al. 1985), tadpoles are probably at even less risk than suggested by these data.

Acknowledgements. We thank Colleen O'Rourke and Jennifer Angel who volunteered their time and efforts to make this study successful. Additional support was given by the U.S. Fish and Wildlife Service. Earlier drafts of the manuscript were reviewed by R. Hall, B. Link, and N. Beyer.

REFERENCES

ASTM ( 1988) Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. E729-88. American Society for Testing and Materials, Philadelphia, PA deLamas MC, de Castro AC, Pechen de D'Angelo A (1985) Cholinesterase activities in developing amphibian embryos following exposure to the insecticides dieldrin and malathion. Arch Environ Contam Toxicol 14: 161-166. Ecobichon DJ, Comeau AM (1973) Pseudocholinesterases ·of mammalian plasma: Physicochemical properties and organophosphate inhibition in eleven species. Toxicol Appl Pharmacol 24:92-100. Ellman GL, Courtney KD, Andres V Jr., Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88- 95. Eto M (1974) Organophosphorus pesticides: Organic and biological chemistry. CRC Press, Inc. Cleveland, OH Hill EF, Fleming WJ (1982) Anticholinesterase poisoning of birds: monitoring and diagnosis of acute poisoning. Environ Toxicol Chem 1:27-38 Gasner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16: 183-190. Kozlovskaya, VI, Mayer, FL, Menzikova, OV, Chuyko, GM (1993) Cholinesterases of aquatic animals. Rev Environ Contam Toxicol 132: 117-140. e 71 Lores EM, Moore JC, Moody P, Clark J, Forrester J, Knight J (1985) Temephos residues in stagnant ponds after mosquito larvicide applications by helicopter. Bull Environ Contam Toxicol 35:308-313. Ludke, JL, Hill EF, Dieter MP (1975) Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch Environ Contam Toxicol 3: 1-21 Magnotti RA Jr., Zaino JP, McConnell RS (1994) Pesticide-sensitive fish muscle cholinesterases. Comp Biochem Physiol 108C:187-194. Mayer FL Jr., Ellersieck MR (1986) Manual of acute toxicity: interpretation and data base for 410 chemicals and 66 species of freshwater animals. US Fish Wild! Serv Resour Pub! 160, Washington DC. Mu Lee, B, Scott, GI (1989) Acute toxicity oftemephos, fenoxycarb, diflubenzuron, and methoprene and Bacillus thuringiensis var. israelensis to the mummichog (Fundulus heteroclitus) Bull Environ Contam Toxicol 43:827-832. SAS (1987) SAS/STAT guide for personal computers, version 6 ed. SAS Institute Inc. Cary, NC. Thompson HM, Walker CH, Hardy AR ( 1991) Changes in the activity of avian serum esterases following exposure to organophosphorus insecticides. Arch Environ Contam Toxicol 20:514-518. Thompson HM, Walker CH (1994) Blood esterases as indicators of exposure to organophosphorus and carbamate insecticides. In: Fossi MC and Leonzio C (eds) Nondestructive biomarkers in vertebrates. Lewis Publishers, Boca Raton FL, pp 37-62. Tsai SC (1978) Control of chironomids in milkfish (Chanos chanos) ponds with abate (temephos) insecticide. Trans Am Fish Soc 107:493-499. Ward, DV, Busch, DA (1976) Effects oftemephos, an organophosphate insecticide, on survival and escape behavior of the marsh fiddler crab, Uca pugnax. Oikos 27:331- 335. Ward, DV, Howes, BL, Ludwig, DF (1976) Interactive effects of predation pressure and insecticide (temephos) toxicity on populations of the marsh fiddler crab Uca pugnax. Marine Biol 35:119-126.

7.4 Preliminary Results of F ollowup Investigation

During the growing season of 1997, Abate 4f.: and Alt(?sid were sprayed on the experimental ponds in a similar manner as described in Chapter 6. In late August and early September, the ponds were sampled for metamorphing southern leopard frogs (Rana utricularia). Preliminary analyses showed that juvenile frogs collected from the Altosid-sprayed ponds had a higher frequency of deformities than those from control ponds (p=0.025). The most common deformities included missing or partially missing hind limbs, but there were also missing eyes and demelanization resulting in a pale tan coloration. No amphibians captured from Abate-sprayed ponds had deformities but the catch per unit effort was significantly decreased in these ponds than in Altosid or control ponds. Because of the concern over deformed amphibians in parts of the midwest and northeast U.S., the study will be expanded and repeated.

72 8.0 CONCLUSIONS AND MANAGEMENT RECOMMENDATIONS

The major findings of these studies are:

1. Although spraying with Abate 4E at an operational rate of 0.054 kg a.i/ha did not affect populations of adult fiddler crabs (U pugnax), it caused a significant (16%) reduction in the survival of juveniles exposed in in situ bioassays. Acetylcholinesterase activity was significantly inhibited (28% lower) in U minax exposed to Abate 4E at the same rate. These results suggest the need to continue to monitor for non-target impacts of Abate 4E on fiddler crabs and other invertebrates, to evaluate whether effective mosquito control can be achieved at lower application rates, and to determine whether this level of cholinesterase inhibition in fiddler crabs results in short or long-term toxicity.

2. Spraying with Altosid did not appear to affect coffee bean snails (Melampus bidentatus) or the percent of female fiddler crab (Uca minax) that were gravid.

3. Observations of significantly lower and less diverse emerging insect populations in one ponded area at Prime Hook NWR cannot be linked to the spray history of the pond. Other factors such as salinity, water depth, organic matter, and predation may be of equal or greater importance than spray history. Emergence trap monitoring revealed that only a single mosquito was captured from the two ponds under investigation, which raises a question as to the need for spraymg.

4. Freshwater experimental ponds sprayed with Abate 4E at 0.054 kg a.i./ha suffered dramatic reductions in the diversity of insect fauna. Ephemeroptera, Odonata, Chironomidae, and Chaoborus appear to be sensitive taxa. Spraying with Altosid at 0.011kg a.i./ha did not affect the numbers of insects, species, families, or specific taxa; there were no consistent effects on several species diversity indices.

5. Preliminary data suggest that frogs may be at risk from the spraying of both Abate 4E and Altosid. An increased prevalence of deformities occurred in the ponds sprayed with Altosid - . . whereas a lower catch per unit effort occurred in the ponds sprayed with Abate 4E. Continuing studies in the experimental ponds will confirm or refute the preliminary findings.

Based on the studies reported herein, the authors make the following management recommendations:

1. Continued spraying of Abate 4E or Altosid on salt marsh habitats should be permitted while the U.S. Fish and Wildlife Service, researchers, and mosquito control agencies investigate alternative methods of mosquito control and evaluate the relative risks of various control methods. These results suggest the need to continue to monitor for non-target impacts of Abate 4E on fiddler crabs, to evaluate whether effective mosquito control can be achieved at lower application rates of Abate 4E, and to determine whether the observed level of cholinesterase inhibition results in short or long-term toxicity.

73 2. Because non-target impacts were observed on freshwater insects from the spraying of Abate 4E and because there is preliminary evidence of adverse impacts on frogs from Abate 4E and Altosid, we recommend that refuges discontinue the use of these larvicides on freshwater habitats.

3. Emergence traps are recommended as an effective tool for refuge managers to monitor insect populations in ponds.

74 Appendix A

Water quality data for Chapter 6 Water Quality Summary- Data for Control Sites

5-15-95 5-22-95 5-30-95 6-6-95 6-13-95 6-21-95 6-28-95 7-5-95 7-12-95 7-19-95 POND TRT PARAM WK1 WK2 WK3 WK4 WK5 WK& WK7 WKS WK9 WK10 1 control TEMP 25.5 25.2 24.2 24.0 23.2 25.3 21.9 25.8 26.2 28.6 0.0. 10.4 11.9 12.4 9.5 6.5 2.3 0.68 0.61 3.7 2.8 pH 7.7 7.7 8.3 7.2 7.0 6.5 6.8 6.6 6.6 6.7 Cond 0.16 0.15 0.20 0.17 0.16 0.19 0.17 0.18 0.19 Hardness 80.0 80.0 84.0 92.0 52.0 100.0 96.0 88.0 80.0 88.0 Alkalinity 82.0 78.6 83.3 93.3 82.5 82.7 87.4 74.6 76.8 83.0 DOC 14.8 15.0 15.5 TOP 0.022 0.022 0.049 TON 0.86 0.95 1.1 4 control TEMP 25.2 21.5 23.6 21.9 20.6 26.3 21.6 23.4 24.5 26.1 0.0. 8.3 6.1 9.4 5.6 2.2 6.0 3.6 5.1 5.9 3.4 pH 7.2 7.0 7.4 6.8 6.6 6.9 6.8 6.8 6.9 6.5 Cond 0.18 0.17 0.21 0.22 0.18 0.19 0.16 0.16 0.18 Hardness 80.0 72.0 84.0 96.0 84.0 96.0 92.0 76.0 76.0 84.0 Alkalinity 80.6 75.6 86.4 99.0 87.9 94.9 89.4 74.7 75.0 86.5 DOC 18.2 14.9 14.5 TOP 0.051 0.053 0.039 TON 1.4 1.2 0.97 5 control TEMP 26.0 23.5 23.8 22.2 22.0 25.8 21.4 25.8 28.5 27.9 0.0. 8.8 6.0 8.9 4.4 3.6 2.9 2.6 5.0 5.6 3.2 pH 7.3 6.9 7.4 6.6 6.6 6.7 6.6 6.6 6.8 6.5 Cond 0.15 0.14 0.18 0.17 0.16 0.18 0.14 0.14 0.16 Hardness 72.0 68.0 76.0 92.0 96.0 92.0 76.0 60.0 68.0 68.0 Alkalinity 80.6 74.5 75.9 83.5 73.2 78.6 75.3 66.3 58.6 69.6 DOC 14.6 14.2 12.1 TOP 0.056 0.088 0.027 TON 1.4 1.4 0.91 9 control TEMP 25.34 25.12 24.55 23.16 22.95 26.65 21.82 24.24 26.32 27.81 0.0. 6.96 6.77 6.65 4.55 3.8 6.06 0.77 0.98 3 2.01 pH 7.08 7.06 7.21 6.92 6.84 7.04 6.8 6.62 6.8 6.6 Cond 0.16 0.145 0.185 0.16 0.163 0.171 0.171 0.145 0.155 Hardness 88 76 76 92 84 92 -· 92 76 72 72 Alkalinity 87.5 72.6 74.9 86.6 71.9 75.1 79.5 62.7 58.4 69.6 DOC 14.3 13.4 13.9 TOP 0.042 0.038 0.03 TON 1.07 0.97 0.97 - ·- 10 control TEMP 25.5 24.2 24.0 21.3 20.4 26.6 21.5 24.6 25.0 26.7 0.0. 4.7 3.5 7.0 1.9 1.2 2.3 0.44 2.3 2.3 0.92 pH 7.0 7.0 7.2 6.9 6.8 6.8 6.8 6.8 6.7 6.6 Cond 0.16 0.14 0.18 0.16 0.17 0.18 0.15 0.14 0.16 ··- Hardness 76.0 76.0 96.0 68.0 104.0 92.0 76.0 72.0 76.0

A1 Water Quality Summary- Data for Control Sites

5-15-95 5-22-95 5-30-95 6-6-95 6-13-95 6-21-95 6-28-95 7-5-95 7-12-95 7-19-95 POND TRT PARAM WK1 WK2 WK3 WK4 WKS WK6 WK7 WKS WK9 WK10 Alkalinity 79.3 74.6 83.1 73.0 79.8 81.3 67.2 62.6 70.4 DOC 13.5 13.4 12.1 TDP 0.031 0.051 0.031 TDN 1.0 1.2 0.94 22 control TEMP 25.6 21.9 25.2 22.2 21.3 26.1 21.5 24.3 26.0 27.5 D.O. 1.4 1.6 4.8 2.1 1.6 1.0 0.64 2.8 3.3 2.6 pH 6.7 6.7 7.0 6.7 6.6 6.6 6.8 6.7 6.7 6.7 Cond 0.15 0.14 0.18 0.16 0.17 0.18 0.15 0.15 0.17 Hardness 72.0 64.0 68.0 80.0 60.0 88.0 80.0 76.0 68.0 72.0 Alkalinity 85.4 65.8 69.1 78.1 69.3 75.7 75.6 62.6 60.9 71.5 DOC 24.9 20.6 18.2 TDP 0.18 0.064 0.056 TDN 2.0 1.3 1.3

