1600 THOMPSON PARKWAY SARASOTA, FLORIDA PHONE: (813) FAX: (813)

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1 1600 THOMPSON PARKWAY SARASOTA, FLORIDA PHONE: (813) FA: (813) /t nonprofit organization dedicated to excellence in marine sciences" SUBLETHAL EFFECTS OF MOSQUITO LARVICIDES ON NONTARGET INVERTEBRATE AND VERTEBRATE LARVAE FIRST QUARTERLY REPORT For the period July 1, to September 30, 1990 FL HRS Contract LPS02 Submitted To: Dr. John P. Smith FL HRS Entomology Services 1217 Pearl Street Jacksonvil le, FL Submitted By: Dr. Richard H. Pierce Mote Marine Laboratory 1600 Thompson Parkway Sarasota, FL (813) OCT MOTE MARINE LABORATORY L1BRAR 1600 THOMPSON PARKWAY SARASOTA, FLORIDA October 10, 1990 ROBERT M. JOHNSON CHAIRMAN OF THE BOARD WILLIAM R MOTE PRESIDENT KUMAR MAHAOEVAN. h 0 EECUTIVE DIRECTOA

2 TABLE OF CONTENTS TABLE OF CONTENTS I NTRODUCTI ON SUMMARY OF SIGNIFICANT ACCOMPLISHMENTS RESULTS AND DISCUSSION ANTICIPATED FUTURE STUDIES ITEMIZED TOTAL HOUR REPORT

3 LIST OF TABLES Table 1. Table 2. Survival of crab (Uea) larvae exposed to saltmarsh conditions: 7/7/90. 4 Survival of crab (Uea) and mosquito (Aedes) larvae after exposure to abate in a saltmarsh ecosystem: 8/7/90. St. Jude, Florida. 5 Table 3. Application 1, August 7, Abate Concentration in Tidal Pool Water Adjacent to Larval Trays. 6 Table 4. Survival of crab (Aratus), snook (Centropomus) and mosquito (Aedes) larvae during and following exposure to a saltwater environment: 9/6/90. No abate applied. 8 Table 5. Number of larvae in each field cone. 8 Table 6. Application 2 September 7, Abate Concentrations in Tidal Pool Water Adjacent to Larval Trays. 10 Table 7. Table 7. Abate Application 2, September 7, Abate Application Impacting Filter Paper and in Adult Uea. 11 i i

4 I. INTRODUCTION This investigation was undertaken to assess the acute and sublethal toxicity of the mosquito larvicides, abate (temephos) and altosid (methoprene), on non-target invertebrate and vertebrate larvae in saltmarsh environments under a mosquito abatement program. Activities from July 1 through September 3a, 1990, represent the FIRST QUARTERLY REPORT period for the project. Activities during this period focused on field studies and techniques for collecting and rearing larvae from the marsh fiddler crab, Uca rapax, and the mangrove tree crab, Aratus pisonii. Four field studies were performed during this three month period, associated with the three full moon high tide events, because that was when the crab larvae were present. The four field studies included two field controls on July 7 and September 6: the July control study of Uca larvae with no mosquito, Aedes or Aratus larvae present and the September 6 study monitoring Aratus, Aedes, and snook. The abate application study on August 7 monitored Uca and Aedes while the September 7th application monitored Aedes and Aratus larvae. Additional. effort was directed toward continued studies for developing viable reproductive co16nies of the two species of crab, Uca rap ax and Aratus pisonii, to provide a reliable and consistent supply of crab larvae for feeding and survival tests, and as a source of larvae for field studies. The lab colonies helped to eliminate the uncertainty of finding gravid females in the marsh prior to each larvicide application, and reduced the amount of stress imposed on the crabs prior to eclosure. II. SUMMARY OF SIGNIF ICANT ACCOMPLISHMENTS 1. Successful development of a viable reproductive colony of the mangrove tree crab, Aratus pisonii, in the laboratory, with subsequent eclosure and rearing of the larvae into the second zoeal stage, resulting in advancements in proper feeding and handling techniques for larvae survival. 2. Completion of four sets of field tests, including two for acute and sublethal toxicity of abate, and two control tests without abate applications. 1

