Nont Arget Effects of the Mosquito Larvicides
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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.