Range TEMP 25.2-26.0 21.5-25.2 23.6-25.2 21.3-24.0 20.4-23.2 25.3-26.7 21.4-21.9 23.4-25.8 24.5-28.5 26.1-28.6 0.0. 1.4-10.4 1.6-11.9 4.8-12.4 1.9-9.5 1.2-6.5 1.0-6.1 0.4-3.6 0.6-5.1 2.3-5.9 0.9-3.4 pH 6.7-7.7 6.7-7.7 7.0-8.3 6.6-7.2 6.6-7.0 6.5-7.0 6.6-6.8 6.6-6.8 6.6-6.9 6.5-6.7 Cond 0.15-0.18 0.14-0.17 0.18-0.21 0.16-0.22 0.16-0.18 0.17-0.19 0.14-0.17 0.14-0.18 0.16-0.19 Hardness 72.0-88.0 64.0-80.0 68.0-84.0 80.0-96.0 52.0-96.0 88.0-104.0 76.0-96.0 60.0-88.0 68.0-80.0 68.0-88.0 Alkallnlty 79.3-87.5 65.8-78.6 69.1-86.4 78.1-99.0 69.3-87.9 75.1-94.9 75.3-89.4 62.6-74.7 58.4-76.8 69.6-86.5 DOC 13.5-24.9 13.4-20.6 12.1-18.2 TOP 0.022-0.2 0.022-0.1 0.027-0.1 TON 0.86-2.0 0.95-1.4 0.91-1.3

Median TEMP 25.5 23.9 24.1 22.2 21.7 26.2 21.5 24.5 26.1 27.7 0.0. 7.6 6.0 8.0 4.5 2.9 2.6 0.73 2.6 3.5 2.7 pH 7.1 7.0 7.3 6.8 6.7 6.8 6.8 6.7 6.8 6.6 Cond 0.16 0.14 0.18 0.17 0.17 0.18 0.16 0.15 0.16 Hardness 78.0 72.0 76.0 92.0 76.0 94.0 92.0 76.0 72.0 74.0 Alkalinity 81.3 74.5 75.4 85.1 73.1 79.2 80.4 66.8 61.8 71.0 DOC 14.7 14.6 14.2 TOP 0.047 0.052 0.035 TON 1.2 1.2 0.97

Water Quality Data collected by CBFO summer 1995, PWRC Experimental ponds Units: Temp (C), D.O. (mg/L), Cond (millimhos/cm), Hardness, Alk (mg/L CaC03), DOC (mg C/L), TDP (mg P/L), TDN (mg N/L) DOC TDP and TDN readings taken on 5-24-95, 6-13-95 7-6-95

A2 Water Quality Summary Data for Abate Sites

5-15-95 5-22-95 5-30-95 6-6-95 6-13-95 6-21-95 6-28-95 7-5-95 7-12-95 7-19-95 POND TRT PARAM WK1 WK2 WK3 WK4 WK5 WK6 WK7 WKB WK9 WK10 2 abate TEMP 27.1 21.6 24.0 21.5 20.2 26.2 21.6 23.5 24.6 25.9 0.0. 6.8 3.4 8.9 3.1 2.4 7.3 1.5 3.9 4.4 2.0 pH 7.2 6.9 7.4 6.9 6.7 6.9 6.8 6.7 6.7 6.6 Cond 0.15 0.15 0.18 0.15 0.16 0.16 0.14 0.13 0.15 Hardness 76.0 64.0 76.0 88.0 88.0 72.0 72.0 64.0 68.0 Alkalinity 78.6 68.1 68.7 74.4 77.2 70.7 56.9 58.2 64.8 DOC 19.8 15.8 15.9 TOP 0.083 0.18 0.046 TON 1.4 1.2 1.1 12 abate TEMP 25.0 24.3 23.4 22.1 20.6 27.5 21.7 24.2 25.5 27.0 D.O. 4.4 3.4 7.1 0.91 1.1 2.3 0.61 1.9 2.1 0.99 pH 7.1 7.0 7.3 6.8 6.7 6.8 6.8 6.7 6.7 6.6 Cond 0.20 0.18 0.24 0.20 0.22 0.21 0.19 0.18 0.19 Hardness 80.0 84.0 80.0 100.0 92.0 92.0 92.0 80.0 80.0 88.0 Alkalinity 77.5 91.9 96.9 102.0 85.8 101.8 97.0 62.2 73.5 86.7 DOC 17.3 16.8 15.4 TOP 0.031 0.06 0.048 TON 1.19 1.31 1.16 19 abate TEMP 26.6 24.31 24.19 22.71 21.88 26.84 22.04 24.7 26.64 28 D.O. 6.66 5.94 7.55 4.51 3.53 3.5 3.62 5.71 7 6.57 pH 7.29 7.12 7.32 6.93 6.84 6.89 6.87 6.89 6.87 6.8 Cond 0.171 0.156 0.203 0.174 0.19 0.188 0.155 0.151 0.164 Hardness 84 80 80 104 84 88 84 76 72 72 Alkalinity 95.6 82.5 84.5 93.2 79.8 87.4 90.8 76.6 66.9 77.3 DOC 14.2 13.1 12.6 TOP 0.04 0.025 0.035 TON 1.19 0.9 0.97 20 abate TEMP 26.53 25.98 24.28 22.34 20.76 28.15 21.76 24.26 26.07 27.01 0.0. 5.55 3.39 5.17 4.61 4.32 10.16 3.56 8.31 9.6 5.59 pH 7.11 6.97 7.08 6.92 6.85 7.13 6.92 6.97 6.86 6.6 Cond 0.166 0.147 0.195 0.172 0.186 0.186 0.156 0.156 0.184 Hardness 76 76 72 100 52 84 100 76 72 76 Alkalinity 86.3 77.8 81.1 91.8 80.5 93.4 89.2 72.9 68.3 76.8 DOC 16.1 13.9 11 TOP 0.044 0.034 0.024 ------TON 1.25 0.97 0.91 ----23 ------abate ... TEMP 25.4 23.66 24.34 22.75 20.98 26.46 21.75 24.8 27.16 26.95 D.O. 5.26 6.05 6.01 3.6 4.17 5.59 2.94 5.81 12.18 6.33 pH 6.95 6.97 7.1 6.94 6.88 7.01 6.9 6.85 7.71 6.95 Cond 0.147 0.136 0.177 0.15 0.164 0.162 0.14 0.129 0.147 Hardness 72 76 80 96 68 96 92 72 68 68

A3 Water Quality Summary Data for Abate Sites

5-15-95 5-22-95 5-30-95 6-6-95 6-13-95 6-21-95 6-28-95 7-5-95 7-12-95 7-19-95 POND TRT PARAM WK1 WK2 WK3 WK4 WK5 WK& WK7 WK8 WK9 WK10 Alkalinity 71.9 67.9 75.6 120.8 70.5 78.8 76.9 66.7 58.9 63.6 DOC 16.5 15.3 14.2 TOP 0.055 1.052 0.114 TON 1.09 1.59 1.06 24 abate TEMP 25.16 23.48 24.37 22.29 21.13 27.04 21.6 24.44 25.32 27.79 D.O. 1.98 5.37 7.29 2.44 1.73 3.93 0.94 3.02 0.189 2.03 pH 6.93 7.02 7.27 6.9 6.82 6.91 6.95 6.9 6.82 6.82 Cond 0.195 0.181 0.237 0.2 0.225 0.227 0.198 0.189 0.213 Hardness 116 92 100 112 80 124 120 92 92 112 88 92 mean Hd 98.7 Alkalinity 98 107.5 114.8 90.5 107.3 112.6 94.7 92 104.8 DOC 20.4 17.2 16.6 TOP 0.193 0.132 0.054 -- TON 1.62 1.39 1.09 Range TEMP 25.0-27.1 21.6-26.0 23.4-24.4 21.5-22.8 20.0-21.9 26.2-28.2 21.6-22.0 23.5-24.8 24.6-27.2 25.9-28.0 D.O. 2.0-6.8 3.4-6.1 5.2-8.9 0.91-4.6 1.1-4.3 2.3-10.2 0.61-3.6 1.9-8.3 0.19-12.2 1.0-6.6 pH 6.9-7.3 6.9-7.1 7.1-7.4 6.8-6.9 6.7-6.9 6.8-7.1 6.8-7.0 6.7-7.0 6.7-7.7 6.6-7.0 Cond 0.15-0.20 0.14-0.18 0.18-0.24 0.15-0.20 0.16-0.23 0.16-0.23 0.14-0.20 0.13-0.19 0.15-0.21 Hardness 72.0-116.0 64.0-92.0 72.0-100.0 88.0-112.0 52.0-92.0 84.0-124.0 72.0-120.0 72.0-92.0 64.0-92.0 68.0-112.0 Alkalinity 71.9-95.6 67.9-98.0 68.7-107.5 74.4-120.8 70.5-90.5 77.2-107.3 70.7-112.6 56.9-94.7 58.2-92.0 63.6-104.8 DOC 14.2-20.4 13.1-17.2 11.0-16.6 TOP 0.031-0.19 0.025-1.05 0.024-0.11 TON 1.1-1.6 0.90-1.6 0.91-1.2

Median TEMP 26.0 24.0 24.2 22.3 20.9 26.9 21.7 24.4 25.8 27.0 0.0. 5.4 4.4 7.2 3.4 3.0 4.8 2.2 4.8 5.7 3.8 pH 7.1 7.0 7.3 6.9 6.8 6.9 6.9 6.9 6.8 6.7 Cond 0.17 0.15 0.20 0.17 0.19 0.19 0.16 0.15 0.17 Hardness 82.0 78.0 80.0 100.0 80.0 90.0 92.0 76.0 72.0 74.0 Alkalinity 78.6 80.2 82.8 97.6 80.5 90.4 90.0 69.8 67.6 77.1 DOC 16.9 15.6 14.8 TOP 0.050 0.096 0.047 TON 1.2 1.3 1.1 ------·--·· Water Quality Data collected by CBFO summer 1995, PWRC Experimental ponds Units: Temp (C), D.O. (mg/L), Cond (millimhos/cm), Hardness, Alk (mg/L CaC03), DOC (mg C/L), TOP (mg P/L), TON mg N/L) DOC TOP and TON readings taken on 5-24-95 6-13-95 7-6-95

A4 Water Quality Summary Data for Altosid Sites

5-15-95 5-22-95 5-30-95 6-6-95 6-13-95 6-21-95 6-28-95 7-5-95 7-12-95 7-19-95 POND TRT PARAM WK1 WK2 WK3 WK4 WK5 WK6 WK7 WKB WK9 WK1D 3 altosid TEMP 26.4 23.72 24.23 22.7 23.29 25.63 21.45 25.96 27.86 27.29 0.0. 5.43 4.93 9.26 5.26 8.06 3.4 3.05 2.3 5.91 3.72 pH 6.9 6.74 7.23 6.8 6.91 6.65 6.73 6.64 6.75 6.49 Cond 0.141 0.133 0.171 0.146 0.174 0.16 0.137 0.128 0.143 Hardness 72 60 68 80 72 76 76 68 52 64 Alkalinity 70.4 49.8 68.7 77.7 61.8 72.7 71.7 54.8 52.7 63.1 DOC 22.4 19.9 17.6 TOP 0.042 0.21 0.048 TON 1.45 1.6 1.1 6 altosid TEMP 25.96 21.33 23.77 21.57 20.32 27.21 21.7 23.83 25.16 26.22 D.O. 4.9 1.95 5.43 1.63 2.5 2.19 1.19 1.86 1.6 0.98 pH 7.05 6.83 7.05 6.76 6.72 6.77 6.79 6.71 6.66 6.62 Cond 0.181 0.165 0.213 0.182 0.196 0.192 0.163 0.162 0.176 Hardness 84 80 76 88 80 96 100 80 72 76 Alkalinity 67.3 82.7 90 94.6 82.9 90.1 92.6 76.8 72.1 82.7 DOC 13.8 13 11.3 TOP 0.025 0.046 0.023 TON 1.1 1.0 0.86 15 altosid TEMP 26.2 23.9 24.6 23.0 21.9 26.4 21.9 25.8 30.1 30.2 0.0. 7.1 6.9 8.1 7.0 6.0 8.6 2.2 8.8 11.1 10.4 pH 7.1 7.0 7.4 7.0 6.9 7.1 6.7 7.2 8.0 7.3 Cond 0.16 0.14 0.19 0.17 0.17 0.20 0.14 0.14 0.16 Hardness 76.0 68.0 84.0 92.0 76.0 96.0 92.0 60.0 72.0 68.0 Alkalinity 96.1 74.1 83.9 91.2 73.5 78.5 85.5 66.1 56.7 70.7 DOC 16.1 14.6 12.7 TOP 0.034 0.03 0.028 TON 1.1 1.1 0.93 16 altosid TEMP 26.0 24.7 23.9 22.5 20.8 25.7 21.5 23.8 25.2 26.7 0.0. 6.2 4.9 8.6 4.1 0.90 1.0 0.26 2.2 2.8 1.4 pH 7.2 7.1 7.5 6.9 6.7 6.6 6.8 6.7 6.7 6.6 Cond 0.19 0.16 0.21 0.19 0.22 0.22 0.18 0.17 0.20 Hardness 80.0 76.0 80.0 100.0 76.0 100.0 >--·--· 96.0 76.0 76.0 80.0 Alkalinity 153.8 79.4 87.0 99.1 66.5 101.0 99.4 83.2 76.8 88.2 DOC 17.3 15.4 13.4 TOP 0.035 0.034 0.029 ---- "- TON 1.2 1.0 0.94 ------"- 17 altosid TEMP 25.0 26.5 24.1 22.9 21.8 27.2 21.8 24.2 27.3 29.4 0.0. 7.0 5.8 7.6 4.2 4.1 4.4 2.4 4.4 7.2 4.5 pH 7.2 7.3 7.4 6.9 6.9 7.0 6.8 6.8 7.0 6.8 Cond 0.19 0.16 0.23 0.19 0.21 0.21 0.16 0.16 0.19 Hardness 88.0 80.0 88.0 116.0 52.0 100.0 96.0 76.0 76.0 92.0

A5 e Water Quality Summary Data for Altosid Sites

5-15-95 5-22-95 5-30-95 6-6-95 6-13-95 6-21-95 6-28-95 7-5-95 7-12-95 7-19-95 POND TRT PARAM WK1 WK2 WK3 WK4 WK5 WK6 WK7 WKS WK9 WK10 Alkalinity 93.9 84.7 92.1 98.1 84.8 102.9 103.2 75.3 77.5 87.7 DOC 21.5 19.3 16.3 TOP 0.053 0.041 0.040 TON 1.5 1.3 1.1 21 altosid TEMP 27.6 23.9 24.5 22.6 20.7 25.2 21.6 23.5 26.9 26.0 0.0. 5.3 3.2 7.3 2.0 1.1 2.3 0.3 0.8 1.8 0.5 pH 7.0 6.9 7.3 6.8 6.7 6.6 6.7 6.5 6.7 6.5 Cond 0.17 0.15 0.20 0.16 0.18 0.19 0.17 0.15 0.18 Hardness 92.0 80.0 84.0 108.0 52.0 100.0 96.0 100.0 76.0 84.0 Alkalinity 85.5 76.2 72.5 92.5 79.3 93.0 90.5 75.4 69.1 73.8 DOC 19.2 16.2 17.4 TOP 0.082 0.037 0.050 TON 1.3 1.0 1.2

Range TEMP 25.0-27.6 21.3-26.5 23.8-24.6 21.6-23.0 20.3-23.3 25.2-27.2 21.5-21.9 23.5-26.0 25.2-30.1 26.0-30.2 0.0. 4.9-7.1 2.0-6.9 5.4-9.3 1.6-7.0 0.90-8.1 1.0-8.6 0.26-3.1 0.79-8.8 1.6-11.1 0.52-10.4 pH 6.9-7.2 6.7-7.3 7.1-7.5 6.8-7.0 6.7-6.9 6.6-7.1 6.7-6.8 6.5-7.2 6.7-8.0 6.5-7.3 Cond 0.14-0.19 0.13-0.17 0.17-0.23 0.15-0.19 0.17-0.22 0.16-0.22 0.14-0.18 0.13-0.17 0.14-0.20 Hardness 72.0-92.0 60.0-80.0 68.0-88.0 80.0-116.0 52.0-80.0 76.0-100.0 76.0-100.0 60.0-100.0 52.0-76.0 64.0-92.0 Alkalinity 67.3-153.8 49.8-84.7 68.7-92.1 77.7-99.1 61.8-84.8 72.7-102.9 71.7-103.2 54.8-83.2 52.7-77.5 63.1-88.2 DOC 13.8-22.4 13.0-19.9 11.3-17.6 TOP 0.025-0.082 0.030-0.21 0.023-0.050 TON 1.1-1.5 1.0-1.6 0.86-1.2

Median TEMP 26.1 23.9 24.1 22.7 21.3 26.1 21.7 24.0 27.1 27.0 0.0. 5.8 4.9 7.8 4.1 3.3 2.9 1.7 2.2 4.4 2.6 pH 7.1 7.0 7.3 6.8 6.8 6.7 6.8 6.7 6.7 6.6 Cond 0.18 0.16 0.20 0.17 0.19 0.20 0.16 0.16 0.18 Hardness 82.0 78.0 82.0 96.0 74.0 96.0 96.0 76.0 74.0 78.0 Alkalinity 89.7 77.8 85.5 93.6 76.4 91.6 91.6 75.4 70.6 78.3 DOC 18.3 15.8 14.9 TOP 0.039 0.039 0.035 TON 1.2 1.1 1.0

Water Quality Data collected by CBFO summer 1995, PWRC Experimental ponds Units: Temp (C), D.O. (mg/L), Cond (millimhos/cm), Hardness, Alk (mg/L CaC03), DOC (mg C/L), TOP (mg P/L), TON (mg NIL) DOC TOP and TON readinas taken on 5-24-95 6-13-95 7-6-95

A6 Appendix B

Taxonomic data for Chapter 6 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order Fomllv s-..1n1Genua Emeraenl lnseciS colec19d Ma 22, 199!5 !ram com"Ol oands. Mean per SOPER 1 1D1111 4 flltal 91otal 101otal 221olal TOTAL nand POND Di ...... Clironomidae Oiranomus SD.1 0 0 1 0 42 89 11.5 11.2 Oiranomus ID.2 0 0 0 0 0 7.3 11.0 - cclatcr 0 0 0 0 0 "0 0.0 o.o IP. 0 0 0 1 0 2 0.3 0.5 - ,-.. 0 1 0 0 0 4 0.7 1.2 -.nttun 1 1 0 0 3 I 1.3 u Pnc:hirononul rnonoclYcmJs 0 0 0 0 0 0 0.0 0.0 0*onaninl females 0 0 1 1 0 2 0.3 0.5 T-...... ,. 1 2 1 1 2 11 1.1 1.2 T-females 0 5 1 8 1 21 ,.1 5.1 - -~1111. 0 0 0 0 0 0 0.0 0.0 CriffltMuo SD.1 1 0 0 0 0 1 0.2 0A Nanodllclus ID. 0 0 0 0 0 0 0.0 0.0 Ortlodlhv

# lndlvlduols 146 58 62 74 !56 527 87.1 40.0 #a-I•• 8 10 11 13 91 221 3.7 1.1 #l1mllles 4 4 5 5 • 1.3 0.8 #ordera 2 2 2 2 '2 3 0.5 o., •

Bl Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order Famllv SDeclas/Ganus Emeraant lnsacts collactad Mov 22, 1115 from ...,nds snra ,.d with Altosld. Mnnnar SD PER 3101111 6101111 15101111 16 lotat 17- 21 total TOTAL '-·d POND lnint.. Clironomldae Clironorrus so. I e 19 5 33 0 6 0 13 10.5 13.0 0woncmJs m.2 1 0 308 0 13 0 Cla ...... 322 53.7 12A.7 c_ 0 0 0 0 0 0 0 0.0 0.0 Dlaotendl-- ..,_ 0 0 0 1 0 0 1 0.2 0.4 Mia~---·-- 2 0 0 0 0 0 2 u 0.1 P-•-t1un 1 5 2 0 1 0 I 1.5 1.1 Parac:tircnorrus monoclrorllJs 0 0 1 0 0 0 1 0.2 0.4 Chircncmlri lemeJes 0 1 0 0 0 1 2 0.3 0.5 T•-rsus on. 2 0 6 1 0 1 10 1.7 2.3 T•-...... ,. lemeles 3 1 7 5 3 1 20 - 13 2.3 ""· 1 0 0 0 0 0 1 0.2 0.4 Cli--'--,cn.1 0 0 0 0 0 0 0 0.0 0.0 Nenodllcius .,,_ 0 0 0 0 0 0 0 0.0 0.0 Ortloc:ledlnee lemeles 0 0 1 1 1 0 3 o.s 0.5 1~• ..,. 5 2 23 2 3 0 35 5.1 ... Lebnnirie sn. 0 0 0 0 0 0 0 0.0 0.0 Prodedus bels 4 0 11 6 29 3 53 ... 10.5 Prodadus lreemari er. so. 0 0 0 1 2 0 3 0.5 0.8 Psect""'--"""" 2 0 2 0 1 0 5 0.1 1.0 Total chlronomldaa 40 14 394 17 59 • 530 11.3 151.0 CUlddae ChaoboruS .... 1 39 8 71 9 37 14 171 21.7 24.5 ChaoboruS cn.2 0 1 0 0 1 1 3 0.5 0.5 Total Chaoborus 31 I 71 I 38 15 181 30.2 2A.2 CUex 0 0 0 0 0 0 0 0.0 0.0 eera•-dae Cera"""""""dae 0 1 0 1 1 4 7 1.2 1.5 lc"'-""'dae 1 0 0 1 0 2 4 0.7 0.1 Ool ...... da. Oolc:tl0POdldae 0 0 0 0 1 0 1 0.2 0.4 -·S1nllt'lffllllidlle S1nll""""'da• 0 0 0 0 0 1 1 0.2 0.4 ITlouldlle Tonuidae 0 0 1 0 0 0 1 0.2 0.4 '"""""de• 1-dll• 0 0 0 0 0 0 0 0.0 0.0 • Ced-.idlle 0 0 0 0 0 0 0 0.0 0.0 Sed...... ,..e • 0 0 0 0 0 0 0 0.0 0.0 e • 1-.... 0 0 0 0 0 0 0 0.0 0.0 - Tllbaridae Tabaridee 0 0 0 0 0 0 0 0.0 0.0 Sdnm"71dae 1-c1a. 0 0 0 0 0 0 0 0.0 0.0 Total Dlntaro 80 24 488 21 ,. .. 28 725 120.1 172.0 Bae11dlle Celbaetls 0 0 0 0 0 0 0 0.0 0.0 Ceeridae Ceeris 0 0 0 0 0 0 0 o.o 0.0 Total Enhamarontara 0 0 0 0 0 0 0 0.0 0.0 HeniDIMI Selddae Salddae 0 0 0 0 0 0 0 0.0 0.0 Genldae Genis 0 0 0 0 0 0 0 0.0 0.0 Total Hemlntaro 0 0 0 0 0 0 0 0.0 0.0 Odonlta Lestdae Lesldae 0 1 0 0 0 0 1 0.2 OA ~dlle LibeUdae 0 0 011 0 0 0 0.0 0.0 ,...... ,,..oridlle ,,..___.oridae 0 0 0 0 0 0 0 0.0 0.0 Total Odonata 0 1 0 0 0 0 1 0.2 0.4 da. 1...... _.ldae r--- 1...... 0 0 0 0 0 0 0 0.0 0.0 Leotocendlle L...,...erldae 0 1 0 0 0 0 1 0.2 0.4 Total Trlchontora 0 1 0 0 0 0 1 0.2 0.4 Colentlola Colern>ola 0 16 0 6 0 1 23 u u

# Individuals 80 42 466 34 99 29 750 125.0 119.3 #s--les 12 11 12 11 13 10 25 11.5 1.0 #famllln 3 6 3 5 4 6 10 4.5 u #orders 1 4 1 2 1 2 4 1.1 1.2 •

B2 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order Family SnaclfflGenus Emernent In acts collected Mn 22. 1195 from """"• •--' with Abete. Ileen-• SOPER 2llllal 12lolal 19111111 20111111 23111111 24tolal TOTAL '-d PONO IDiDtere ClironorndH OircnorlllS SD.1 8 34 0 83 5 10 1'° 213 31.5 OircnorlllS SD.2 0 1 0 0 0 0 1 G.2 O.A Cll-colalar 0 0 0 0 0 1 1 G.2 O.A DI-SD. 0 0 0 0 0 0 0 0.0 0.0 ,-.. 0 0 0 0 0 0 0 0.0 0.0 '1Un 0 2 0 0 0 0 2 0.3 0.1 Panlctnnorru mcnoc:hronus 0 0 0 0 0 0 0 0.0 0.0 0nncniri females 0 0 0 0 0 0 0, 0.0 0.0 T - 1 1 2 1 0 1 • 1.0 o.t T.-females 0 2 3 2 2 0 • 1.5 1.2 ISD. 0 0 0 0 0 0 0 0.0 0.0 l~so.1 0 0 0 0 0 0 0 0.0 0.0 NllnodldusSD. 0 0 0 0 0 1 1 o.2 O.A Clrtlac:lldlrae f- 0 1 0 0 0 0 1 o.2 0.4 Al>la-...aSD. 0 0 2 0 0 0 2 G.3 0.1 Labnnlna SD. 0 0 0 0 0 0 0 0.0 0.0 Proc:la

fl lndlvlduals 22 52 81 124 35 47 381 I0.2 37.0 11.-1.. 9 8 7 10· 8 11 23 1.5' 1.1 #famlllN 6 3 .. 6 .. 7 12 I.O 1.5 #ordars 2 2 3 .. 1 2 5 u 1.0

B3 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

I Order Famllv Soeclea/Genua f Emement Insects cOllected June I, 1191 from control DOnda. i - I __..,_I _j______- i I i iMean_,. iSDPBt 1.. total 11 total total i5 9 total i10 total :22 total :TOTAL 1DOnd 1POND e OiDtera ! Chironomidae Chironomus SP.1 1 ol 0: 3 0 0 oi 3' a.so· __ J,~ I --- I Cryptochironomus sp. 1 ol o: o, O: Oi O' ·- oj o.ooj 0.00 ! Oicrotendioes sp. ! 01 0- 2 0 4 0 -- 1.00 U7 ! iKlefferufus dux I o1 o: O' o· o' • ------0 0 0.00 0.00 I -- Miaotenclioes oedellus 01 0 0 O: o, 0 0, 0.00 0.00 I Pol•"""ilum c:J. lalcifonne I ol Di Oi 0' oi Oi ol o.ooi 0.00 I ! PnlvnRriilum tnnonum 4! 4' 4 0' 13' 1 ---- -~ --- 1 2.17 2.04 I --- -- QI I Pol•NKfilum tritum I 21 1i 0' 01 01 31 a.so, 0.14 I I o: Parachironomus monochromus] ol OI 0 o\ ol ol o.ool 0.00 I i Chironominl females I 1i 1! 4 1' 0 7 1.17 ------0 ------· 1.47 Tanvtarsus sp_ 2i o: 1 Oi o, o, 3i 0.501 0.14 Tanvtarsini females I 31 1i 31 ol 1! ol - 1.»1 1.37 0, o: •' Cricotoous sp.1 i 21 o: OI oi 21 0.331 G.12 Psectrocledius SD. ol o' 0 O! O· 1! 1; 0.17, 0.41 ·r Orthoclacfinaa females I ol 0: o: 0 0 ol 0 0.00 1 0.00 Ablabesymja sp. 1 15f 71 5i 21 1! o: I --- 301 1.001 1.51 Clinotanvnus oinauis i o, 0, 1i O: O: oi 1' 0.17 0.41 11 O! I i 'Labrundinla sp_ I 2' 0' o' ____o~ -- 3 -- 0.10_ 0.14 I Larsia so. 01 0' 0' 0, 0 0: 0 0.00 0.00 ! Prodadius bellus i 251 1/ 6! oi 41 3j 39I ,.soi t.31 : ! Prodadius lreemani sp_ 1T QI 2 O' 0' ____Q~_ 3 1 -- 0.5o 0.14 I ------' Psectrotanvcus dyari I ol o! o: o! O' 01 01 o.ooi 0,00 I Total Chlronomldae i sai 14' 34, 2! 11 1 5j 1221 20.33[ 20.n I I I el !Culicidae Chaoborus sp.1 12' 13 ------2 ----- 15 - ______15_ 15 10.83 5.04 i Chaoborus sp,2 I oi 11 01 o, 3 1: 5! 0.831 1.17 ! Total Chaoborus I al 13! 13: 2i 11' 1,: 70 1 11.87 1 __ 5.~2 I o; I Culax I ol o, ol o, o: ol o.ool 0.00 ! JCeralDPO!IOnidaa Caratnnnnnnidae oi 01 0' o' O, ol o; o.ooi 0.00 Eohvdrklae E"""dridae ! 1! o: o: 0 o. 1, 2' 0.33: 0.52 !Doll- - Dollmooadidaa 1 ol o' o! 1· 01 ol 11 o.~r! __ 0.41 !StratiomuildaA Stratiomyiidae i ol 0: oi 1' o' 01 1: ---co.11, 0;41 !TIIXJldae TiDUlidaa tvoe #1 i o' o: o: 0: o' o: ___o_· __o.:...oo; 0.00 I I Tipulidae tvoe #2 I o! 0 o; o, o: Oi 0 o.oo 0.00 ! i Tipulidaa tvoe #3 I oi 0, o: oi o' 1: 11 o.11i 0.41 l5vmhidae i5yrphidae I 0 0 0 0 0----~------1 0.17 0.41 1Secidonwiidae ISeadom,iidae I o: o' o: 0! O' DI 01 o.ooi 0.00 :Psvchodidae Psvchocfidae o! o; o, o, 0, oi O] o.ool 0.00 I --- ITabanidae iTabanidae 0, 0 0 0 0 0 0.00 . ··------. -- ____ L.__ 0.00 I !Sci<>mv7idaa iSciomYZidae 01 0 O' o: QI 01 01 o.ool 0.00 I ! I TotalDlpters Hi 'ZT' 47 1 6 29 24' m' 33.00' 20,~ 1 Callibaatis I o' 8' E Baatidaa o: 0 0 o: •' 1.331 _3~~ !Caenldae 1Caenis i Oi 0 0 0 0, o, 0 0.00 0.00 I i ITotal Ephemempters ol • 0 0 O' 0, • 1.33 3.27 H""'""""' /Salcfidae 1Saldidae i ol 0 o· o, 0 01 ol o.oo~ 0.00 : -- - IGemc1aa Ganis o! o: 0 0 11 1' 2: 0.33i 0.52 Total Hemlptera I oi 0, o' 0 1 1' 2 0.33 0.52 ; -- Odona1a Lellidae ILestidae oi 2 o· 0 Q, o, 2, 0.33• 0.12 ILibellulidae Libellulidae i oi 01 Qi o! 31 o! 31 o.sol 1.22 : Ieo.nm1on1dae Coanarionldaa o: 0 1 0 0 0 - 1 0.17 0.41 Total Oclonata ol 2' 1' 0' 31 ., 1.001 i O: 1.21 T- H..-ldaa IHvdnllllllldaa I ol o: Q: 1: o: 0 ______11 0.171 _---- 0.41 : o: 1 ___ o_ iL iL-"""ridae 0 -- 0 ---- 0 ---- - 1 0,17 0.41 i Total Trtch-ara I oi 01 1' 1, O! Oi 21 0.331 0.52 Collembola I Collembola i al 2! o' 7 6 3' 21' 4.33 1 3.14 I ' [ : I I i I # lndlYldu1ls I 73i 39' 49, 14 39 2Bi 242, 40.33' 19.H ! ,._,, ... : 131 10· 15' 8 10 10 29' 10.17· 3.0I I 5! 1 i lfamllle1 ..1 4' el 5' 7' 11 5.171 1,!? • #orders I 2i 41 31 3- 4i 3, II 3.17 1 0.75 B4 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order family Snac:la/Genus ; Emergent Insects collected June I, 1195 from - • ..,..,... with Alloslcl.

I : I i iMeanper iSOPER 3 total 16 t0lal : 15 t0lal : 16 total ___[_1_7101111_ '.21 total TOTAL -~- ·PC)NO e Ointana Chironomidae Chironomus SD.1 O! 0 2 O, o. 0 2 D.33 0.12 i SI). o: o: o: 0 0 0 -- D D.DOI D.DO Oiaalencioes IP- O• -·-·------0 - 0-- O' 0 0 D D.DO D.00 0, iKietlaruus dwc 0 0, 0 0 0 --- D D.DO, D.DO M;,,,,,,.....-1.-ilus ol 01 o: ol 0 __Qi_ D: 0.00: 0.00 ,_ -- rJ. fak:iforme f o· o: 01 0: 01 1, 0.17 D.41 Pnlvnwilum trinnnum o: 1 i o: 31 Oi 2i ., 1.00, 1.21 ,_ bilum oi 01 1' ot O' o: 1 0.11! 0.41 Panlc:hilonomus rnonochr0mul: o: 01 O, oi ol 01 0' o.ooi 0.00 Chironomini females 1 ! Oi 1' oi o: o: 2, 0.33 D.52 I T-ID- oi 1 i O· oi 01 oi -- - 1' 0.111 - __o.~ Tanvlanin females ol 1: 1, 1 ', Oi o, 3: 0.50 0.55 Cric:olDl>us IP. 1 I 01 oi 1: ol OI o1 1 I 0.171 'D.41 Psedrodadius SI). ol oi o, ol o' 1 1' 0.17' 0.41 - - - 0l1hodacinlle femaleS 01 01 0 01 0 o, O• 0.001 0.00 . ID. Oi o' O' o! oi O' o! o.ool 0.00 0 o· ___ 0.00 __ ClinotaMllls nmnuis 0 --- -- 0 ------0 0 0 0.00 I -- Labrundlnia SD. 01 01 0, 0 0, 0 0 0.001 0.00 I Larsiaso. o: ol oi 1: 01 0' 1 I D.171 D.41 ---- ·- Prodadius betlus 3! 01 2' O! 4i 1, 10: 1.171 1.13 i Prodadius freemani IP- o! 1: 0' ol 01 o: 1' D.17' D.41 : p s dyari 01 o: o: QI o: oi 0 o.ool D.00 Total Cl*onomklae &I 41 Ii 11 4i 4, 301 I.DOI 1.51 ieui;ac1ae ChaobonJSsp.1 14 14' 1 31 i 17' 11 1 1.50, 7.23 ChaobonJS SD.2 O! ol o, o! o' 1 1' D.17 1 D.~ Total Cllllobonls 141 141 1' 3i 2, 11 12: 1.17 7.47 Culex 0' 0 O' oi ol O! oi o.ool D.DO Ceratnnnnonldae Cera ol 1 0 _____ ._r 0 0 2 0.33' 0.52 EPhvdridae Ellhvdridae 11 0' o• 01 o, 0 1 D.17 D.41 Dolichorx>didae 1 I 0 0, Di 0 __ o~ 1 ur~I---- D.41 0 0 1- 0 0 1 D.41 Stratiornvlidae s ---· _p~ -~;17= __ -- - Tioulidae y- #1 o, - - 01 o, o• O: 0: o; 0.001 0.00 TIDUlldae"""' #2 oi oi 1 i oi oi o' 1 i 0.17 1 0.41 Y-ooulidae """' #3 o: O' O! o: o: DI o: D.DO' D.00 ,Sm>hldae Svmhidae 01 oi O! o: o: 0 o: D.DO' 0.00 Secidomyildae " Oi 0: 0 01 o: o: o' D.DO D.DO ,_ Di oi O' o' o, o! oi o.ool Psvchodidae ---·-D.DO Tabanidae Tabanidae o: ol o' oi 0 0 o' 0.001 D.DO ,_' Sciom-. o: o' o: o: o' 0 _ ____ o· _ o.oo· __ 0.00 Total 111ntan 21· 19 10 10 I' 22 .. 14.17 . I.ID E ,a Baetidae Callibaetis 1, o: 1, oi o: o: 21 D.331 0.12 Caenidae Caenis o' o' 0 o· 0 0 D D.DO D.00 ------Total S:nhamernnten 1 i D' 1' DI 0, D 2. 0.331 0.52 O· 0 0 o· 0 Saldidae Saldidae 0 0 o.oo' ------0.00 0 0 0 1 1 1 Gerridae iGerris ·------3 --··------0.50 o,~ Total Hemlpten, o: o, D 1 1 1 3 D.50' O.IS I 1 ! 0 O' O' 1 Odonala Lestidae Lestidae 1 • 0 2 0.33 ·-0.52 Libellulldae Libelluidae O' o1 0 1; o: O' 1: 0.17·! 0.41 ,_ Coennrionidae O' Oi o: oi 1. o, 1 D.17' 0.,1 Total Odonata 1 i 1 i o, 1' 1' O· 4 D.17, 0.52 TrlchoDlera Hvdrootilidae o' ol o: O' Oi Q! o' o.oo: --D.DO Leotoceridae L_,,.,__ 0 ol o: o' o: 0 o, 0.00, 0.00 o: o· 0 D o.oo' D.00 Total Trtc:t,optera o: o' o' - - -- ·- Collembola Collembola 3/ 41 7; 14 11 2 41 1.13 4.79 i i : I l I I Individuals 26 24' 18 26 19 25 131 23.00 3.51 -- ·-· - -- . - 9· a! 10: 9' 5 7 21 a.ooi 1.71 I ··-·lfamllles 7 5' 5 7: 5, 4 ___1~---- 1.50' 1.22 I #orders 4 3 3 4 • 3 5 3.50 0.55

BS Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

FamllY 5...,.1e11Genu1 )Emeraent lnaec:11 collected J-I, 1HI from DOncl1 , ..... ued wllh Abate. I I i 1 ! i !Mean_. ,SDPER 1-----+------+------+=-='----c'-=-==---'-'-'--'-'=-----"c.c...c='-----"=-==----J.::..:...:=----J.:-=--.:='---+c:=-----'--!=~---i2total i12tota1 i19total :20total ,23total )24total iTOTAL ;,_ !POND FOi"'ipc::'tera==------+'ecChi:.::;'rcnorrn==·dae==---+Chi=·ro,='°''-"mu=s=-:s=-=-p·1___ ~i----'-Ot--i ---~ot-' ___ _::o.:... 1___ _;1:..c· ____:0=+1j ____O:c,l _____,2,.______:0c,_.1!l______o,~1 1,.._-ronomus sp. ol oi o, O: 01 1 i 1 0.111 o.41 OiaotandiPH IP. 01 01 OI 0' 0: ol O 0.00 0.00 1-----+------+'-"Kiefferul==u•=-d=wc:;______;:_o,_I ----"-ot-,'-----=o-'--, ___ ....,oc.;_, ____;o::;_, ______!j1 I ______1'-'-- ____;:;o.'-'-11'-'1 ___--=o:::.4.:.j1 1-----+------+=Miaolendi==~~-pe=,s~:Dl!dal=='=u•=------~ot-'---~ot-: -----=o+i ___ ....,o=------=o"'-, ____ou ____=-01--_--'o~.oo"'-_----=o.oo 1 IPolvPediium ct. falciforme Oi o, o, O, Oi ol oi 0.001 o.oo l------l------l-lPolyped==='um,,_,..,_,tr1,.,.,1aonum="'-"-----''------=-ol'------=-0_1 ----=ol~__ ___,o'-'-, _____o_, ___ --~1 -----=-o!'--_ -~.oo: _ o.oo

i l--ium trilum I 01 0' 0 0 0 01 0 0.00. 0.00 Parachironomus monochromus ol oi 01 o 01 oi oi o.ool o.oo

1 I------L-----1-Chironami=·==·n1'-'1ema1==•="-s______: ___--=-o'--i ----=-o,_i ----=o_, ____ o=-· _____o=-'------~- __ ___ o_'-~--- o_oo 1TanvtarsuSSP 0, 0 0 0 1 0 1 · 0.171 0.41 l------+------I-T!!Tanvtani==·n1=--1=ema=l=es,.____ --Jl ___---=..oi'-- __---=..o!--l __-=o+-I ____,o,._,' _____,D::+------'o::.i1l ____:o.,__ _ _.:O::,.oo,,,1c..__l _ ____c!o.~ 1,...,...,._,, SP. 1 I oi ol D' Di D Di Oi 0.00, o.oo 1------+------+'Paedrodc==::•==d:::iu=-••::i:•P::._------r----.:;.,0!1-----'-ol!------"o... 1 -----=o"-i----'o+------'o::.i'-----=-o+- _ _.:o:::.oo:=.-_._--=~·~ Clnhodadinaatemales I Di ol ol 01 o' ol ol o.oo' o.oo 1------I------J.:iAbl=abes=,=vmi.,a=,:.;;10"".="------+1----0"",l ____ o"",:----=-o..-, 1 -----=o.... , ----'o+,i-----'D=+,1- ---;----'o=.oo:.=._i_-- o.oo

!cnnnhlnvnl.ls ninnuis ' 0 1 oi oi o. o· oi oi o.oo, o.oo 1------l------+'Labrund==::.:'"'=·•.=i•Pr:c-____-+1 ____0=-l ____o"",l ___---=..01 ___ -=o.:...1 -----=0+1 ___ _o!_ _ __o,'-'----""o.~oo~i____ _o.~ 1------1------+'L,,,a,_,,rs,=··=IP------~-----'o=-+1, ____ o"",l ___---=-0' ___ _;:;o-c- __---=o_,_. ___...::O:.,.' ____o=i''----'o=.oo:.=.,.... ___,o.oo Prodadiusbellus 2, 4i 191 58' 201 121 111 11.111 20.42 1-----...l.------+'-'Prod=adi=·=us::..:treema=="'::.:·s::.,:1P.:_------,·'----=-o','-----'-o1_, ----=-o"'-1------=o"-: ____ _co_.'.__ . _____oj ___ o! ____ o.ooi o.oo I IPsedl"Otanvous dyar1 11 o: 0' 2, 01 01 31 o.ao, 0.14

TotalChlronomldae : 3! 4 1 19 1 11 1 211 1cl 123 20.ul 21.21 /Culicidae Chaoborussp.1 0' 01 0' o o: 1 ____:_1 ___0=-.1.:..:7 ___ ..:0.41 Chaoborus ID.2 0 O: 0 0 01 01 01 0.001 0.00 1------1------1-T!!ota~IC:,,hc:•:=obo=rua='-'------'!----o=-,i____ o.:;.,1 ___--=-o1..-1 __--=o"-: -----=o:.+---___1!.J.l ______!,1J...I __...::0:,.:.1,.,_7.:_! ___ -~'~1 euiex I o! 01 o, 01 o 01 01 o.oo· o.oo CeratoDaaonidaa 1- ·dae 01 o1 o, 0 1 o1 o' o 1 o.oo o.oo

'-----...LSEr>hvdri=~·=,daa!!..., _ _j_E!=j:Dhvdri=~·daa= ______0::.,1 ___--::,2:'-- __--=-o ____,Oc...' ____,o,._, ___..,!0~'--- ____;2:_ 1__ --=0·=3;:.,3 __ -°~~ idae ! oi o! o: o: 01 01 ol 0.001 o.oo Stratiomylldaa Stratiomviidae : ol o! ol o; - o: -11: oi o.oo: o.oo TiDUlldaa Ot 0.00 i 0.00 ------·--- ·.:....:. r.. ,.._tvo.o#2 oi o: o: o' o: o o: o.oo 0.00 Tiouidaa tvo.o #3 01 oi oi Oi 01 oi o o.ool o.oo 1-----...J!:'.,Svmhi===·c1aa==-__.,..s,,.;vn>hid==· •=------o"-'---~o _____ ..:.o_' ____ o _ o! _____ o __ OI ______0.00 0.00 ISecidomyiidaa iSecidDml'iidae : 01 O O O: 0, OI Oi 0.001 0.00 PSYchodidaa Ps....+uv

iTotalDlptera 3f &i 11 1 11 221 1Si _-.::121'°'-'-•1 __-=-21==.oo~' __ _ 20.n

,. Baatldae JCa11ibaatis O Oi O: 0 01 Oi 01 0.00 1 0.00 Caenidaa !Caeris o: o, o, o o. 0 1 o: o.oo o.oo 1-----+'-----+'T~ota~IE~,D~lh•==-m~•~~~•Dt~•~ra=------'o=----'o~,____ o=-----"-o ___ _;:;o ____;o,_· _ __o:_ __ __:Oc:.oo:::.______~ J!.Hemi=~;n1=-~--lcs!:!!a::.:ldidae==·=----+'Sal=di=daa='-'------'o'-'-,----'o=------o=-,------=-o------"o_,___ ..:0"-1 _____,o:i.l __...-=,o·=oo:.;.1 __ __ o.~ Genidae Genis 2' O: 0 0 0 0 2 0.33 0.12

1 1------1------+T,_,,ota=:IHe=m"',1p1::•=-ra=-----'----'2=-i ____ oc.'----o=-·------=-o ___ ----"'0______'!___ ---=2'--'____ ....,o::.:.3=3_' ____ _o.~ 0dDnala Lastidae ILestidaa I o, 0 1. o, Oi O 1 0.17 0.41 ube11u11c1aa Libeilulidae , o' o i o I o i o o i o o.oo i o.oo J_____ ../QCoe!!!'!!!i!"""""'~·daa~_.j!~-!!i!!~!:!!..------__.:o~i ___ _:o~!---_.!!.o:._1 ___ _!!o-c-----~---· o _ ___ !,! _ _--~~~ _ o.oo Total Odonate , o' 01 11 o: 01 01 11 0.111 0.41 l-!T=-=:!:-~-...:.H~iYt:'d=l'DDli~·11e1~ae~--l!IH¥=droDll=:::::lidae=. ______...;o::.i.l ____;o::.i.1l ____0=------=-o,._ __ --=o+- ____;o:..>i ___-=-o,___--'o"'.oo'-"-· ___;o.~ 1-----...:.L~LeDl=DCerl=daa=--+'L=-==..=------'o'-·----'O'--. ____ O=-'---·-----=-o----·-- o' -- ·--··· _ ~-- --· 0 o.oo o.oo iTotalTrlchopt1ra 0' 01 o: Oi 01 o, OI o.ooi 0.00

~CDl~le!!mbol~!!•--l------+CDl=lal~m!!!lbol=•-----~!____ 6::..~----=-,1 i___ -=.5,,_' ----'1 ___ --=2:+-d ____:0~'----'1c::.1t- 1, __..:2.::.:IOc::'"."' 1: __ -~ ._____ ...J_ ____--l.1 ______.' _____;' ____1____ i- ______.._' ___-+!----i-----~'---·- #lndlvldual1 i 11: 71 25: 12' 24! 151 1441 24.00 19.12 1------l------+-----=•:.!•:=-:::le::1__;_ __..; ____.::.,4: ___ -..:..3' ___-=.3,._' ____;4 ____ 4".-----"-'----·--'1"'2-_...... :;3.'-'-17'-----~ 1-----...l------1----=•..::'•""m""11=1e1::____ 1___ ...;3::..'---'""'3""1 ____ 3=-'----=-2:----~3-i ___ _;2c_' ______;:1_...l ___,,,2.:::.11L o.~2 lord1ra 31 2: 3, 2 2• 1 4 2.17 0.75

B6 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order Famllv S....,ln/Genus li:.....--Colecled.,..,.13 1995fnlmcont'ol""""". Mean- SOPER 11otal 4- 5 total 9- 10- 22 total TOTAL ,-nd POND lnint,,n, !CNrononidae CNran0n'lls SD.1 0 0 0 0 0 0 0 0.00 0.00 ! ,SD. 0 0 0 0 0 0 0 0.00 0.00 I l"""""""'•lnctials 0 0 0 0 1 0 1 0.17 0.41 ,_v_·-· INIMISUS or. SD. 0 0 0 1 0 0 1 0.17 0.41 Dlcrotendoes cf. t11Dnu 0 0 0 0 1 0 1 G.17 0.41 El idoctil 01 ICITlJS ninr1cans 0 0 0 0 3 0 3 0.!10 1.22 l(Jeffen,u,u 0 0 0 1 0 0 1 0.17 0.41 ,__,,. 0 0 0 0 0 0 0 0.00 0.00 ,...... ,lrn cf. fllldfarme 2 0 1 0 0 0 3 0.!10 0.14 1...... ,...,_,,_ 3 0 6 0 2 0 11 1.13 ,_ 2.40 m.m 0 2 0 1 1 0 4 U7 0.12 Panlc:tnnorrllS monodYOnls 0 0 0 0 0 0 0 0.00 0.00 ParactircnorrlJS ...... 0 0 0 0 0 1 1 0,17 0.41 PseudOctironOn' SD. 1 0 1 0 0 0 2 0.33 0.52 Oironcniri ferMles 2 1 0 0 2 0 5 0.13 0.11 T...... ,..,..,_ 1 0 1 0 0 0 2 o.33 0.52 T•...... ,.ferMles 9 0 1 0 2 0 12 2.00 152 lr,,,.,,,.,.,... so.1 2 0 0 0 0 0 2 o.33 0.12 f,..,.,.,,.,,....,.SD.2 1 0 0 0 0 0 1 0.17 0.41 PsecnldadlUS SD. 0 0 1 0 0 0 1 0.17 0.41 Srriflla lfamalel 0 0 0 0 0 0 0 0.00 0.00 OrtlOCladnae females 0 0 0 0 0 0 0 0.00 0.00 l&--....aso. 36 5 7 7 3 0 51 t.17 1117 0 0 0 0 0 0 0 0.00 0.00 Ir. """"'""'a '"'""'""auttiDerris 0 0 0 0 2' 0 2 0.33 0.12 llltnnlria SD. 3 1 0 1 0 0 5 0.13 1.17 l.atlia SD. 0 0 0 0 2 0 2 0.33 0.12 ProdadlusbellS 24 0 11 1 2 2 40 U7 1.37

ProdadlUS freemari SD. 0 0 0 0 0 0 0 0.00 0.00 I '"""" 0 0 0 0 0 0 0 0.00 0.00 I Total Chlronomidae 14 2t 12 21 3 151 2U3 29.70 .. • ! T s 0 0 0 0 1 0 1 0.17 0.41 Q.tcldae OlaObonsSD.1 7 5 13 7 7 11 50 U3 3.01 - Chaoborus m.2 n 0 0 0 .0 0 0 0.80 0.00 Total Chaoborus 7 5 13 7 I 11 51 l.fO 2.15 I CUex 0 0 0 0 0 0 0 D.00 0.00 Cera'"""""'""'• Cera"""""""da• 2 0 0 1 0 0 3 0.!10 0.14 ,...... 1.. -.... 0 1 0 0 0 0 1 0.,7 0.41 ,Doi...... ,,,..... Oclehmxxtdae 0 0 0 0 1 0 1 0.17 0.41 '5'"111 ...... 5'"111-• 0 0 0 0 0 0 0 0.00 0.00 TIDUldae ITilluldae"""' #1 0 0 0 0 0 0 0 D.00 0.00 ITIN•dae"""'#2 0 0 0 0 0 0 0 0.00 0.00 ITilluldae"""'#3 0 0 0 0 0 0 0 D.00 0.00 I TIDUldae,._#4 0 0 0 0 0 0 0 0.00 0.00 IS\6nhldae ,...... 0 0 0 0 0 1 1 D.17 0.41 I • • 0 0 0 0 0 0 0 D.00 0.00 ,...... 1-... 0 0 0 0 0 0 0 D.00 0.00 Tabaridae Tabaridae 0 0 0 0 0 0 0 D.00 0.00 Scl""""'dae '"'"""""'dae 0 0 0 0 0 0 0 0.00 0.00 Total Dlntera 93 15 42 20 30 15 215 35.13 29.18 Baeldae Calbaels 1 7 19 0 2 2 31 5.17 7.11 Ceenldae Ceenis 0 0 0 0 0 0 0 0.001 0.00 Total Eah-rontara 1 7 11 0 2 2 31 5.17 · 7.11 ,...... Seldldlle 1 0 0 0 0 0 1 0.17 0.41 Gerndae Gems 1 0 0 0 0 0 1 0.17 0.41 -·Total Hemlotera 2 0 0 0 0 0 2 G.33 0.12 Odane1a Lesldaa Lesldae 0 0 0 0 0 0 0 0.00 0.00 UIJeUdae ~dae 0 0 3 0 1 0 4 D.17 1.21 leo.,c,iaridae 0 0 0 0 0 0 0 0.00 0.00 Totai Odona1a 0 0 3 0 1 0 4 U7 1.21 T__,, IHv

# Individuals 147 46 128 139 79 33 572 15.33 4UI #s....,in 17 a 12 9 17 6 32 11.50 4.51 #lamllln 7 5 5 4 6 5 11 5.33 1.03 # orders 4 3 4 2 4 3 5 133 0.12

B7 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order Fomllv I-In/Genus Emeraent lnuc:ts collected June 13 1H5 from n,,nds s• ,...... with Altos Id. Maan Der SD PER 6-1 15 total 16lolal 17tolal 21 total TOTAL IPond POND 3 - ...... _.. Clironomdlle Cltonornaso.1 0 2 0 0 0 0 2 0.33, 0.12 ;SD. 0 0 0 0 0 0 0 0.00 0.00 1"--"--tnc:Nals 0 0 0 0 0 0 0 o.oo 0.00 DI~ neMlSUS

fl Individuals 92 95 10S 118 164 71 145 107.50 31.74 #s....,les 7 12 7 7 6 a 20 7.13 2.14 #lamHles 4 5 3 5 4 5 7 4.33 D.12 #ordere '2 3 3 3 3 3 4 2.13 0.41

BS Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Onler Famllv s-lu/Oenus Emeraent Insects collected June 1"- 1H5 from -d• •A--" with Abete. Mean.,., SD PER 2 tollll 12111181 19- 20- 23- 24101111 TOTAl I_,.. POND ,,..,_,. Oirancmldlle 0w'cnonlls Sll.1 0 0 0 0 0 0 0 0.00 0.00 ,_ '""· 0 0 0 0 0 0 0 0.00 0.00 l,.,.,,,..__lnc:Nals 0 0 0 0 0 0 0 0.00 0.00 --~.SD. 0 0 0 0 0 0 0 0.00 0.00 D1-cf.'11'11nls 0 0 0 0 0 0 0 0.00 0.00 EndacliranOnllsriarlcsns 0 0 0 0 0 0 0 0.00 0.00 l

Psectoclldus SIi. 0 0 0 0 0 0 0 0.00 0.00 Srri111 (femalel 0 0 0 0 0 0 0 0.00 0.00 Clr'tlodl(tnae females 0 0 0 0 0 0 0 0.00 0.00 I-•SD. 0 0 0 0 0 0 0 0.00 0.00 -- ·---•s 0 0 0 0 0 0 0 0.00 0.00 lr..-a~-.....s 0 0 0 0 0 0 0 0.00 0.00 Llll>nldria SD. 0 0 0 0 0 0 0 0.00 0.00 Larsiasn. 0 0 0 0 0 0 0 0.00 0.00 Pradadus-- 11 1 51 18 35 12 121 21.33 11.38 Pradadus freemari SD. 0 0 0 1 0 0 1 0.17 0.41 ...... 0 0 0 1 0 0 1 0.17 0.41 Total Chlronomld•• 12 1 51 20 37 14 135 22.50 11.28 T-•...... -ris 0 0 0 0 0 1 1 0.17 0.41 ruddlle 0.al>olla SD.1 1 0 0 0 0 0 1 0.17 0.41 0.al>olla SD.2 0 0 1 0 8 1 10 1.87 3.14 Total Chaoborus 1 0 1 0 I 2 12 2.00 3.03 CUex 0 0 0 0 0 0 0 0.00 0.00 Cera'"""""""'• Cen,_dlle 0 0 0 0 0 0 0 0.00 0.00 li::-dlle li::-e 0 0 0 0 0 0 0 0.00 0.00 Dclc:hooocldlle 0c1-c111. 0 0 0 0 0 0 0 0.00 0.00 St"lllnnNildlle sn1...... ,e 0 0 0 0 0 0 0 0.00 0.00 Tlludae 1...... _,1 0 0 0 0 0 0 0 0.00 0.00 ITllddlle"""'#2 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 0.00 0.00 -·..... #3 ITllddlle...,. #4 0 0 0 1 0 0 1 0.17 OA1 IS\/rnlidlle 1-. 0 0 1 0 0 0 1 0.17 0.41 l~dlle • 0 0 0 0 0 0 0 0.00 0.00 l.,_dlle ,--...... 0 0 0 0 0 0 0 0.00 0.00 T-dll• Tabandee 0 0 0 0 0 0 0 0.00 0.00 • Sci--• 0 0 0 0 0 0 0 0.00 0.00 Total Dlntara 13 1 53 21 45 11 1'9 24.13 20.00 ,_ Baetdlle C.llbaets 0 0 0 0 0 0 0 0.00 0.00 C.eridlle C.eris 0 0 0 0 0 0 0 0.00 0.00 Total EDhemerontera 0 0 0 0 0 0 0 0.00 0.00 1- llallidlle 0 0 0 0 0 0 0 0.00 0.00 Gln1dae Genis 0 0 0 0 0 0 0 0.00 0.00 -·TotalHomlatara 0 0 0 0 o' 0 0 0.00 0.00 0danata Lesldle LNtdu 0 0 1 0 0 0 1 0.17 0.41 l.lleUda• l.l,IUdle 0 0 0 0 0 0 0 0.00 0.00 leonsianldlle lr..-an1<1ae 0 0 0 0 0 0 0 0.00 0.00 TotalOdonata 0 0 1 0 0 0 1 0.17 0.41 Trlc:txdn 1...... -,dll• 1--c111• 0 0 0 0 0 0 0 0.00 0.00 L.....,_""da• L-.. 0 0 0 0 0 0 0 0.00 0.00 Total Trlch""'- 0 0 0 0 0 0 0 0.00 0.00 15.32 CallmlJoll Colemball 34 33 39 9 a 6 121 21.10

#lndMduels 47 34 93 30 53 22 271 41.IO 2U3 ,.-1 .. 4 2 5 5 5 5 14 U3 1.21 #famlll• 3 2 5 3 3 3 II 3.17 O.H #onl- 2 2 3 2 2 2 3 2.17 0.41

B9 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Ordlr Fe...., - Emergent Insects collectad June 21 from control ponda. Mean aer SD PER tl01al 41111111 5total 10- 22tolal TOTAL ""nd POND OIDl8rl Cliranonidee Clirononu; SD.1 0 0 0 0 4 4 0.10 1.79 Oler-SD. 0 0 0 0 0 o' 0.00 0.00 - ...... er.SD. 0 0 0 1 0 1 0.20 OAS Ol~cf.l1tanlls 1 0 0 0 0 1 0.20 0.45 Elnfeldla SD. 0 0 0 1 0 1 0.20 OAS El ldod*OI IOl'IU ri!11c:ans 0 0 0 2 a 2 0.40 OM ilD. 0 0 4 1 0 5 1.00 1.73 KlefftllWS u 0 0 0 0 0 0 ILOO ILOO Ml ...... - .. 0 0 0 0 0 0 o.oo ILOO ,--1- 0 0 4 7 0 11 2.20 3.11 m.m 0 2 16 3 0 21 4.20 1.72 Pllracl*onornll so. rr. camau 0 0 1 0 0 1 IL20 OAS PanclironarnlS IIICIIIOCfflllf 0 0 0 0 0 0 0.00 ILOO Clironaniri females 2 1 t 1 1 •· 1.20 0.45 T..-..... SD. 1 0 a 0 a 1 0.20 OAS Tenllllnlnl ,..,_ 2 1 1 0 0 4 O.IO U4 '""'°""""•SD.1 0 0 0 0 0 0 0.00 0.00 11".rir""""•SD.2 0 0 0 0 0 0 0.00 0.00 Oltlodllclnae lemeles 0 0 0 0 0 0 0.00 ILOO IANooho,""""81D. 9 5 5 4 0 23 uo 3.21 -~- 0 0 0 0 1 1 0.20 OAS labnniria SD. 0 0 0 0 0 0 0.00 0.00 Larsia SD. 1 2 1 1 0 5 1.00 IL71 Peramern IP, 0 0 0 1 0 1 0.20 U5 Prodllclus ~ 1 0 0 2 0 3 O.IO ut Prodllclus hemali SD. 0 0 0 0 1 1 0.20 0.45 Prodllclus Sl.tiellei SD. 0 0 0 0 0 0 0.00 ILOO ...... 0 0 0 0 2 2 0.40 ut T 0 0 0 0 2 2 0.40 0.19 Total Chlronomldae 17 11 33 24 11 11.20 1.31 0.ddH Cheoborus SD.1 1 2 2 5 2 "12 2.40 1.12 Cheoborus SD.2 0 0 0 0 1 1 0.20 0.45 Total Chaoborus 1 2 2 5 3 13 2.IO 1.52 CUex 0 0 0 0 0 0 0.00 ILOO cera-• cera-.c1ee 6 2 2 1 0 11 2.20 2.21 Ii:-.. ,-...- 2 0 0 0 0 2 -· 0.40 .._ .. Dolchmaddae [)olrtvvv.tdee 0 0 0 0 0 0 0.00 ILOO sn...... sn-• 0 0 0 0 0 0 ILOO 0.00 l11n,•dee 11'Wdee"""'#1 0 0 0 0 0 0 0.00 ILOO 11'Wdee"""'#2 0 0 0 a 0 0 ILOO ILOO l'llnuldee"""' #3 0 0 0 0 0 a 0.00 ILOO - r .. •dee"""'#4 0 0 0 0 0 0 0.00 ILOO ITWdee"""'#5 0 0 0 0 0 0 ILOO ILOO 1-.. 1-... 0 0 0 0 0 0 ILOO ILOO Cecf-...Udee CecfdeUdlle ~dee 0 0 0 0 0 0 0.00 ILOO leoen..tonidee leo.vtondee 1 2 0 1 0 4 O.IO IL14 Total Odonal• 1 2 0 1 0 • 0.10 0.14 T...... 1...... de. 0 0 0 0 0 0 0.00 . 0.00 L-• L_,...... ,..• 0 0 0 0 0 0 0.00 ILOO Total TrichoPlera 0 0 0 0 0 0 0.00 0.00 1...... __c1e. r.--11w.1s 0 0 0 1 0 1 IL20 G.45

~ Colentloll 75 27 50 98 57 307 11.40 21.73

# Individuals 106 59 98 131 94 ... 17.IO 25.13 #s-in 14 10 12 17 11 21 12.IO 2.77 #famillft 8 6 5 7 5 • 1.20 1.30 #orders • 4 3 5 3 5 3.10 0.14 B 10 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate. ,_ 0nler F_, Emeraent Insects collected June 21 1H5 from ponds soravad with Altoslct. "_ SDPEJt 3to1111 15to1111 ... 18to1111 17- 211Dta1 TOTAL loond POND •Dintar'II Clironomldae O*'llnOll'IAID.1 0 0 0 0 0 0 0 0 0 D ·- 0 D 0 ·-. D 0 0 0.00 0.00 nervosusar.so. D 0 0 0 0 0 0 0.00 0.00 cf.'11olnls 1 1 0 0 1 0 3 D.50 0.55 en.la so. 0 0 0 0 0 0 0 0.00 0.00 EndDctinlnonl,s ,...,,,.._ 0 0 0 0 1 0 1 0.17 0.41 ilD. D 5 1 D 2 0 I 1.33 U7 ICillllanuu 0 0 0 0 1 D 1 0.17 0.41 ,-. 0 0 0 0 D 0 0 o.oo 0.00 3 5 0 D 0 0 I 1.33 2.11 1--...t1t.m 5 2 0 0 0 0 7 1.17 2.04 Pwaclwolal'IJS so. rr. carlnnls 0 0 0 0 0 0 0 0.00 0.00 Parachlronam.lS monoclrorrus 0 0 0 0 0 0 0 0.00 0.00 Q'Rnclliri , ...... 4 2 2 0 0 0 I 1.33 U3 T-...ID. 0 0 0 0 2 0 2 0.33 0.12 T_f.,,... 1 1 D D 2 D 4 U7 D.12 ;,..,._._"IID.1 D 0 D D D 0 D 0.00 0.00 l,..,.,,...... "IID.2 D 0 1 D D 0 1 D.17 0.41 ~.,...... D 0 0 0 0 0 0 0.00 0.00 . IID. 9 0 0 0 0 1 10 U7 3.11 ,nin

# lndlvkluele 152 74 237 IO 232 116 n1 141.13 72.14 ...... 18 10 • 5 14 10 21 10.50 UI #famlllea • 5 • 4 5 II 12 1.33 2.11 #ordera 5 4 3 3 3 5 • :s.n o.n Bll Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

Order F....., -es/Geris Emeraont lnHCts colloctocl Juno 21 1195 lrom nonds •• ,.,-t wtth Abato.

Mean Der SOPER

2 total 12- 1910111 201Dtel 23tlllal 24total TOTAL 1 ...... PONO IDIDlerl Clironorrldee O*ancn'IISSD.1 0 0 0 0 0 0 0 0.00 0.00 ID. 0 0 0 0 0 0 0 0.00 0.00 Ol-l'leMISUS ar. SD. 0 0 0 0 0 0 0 0.00 0.00 DI_,, cf. t1l0mlS 0 0 0 0 0 0 0 0.00 0.00 EWelcla SD. 0 0 0 0 0 0 0 0.00 0.00 Endoclironornls riar1cans 0 0 0 0 0 0 0 0.00 0.00 - -· ,ID. 0 0 0 0 0 0 0 0.00 0.00 IQellerwscuc 0 0 0 0 0 0 0 0.00 0.00 ,nadailJS 0 0 0 0 0 ,_ 0 0 0.00 0.00 - 0 0 1 0 0 0 1 0.17 0..41 0 0 0 0 0 0 0 0.00 0.00 Panld*onorTIII- ID. IT. car1na\ls 0 0 0 0 0 0 0 0.00 0.00 Panld*onorTIII rnonoctTI,mJs 0 0 0 0 0 0 0 0.00 0.00 Clnnolriri females 0 0 1 0 0 0 1 0.17 D.41 T...... ID. 0 0 0 0 0 0 0 0.00 0.00 r-,ema1es 0 0 0 0 0 0 0 0.00 0.00 Cl'k:"""""' 111.1 0 0 0 0 0 0 0 0.00 0.00 ICr1ct>tMt&111.2 0 0 0 0 0 0 0 0.00 0.00 Clrt1odlldnle lemales 0 0 0 0 0 0 0 0.00 0.00 i&Na,,_,_O ID. 0 0 1 0 3 0 4 D.17 1.21 anota-~-·· 0 0 0 1 1 0 2 0.33 0.52 Llbnnlri1I SD. 0 0 0 0 1 0 1 0.17 0.41 LanllSD. 0 1 0 0 0 0 1 0.17 0.41 f'llnlmelfn8 SD. 0 0 0 0 0 0 0 0.00 0.00 Plocladus bells 7 1 8 53 21 3 13 15.50 11.15 Ploclaclus lrNmllri SD. 0 0 0 0 2 0 2 0.33 0.12 Ploclaclus IUllettei so. 0 0 0 0 0 0 0 0.00 0.00 'dvar1 0 0 0 11 5 2 11 :S.00 4.31 T"""""" IJU'ldpemis 0 0 0 0 7 1 I 1.33 2.10 Totol Chlronomldoo 7 2 11 15 40 • 131 21.13 25.20 0*:ldo• OlaoboNsSD.1 0 1 0 0 0 0 1 0.17 D.41 Cheobonll ID.2 0 0 0 0 0 0 0 0.00 0.00 Total Chooborus 0 1 0 0 0 0 1 0.17 0.41 Cl.x 0 0 0 0 0 0 0 0.00 0.00 cera-• cera...... ,.,.dee 0 0 0 0 0 0 0 0.00 0.00 lo:-..-. IEDIMttdo• 0 0 0 0 1 1 2 0.33 0.52 Dolc:hODOdldee Dol...... ,,,,.dee 0 0 0 0 0 0 0 0.00 0.00 Snlmnwlldee Sn•--· 0 0 0 0 0 0 0 0.00 0.00 ITIDuldee ITIDl.tdle"""' #1 0 0 0 0 0 0 0 0.00 0.00 ITIDl.tdee"""'#2 0 0 0 0 0 0 0 0.00 0.00 ITIDl.tde•"""' #3 0 0 0 0 0 0 0 0.00 0.00 ITlouldle....., #4 0 0 0 0 0 0 0 0.00 0.00 TDJldee...,.#5 0 0 0 0 0 1 1 0.17 0.41 ...... 1-.... 1 0 0 2 0 0 3 0.50 0.14 • e 0 0 0 0 0 0 0' 0.00 0.00 • 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 0.00 0.00 Tllbanldle T..... ID. 0 1 0 0 0 0 1 0.17 0.41 Sci-• 0 0 0 0 0 0 0 0.00 0.00 Totel Olotora I 4 11 S7 41 I 138 23.17 25.31 -- Calbaels 0 0 0 0 0 0 0.00 0.00 Baeldoo 0.00 0.00 CaeridH Caeris 0 0 0 0 0 0 0.00 0.00 Total Eph-rootora 0 0 0 0 0 0 0 2 0 0 0 0.33 0.12 H- Salcido• Salcldle 0 0 0 0 0 0.00 o.oo Gentde• Gents 0 0 0 0.33 D.12 Total Hemlotera 0 0 2 0 0 0 0 0 0 0 0.00 0.00 Odanl1I Leddee Lnldoo 0 0 0 0 o.oo 0.00 LlbeUdoo I.JlleUdoo 0 0 0 0 1 0 0.33 0.12 eo.a10ridee 0 1 0 • 0 0.33 0.52 Total Odonato D 1 0 0 1 0 0 0 0 0 0 0.00 0.00 T- • 0 0 0.00 D.00 L-. L...... ,_ D 0 0 0 D 0 0 0.00 0.00 Total Trlchnatera 0 0 0 0 0 0 0 0 o.oo 0.00 ....- ...... de • T.....,._IIIWlls 0 0 0 1& 48 83 23 95 6 211 35.17 23.23 Colln'DCIII Coleneall

14 354 51.00 34.51 # Individuals 24 53 76 90 97 10 & 11 I.DO 2.21 #SDOCIH 3 8 8 5 4 4 3.17 0.12 #lomllln 3 5 3 3 • • 2 3 3 2 3 2 :a.so D.55 #ordera • B 12 Emergent insect summary data for control pond, and experimental ponds sprayed with Altosid and Abate. 0nllr F....., -entln-collHINJ,...• 1•11wacon1ro1-.._ ....,_ IDPEIII 1- 4111111 5111111 9111111 10111111 22111111 TOTAL 1...... POND 1,-,. ~ D*arDIUm.1 0 0 0 0 0 0 0 o.ao 0.IO 0 0 0 0 0 0 0 0.00 O.IO -····d.m.na 0 0 1 0 0 0 1 0.17 U1 EndocNICIIICIII ... nlC111CIIII 0 0 1' 0 0 0 1 0.17 U1 11 1 3 1 0 0 5 0.13 1.17 IOelllll&llu 0 0 0 1 1 I s 0.50 0.11 0 0 0 0 o' o: 0 0.00 0.00 0 1 3 2 3 0 • 1.50 UI ------11un 0 33 241 0 0 0 57 1.50 ,... Penl~ ID. rr. cannau 0 0 0 0 0 0: 0 0.00 0.00 hraclnnanulllllnOClnnll 0 0 0 0 0 0 0 0.00 0.00 0*1lnalnlnl,.,... 0 0 0 1 1 4 • 1.00 1.55 T 0 0 0 0 0 0 0 0.00 O.IO T_...... ,.,.._ 0 I I 0 1 0 s uo 1.15 .I 0 0 0 0 0 0 0 o.ao o.ao ,_•ID.2 0 0 0 0 0 0 0 O.IO o.ao Ortlodldnal , ..... 0 0 0 0 0 0 0 0.00 o.aa •• -• -~-ID. 2 3 7 5 4 I 22 117 2.11 l.,,,,,_.nirllus 0 0 0 0 0 I 1 0.17 0.41 L.ebNldril ID. 0 0 1 0 1 0 2 us 1.12 LenialD. 0 1 2 0 0 0 s 0.50 U4 Prodldusllakll 0 0 0 3 II 1 10 1.17 U2 Prodellul"-nllD. 1 0 0 0 0 0 1 0.17 U1 ...... 0 0 0 0 1 2 s 0.50 G.14 T--N- 0 0 o, 0 I 1' 2 G.33 G.52 Total Chl....nidH 3 40 43 13 II tt 121 21.50 1U4 Qicldle ~-., t 15 3 4 5 3 31 UT 5.00 ~-.2 0 0 0 0 0 0 0 0.00 O.IO TolllC...... 1 11 :, 4 I :, 31 5.17 5.00 CUIX 0 0 0 0 0 0 0 0.00 0.00 - • eere-• 8 1 1 6 0 0 14 2.33 2.11 Ealwa1dle .,...... 0 1 0 0 0 0 t 0.17 G.41 I 0 0 0 0 0 0 0 0.00 0.00 &ntanMldlle sn- 0 0 0 0 0 0 0 0.00 0.00 ..,;..,... 0 0 0 0 0 0 0 0.00 0.00 0 0 1 0 0 0 1 0.17 , -do• -·- ... • - -· 2 0 1 1 0 0 4 UT 0.12 - 0 0 Oi 0 0 0 0 0.00 0.00 0 0 0 1 0 0 1 0.17 Ut Taclrml TlllednN 0 0 o' 0 0 0 0 0.00 0.00 . 0 0 0 0 0 0 0 0.00 0.00 Total....._ 12 IT • 25 34 14 111 30.17 11.11 Baeldle ~ 1 9 a 1 1 13 33 5.IO 5.21 Cllenldoe Cllenls 1 5 5 1 0 0 12 2.00 2.37 Total E..-rolllen 2 14 13 2 1 13 45 7.50 U1 selcldoe S8kldle 0 0 0 1 0 0 1 0.17 OA1 ·- Gents 0 0 0 0 0 0 0 0.00 0.00 ~· Total Hemlatoro 0 0 0 1 0 0 1 0.17 U1 Cldcn1a LesldH LestdH 0 0 0 0 0 0 0 0.00 0.00 UbeUdH &...- 0 0 0 0 0 0 0 0.00 0.00 0 0 1 4 2 0 7 1.17 1.10 Tolllcw-la 0 0 1 4 2 0 7 1.17 1.IO 1- 0 0 Di 0 1 Oi 1 0.11: U1 1 0 0 0 ol t o.17 Ut - ~~- 0 Tolll Trlc.,_.,. 0 1 0 0 1 0 2 0.33 0.52 ,...... :--.ac1oe ·-....., 0 0 01 0 0 o• 0 0.00 0.00 Calembola COleMIOII 140 53 S3 220 147 117 780 121.17 57.14

#lndivlduols 154 125 146 252 175 144 ... 111.00 45.14 a 13 17 15 14 10 21 12.113 131 ··-·-#fomllln 7 a 9 10 6 4 7.33 2. IS #onlen 3 4 4 5 5 3 "• ..oo 0.11

B 13 Emergent insect summary data for control pond, and experimental ponds sprayed with Altosid and Abate. Order Fa"""' ·- Emeraent Insects collected Julv 5 1H5 from oonds spraNd with Altosld. Mean par SD PER -- 3 total 6tolal 15 total 16total 1710181 2110181 TOTAL loond POND l11in1M11 CliranOllidH Cltonorrus ID. I 0 0 0 0 0 0 0 0.00 0.00 ,_ neMIIUSr,:.SD. 0 0 0 0 0 0 0 0,00 0.00 tf.'111lrnJS 1 3 0 0 0 0 • G.17 1.21 Elldochia,onus ...... 0 0 1 0 0 0 1 0.17 DAI iSD. 0 0 0 0 0 0 0 0.00 0.00 Klellarwsu 0 1 0 0 1 0 2 0.33 0.52 ·- ·- 0 0 0 0 0 0 0 0.00 0.00 1-.-'1_ 2 2 0 0 0 3 7 1.17 1.33 l~t1t.m 3 •o 1 0 2 0 •• 7.17 15.a ParactwononUI sp, II'. ca/1nl"5 0 0 0 1 0 0 1 0.17 o.•1 0 0 0 0 0 0 0 0.00 0.00 Pwllcliranorrus - Cl*'anonirif- 1 2 1 0 2 1 7 1.17 0.75 T...... SD. 0 1 0 0 1 1 3 0.50 O.S5 T-females 0 1 1 0 0 0 2 0.33 0.52 i~,.SD.1 0 0 0 0 0 0 0 0.00 0.00 !~so.2 1 0 1 0 0 0 2 0.33 O.S2 Ort1odllc1Me ,.,.1es 0 0 0 0 0 0 0 0.00 0.00 ...... SD. 5 2 0 0 3 1 11 1.13 ,...... 0 0 0 0 0 0 0 0.00 0.00 L.abnmlil SD. 1 2 0 0 1 1 5 0.13 0.75 Lanie SD. 0 0 0 0 0 3 3 0.50 1.22 Procll<>Mdldle 0 0 0 0 0 0 0 0.00 0.00 5"al""""'dle Sn1mNlldle 0 0 0 0 0 0 0 0.00 0.00 TWdle !Tllddle 0 0 0 0 0 0 0 0.00 0.00 IS\odidle 0 0 0 0 0 0 0 0.00 0.00 • -·CedtWINlidle Ced"""""'dl• 0 0 0 0 0 0 0 0.00 0.00 Seddnnllllldle l...... ,_..dle 0 0 0 0 0 0 0 0.00 0.00 Psw:hodldle I-die 0 0 0 0 0 0 0 0.00 0.00 Tabaclnle Tlba

0rdlr Fa,_ ISMdesK.enus Emeraent Ins acts colllCted Julv 5 1 N5 from nnnds •ft- ..... with Abate. ...n.,., SD PER 2 IOIIII 12 to1a1 19lolal 201otal 231otal 24 total TOTAL ,...... POND ,...... ,, Oircnorridle awonan..so.1 0 0 0 0 0 0 0 0.00 0.00 Olcrotencl"'"' nervosus N'ldnll SD. 0 0 1 0 0 0 1 0.17 0.41 Lan11m. 0 0 0 0 0 1 1 0.17 U1 Prudlclus belJs 3 0 5 11 16 2 37 1.17 1.11 Prodadus freemani SD. 0 0 0 0 3 0 3 0.50 1.22 ,_ri 0 0 0 16 3 4 23 3.13 1.21 T•...... - ...... , 0 0 0 0 2 3 51 0.13 1.33 Total Chlronomidae 3 0 7 29 30 10 71 13.17 13.11 CUlcidle Chaobcrus ID.1 3 1 0 0 0 1 s 0.13 1.17 Chaobcrus m.2 1 1 0 0 3 0 5 0.13 1.17 Total Chaobonas 4 2 0 0 3 1 10 1.87 1.13 rum< D 0 D D D 0 0 0.00 0.00 die Cen"""""'"'dl• D D D D D 0 0 0.00 0.00 1-. D 0 0 D 0 0 0 0.00 0.00 die -·Dolehlldnle D D D D D 0 0 0.00 0.00 Sci"""'7idle 1...... -... D D 0 0 0 ol o: 0.00 0.00 Total Dlntera 7 2 • 29 3' 11 11 15.17 13.0I C.llbllls 0 0 D D D 0 0 0.00 0,00 C.enldle CNnls 0 D 0 D D 0 0 0.00 0.00 ·-· Total Enhernerontera 0 0 0 0 0 0 0 0.00 0.00 H__,, Salcldle sutdle D 0 0 0 0 0 0 0.00 0.00 Gwrldle Gonls D D 0 0 0 0 0 0.00 0.00 Total Hemlntera 0 0 0 0 0 0 0 0.00 0.00 Odona1II L- Leddee D D 0 D 0 D 0 0.00 0.00 UbeUdle UbeUdle D 0 D D D D 0 0.00 0.00 11".-..ifYidle • 0 0 0 0 D 0 0 0.00 0.00 Total Odonat1 0 0 0 0 0 0 0 0.00 0.00 1 0.17· T- IMwn-,...ldle ,...... 0 1 D D D 0 0.41 2 0.33 0.12 L-• L-...e 0 0 2 0 0 0 Total Trlchontera 0 1 2 0 0 0 3 0.50 0.14 i<:oleoPtara IMwmimildle ···1so. D 0 D 0 0 0 o· 0.00 0.00 93 51 98 37 53' 11.00 41.11 ~ ColelTtlola 79 176

# Individuals 86 179 103 80 132 48 121 104.17 45.70 #s-1.. 4 4 6 5 8 6 11 5.50 1.52 #famllles 3 3 4 2 4 3 7 3.17 0.75 #orders 2 3 3 2 2 2 3 2.33 0.52 e

B 15 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

- Onllr F•...., Emeroent Insects collected Julv 11 1115 from control "' nds. h•n- SD PER 1 tDtlll 4 lolal 5 tobll 9tDtlll 10 follll 22101111 TOTAL 1~nd lpoffD IDirJwa CNrcnomldle O*onanll_,._ 1 0 0 0 0 0 1 0.17 0.41 ln-..o.... bnl~lls 0 0 0 0 0 0 0 0.00 0.00 Dt-sner;osus

# Individuals 531 158 287 320 268 389 1133 322.17 124.04 ...... , ... 14 14 19 18 17 17 32 11.IIO 2.07 #famllln 7 • 8 8 SI 7 11 7.13 0.75 #orders 4 5 4 15 3 4 • 4.17 0.71 B 16 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

0rdlr f""""' l""-les/Gen.lS Eme111ent Insects collected Julv 11 1ffl from nonds SN ,..., with Altosld. lleannar SD PER 3-1 6- 15- 16111111 17- 21IDIIII TOTAL iDOnd POND IDinlllnl Cl*anomldee 0 0 0 0 0 0 0 0.00 0.00 Cl*onarrllS -- 1,,_..,1nctn1a 0 2 0 0 0 0 2 0.33 0.12 01-nerwsus "'· ""· 1 0 1 0 0 0 2 0.33 0.52 01~-d. '1t0rlllS 1 0 0 0 0 0 1 0.17 0.41 Elnleldla m. 1 0 0 0 0 0 1 0.17 0.41 El ldocli 01 IOIIIIS rinricans 2 0 2 0 0 0 4 0.17 1.03 ""· 0 0 2 0 0 0 2 0.33 0.12 Ml--- 0 1 0 0 0 0 1 0.17 0.41 d. faldforme 0 0 0 0 0 0 0 0.00 0.00 ,_ tt-- 0 2 0 1 0 0 s o.so 0.14 ,_ .. 0 7 0 1 0 0 I 1.33 2.10 _ ... _,.._calinallS 0 0 0 0 0 0 0 0.00 0.00 - 0 0 0 0 1 0 Parac:l*onomUs rnonocll'onllS 1 0.17 0.41 PHudOclir'OnOmUOII, 0 0 0 0 0 3 3 0.50 1.22 Zawelela rnannon,111 0 1 0 0 2 0 3 0.50 0.14 ctnnclliri females 1 6 0 1 1 0 • 1.50 2.21 Tlll'Mlll'IU!l!ID, 3 3 2 2 1 2 13 2.17 0.75 Tllfflltarslnl females 5 3 4 0 0 4 11 2.17 2.11 1..,,,_..... ,1 0 0 0 0 0 0 0 0.00 0.00 Crt ...... ,2 3 0 1 0 0 0 4 0.17 1.21 01'1hadadlinae females 0 0 0 0 2 0 2 0.33 0.12 1...... 1 0 3 0 0 0 4 0.17 1.21 a--- ...... •s 0 0 0 0 0 0 0 0.00 0.00 l.atlnnlnia ""· 0 2 1 1 1 0 5 0.13 0.75 Laniasn. 2 0 0 2 0 9 13 2.17 141 Prodadlusbt*IS 0 0 0 0 1 1 2 0.33 0.52 Prodadus freemali .,,_ 0 0 0 0 0 0 0 0.00 0.00 Prodadlus SLtielbli ...... 0 0 0 0 0 0 0 0.00 0.00 Ta-·---• 1 0 0 0 0 0 1 0.17 0.41 Total Chlronomldae 21 27 11 I • 11 100 11.17 7.21 Culddaa Chaobcrus sn.1 0 0 1 1 0 0 2 0.33 0.52 Chaabcl\ls cn2 0 0 0 0 0 0 0 0.00 0.00 Total Chaoborus 0 0 1 1 0 0 2 0.33 0.52 CIAex 0 0 0 0 0 0 0 0.00 0.00 Mansoria sn. 0 0 0 0 0 0 0 0.00 0.00 Cent-de• Ceni...... ,dee 5 3 0 1 2 8 11 3.17 2.13 .,...... ,_,,.. 0 1 0 0 0 0 1 0.17 0.41 • Ool""""""'de• Dol ...... dae 1 0 0 0 0 0 1 0.17 0.41 S1ralnnwlidee stat--dee 0 0 0 0 0 0 0 0.00 0.00 ...... de ...... #1 0 0 0 0 0 0 0 0.00 0.00 -·- ...... de ...... #2 0 0 0 0 0 0 0 0.00 0.00 Tlno•dee"-#3 0 0 0 0 0 0 0 0.00 0.00 ...... de ...... 0 0 0 0 0 0 0 0.00 0.00 ...... de ...... #5 0 0 0 0 0 1 1 0.17 0.41 """""de• 1...... de. 0 0 0 1 0 0 1 0.17 0.41 CedNWNAldee lr"...... idee 0 2 0 0 0 0 2 0.33 0.12 SeciNWNAldee • 0 0 0 0 0 0 0 0.00 0.00 l--....odee 0 0 0 0 0 0 0 0.00 0.00 Tabednae TabllnJsm. 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 o.oo 0.00 • Sci-• 0 0 0 0 0 0 0 0.00 0.00 Total Dlntera 27 33 17 11 11 21 127 21.17 1.43 Baddie Clllbllela 41 12 15 11 14 18 111 11.50 11.21 Caendae Caenls 0 0 0 0 0 1 1 0.17 0.41 Total Eoh-rontera 12 15 11 1• 18 112 18.17 11.29 "0 0 0 0 0 0 0 0.00 0.00 - Genldae Gents . 0 0 0 0 0 0 0 0.00 0.00 -· -Total Hemlntera 0 0 0 0 0 0 0 0.00 0.00 Odana111 Lesldee Lesldae 0 0 0 0 0 0 0 0.00 0.00 Ubea.tdee l.lleUdae 0 0 0 0 0 0 0 0.00 0.00 ,..-....,dee - 2 4 7 3 1 2 11 3.17 2.14 Total Odoneta 2 4 7 3 1 2 11 3.17 2.14 1- ...... de. ,..._,dee 0 1 0 0 0 0 1 0.17 0.41 • Loatncerldae 1 1 0 0 1 0 3 0.50 0.55 Total Trlchontara 1 2 0 0 1 0 4 0.17 0.12 534 304 138.70 ColenC>alll ~ 153 336 489 360 2178 312.17

# Individuals 224 387 528 385 581 353 2431 401.33 123.00 #s-ln 17 17 12 12 12 11 35 13.50 2.74 #famllln 7 9 5 7 6 7 14 1.13 1.33 #orders 5 5 4 4 5 4 5 4.50 0.55 B 17 Emergent insect summary data for control, and experimental ponds sprayed with Altosid and Abate.

,_ 0rclllr F- Emeraent Insect• collected Julv 1t 11195 from ponds•"' ,_ with Abate. M•n- ID PER 2- 12-1 19- 20lolal 23 lolal 2•- TOTAL ,...... POND i~ O*anomdl• O*arlOIIIII-- 0 0 0 0 0 0 0 0.00 0.00 tncNels 0 0 0 0 0 0 0 0.00 0.00 nerwsusr,.111. 0 0 0 0 0 0 0 0.00 0.00 cf.'1101111S 0 0 0 0 0 0 0 0.00 0.00 - Elnl-11>. 0 0 0 0 0 0 0 0.00 0.00 Endoct.-011011111 .,....,._ 0 0 0 0 0 0 0 0.00 0.00 - 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 0.00 0.00 - cf.fat:lfonne-- 0 0 1 0 0 0 1 0.17 OA1 0 0 0 0 0 0 0 0.00 0.00 - ~-.iam--- 0 0 0 0 0 0 0 0.00 0.00 Parac:l*anolnll ...... car1na"5 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 0.00 0.00 Pareclironomul - ~ ... 0 0 0 0 0 0 0 0.00 0.00 Zaftla fflllfflOl'UI 1 0 0 0 0 0 1 0.17 OA1 O*-.inf- 0 1 0 .0 0 o· 1 0.17 DA1 Tll!VllnUS IP. 0 0 0 0 0 0 0 0.00 0.00 Tel!Yllnlri ,_... 1 1 0 0 0 0 2 o.33 0.52 °""*'.1 1 0 '0 1 0 'O• 2 0.33 0.52 0leoborus ID.2 0 0 0 0 0 0 0 0.00 0.00 Tolal Chaobonla 1 0 0 1 0 0 2 0.33 O.S2 Q'8X 0 0 0 0 0 0 0 0.00 0.00 ...... ID. 0 0 0 0 0 0 0 0.00 0.00 0 1 0 0 0 0 1 0.17 G.41 0 0 0 0 0 0 0 0.00 0.00 • die 0 0 0 0 0 0 0 0.00 0.00 ~-· • ~- -· • • 0 0 0 0 0 0 0 0.00 0.00 TiddN ~--•1 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 0.00 0.00 --#2...... _#3 0 0 0 0 0 0 0 0.00 0.00 ...... 0 0 0 0 0 0 0 0.00 0.00 Tllddle...,. #5 0 0 0 0 0 0 0 0.00 0.00 ,_ 0 0 1 2 1 0 G.17 0.12 • ·- • 0 0 1 0 0 1 '2 0.33 0.52 • 0 0 0 0 0 0 0 0.00 0.00 0 0 0 0 0 0 0 0.00 0.00 Tabllclrae T...... ID. 0 0 0 0 0 0 0 0.00 0.00 o.r-so. 1 0 0 0 0 0 1 0.17 OA1 • 0 0 0 0 0 0 0 0.00 0.00 Total DID1era 10 • • • s 31 I.SO 2.17 BaetdN calbaels 0 0 0 '0 0 0 0 0.00 0.00 Ca- Ca.a 0 0 0 0 0 1 1 0.17 U1 Total ED"-raDI.,. 0 0 0 0 0 1 1 0.17 0.'1 ,._ SakldM Salcldae 0 0 0 0 0 0 0 0.00 0.00 Genldl• Gans. 0 0 0 0 0 0 0 0.00 0.00 Total HamlDI.,. 0 0 0 0 0 0 0 0.00 o.oo 0donnl LNtclH LNICIN 0 0 0 0 0 0 0 0.00 0.00 UbeUdl• LlleUdle 0 0 0 0 0 0 0 0.00 0.00 ~oridle • 0 0 0 1 2 0 :, O.ID O.M Total Odoneta 0 0 0 1 2 0 :, 0.10 0.M r- ...-da. • 0 0 0 1 0 0 1 0.17 U1 L-. L-. 1 0 0 0 0 0 1 0.17 G.41 Tolal TrlchoDI.,. 1 0 0 1 0 0 2 0.33 0.12 COlln'Doll COlln'Doll 115 129 129 532 538 381 1121 30'-17 *-"

# lndlvlduels 121 135 135 138 1149 387 1170 311.17 205.00 IS 5 5 • 4 11 5.33 1.51 • #lemllln 5 3 4 ' 11 U3 1.03 ··-·· • • • ...... 3 2 ') • 3 3 • 2.13 D.71 B 18