5 3. Field testing of modifications to the larvae monitoring trays and in the techniques used for observing the larvae to reduce physical stress that contributed to the high mortality of larvae during earlier field application studies. 4. Field studies of the survival of larvae from two species of crab and snook fry, simultaneously with mosquito (Aedes) larvae, in the absence of abate application. 5. Surface water concentrations of abate were found to be much higher than subsurface samples, indicating greater impact on organisms in contact with the water surface. III. RESULTS AND DISCUSSION: A. Laboratory Reproductive Colonies of Crabs Colonies of Uca rapax and Aratus pisonii were establ ished in the lab to observe their reproductive cycle and to provide larvae for lab as well as field studies. Gravid females collected from the st. Jude saltmarsh in June, along with additional females and males were placed in a microcosm salt marsh habitat in the laboratory. The Uca adults were.fed a commercial fish food preparation, Tetramarine, mixed with water to make a paste. The first Aratus colony failed, because the mangrove 1 eaves were not adequate food. Algal materi a 1 from the St. Jude mangrove floor was then used which proved to be adequate to sustain the Aratus colony. Uca and Aratus larvae were fed rotifers, which in turn were fed yeast and mixed phytoplankton. Because of high mortality of larvae fed on rotifers after the second zoeal stage, various diets using sea urchin eggs, brine shrimp and commercial invertebrate food were tested. B. Larval Field Studies July 7, 1990 Field Control Study The July full moon high tide was used as a field control study to assess the survival of Uca larvae transported and exposed to saltmarsh conditions, without larvicide application. This was necessitated because the lunar high tide 2

6 did not result in sufficient standing water in the saltmarsh for mosquito larvae to develop. Two larval trays were set at each of the field control and test sites. Sets of five Uca larvae were placed in each of three cones in each tray resulting in 30 larvae at each field study site. Additional larvae were placed as a bulk exposure sample in an adjacent cone to be retrieved for sublethal effects studies. The results, Table 1, show a 1 hour suryival rate of 87% and 83% for the control and test sites respect i ve 1 y. Water s amp 1 e analyses showed no detectable abate at either site. The 4-hour survival count was not available, because of a severe thunder storm which precluded our ability to count the larvae. Subsequent observations revealed that the severe impact of the raindrops had killed or displaced many of the larvae in the sets of five. The remaining bulk larvae were returned to the lab for continuing sublethal effects survival observations. Upon return to the lab, 20 larvae from each field site (control and test) were placed in petri dishes and the mortality was monitored daily. The results, Table 1, show approximately 75% survival through the first zoeal stage and through the mol t into the second zoeal stage at day 8. Extensive mortal ity. occurred one day after the molt, probably due to improper nutrition. August 7, 1990 Abate application study An application of abate accompanied the field tests on 8/7/90. Three sets of five larvae each from Uca and the mosquito (Aedes), were placed in the floating trays. Two trays were placed at the test and control sites, resulting in 30 larvae to monitor for the acute toxicity at each site. Additional larvae were placed as bulk in an adjacent cone, to use for replacing larvae that died during the I-hour acclimation, and for continued studies of sublethal effects, following the acute toxicity tests with the sets of five larvae. The results, Table 2, show that by three hours after application, all test site mosquitoes were dead, while 29 of 30 Uca were still alive, and at 6 hours, 20 of 30 Uca (67%) had survived. The control site exhibited all mosquitoes al ive and 26 of 30 Uca al ive at 3 hours; however, at 6 hours, all mosquitoes were dead, and 23 of 30 Uca (70%) remained. Subsequent analysis of the water (Table 3) showed that abate had been applied to the control area as well as the test site, resulting in two test sites for this application episode. 3

7 Table 1. Survival of crab (Uea) larvae exposed to saltmarsh conditions: 7/7/90. Number of Uca larvae per Time Control Site Test Site 0 (Eclosure) Tray-l Tray-2 Tray-l Tray hr hr. Thunderstorm, impossible to count 6 hr. Transport exposed larvae to 1 ab for subl ethal study Control Site Test Site Molt

8 Table 2. Survival of crab (Vea) and mosquito (Aedes) larvae after exposure to abate in a saltmarsh ecosystem: 8/7/90. St. Jude, Florida. Number of Uca larvae per tray ill Time Control Site Test Site Tray-I Tray-2 Tray-J Tray-2 II 8. II 8. II 8. II :00 AM :00 Acclimation :00 Abate application 2: : o- Il :00 Return to lab for sublethal study 15 Field Control Field Test Lab Cont ro 1 10:00 PM

9 Table 3. Appl ication I, August 7, Abate Concentration in Tidal Pool Water Adjacent to Larval Trays. Time Control Site Test Site (.g/l ) { glll Spi ke 109%' Pre App nd ± IHr ± 6. 62' ± Hr ± ± Hr ± ± Hr ± ± Standard recovery 2 nd = Not Detected (limit of detection O. OI g/l), Mean ± Standard deviation (n=3 for test, n=2 for control) 6

10 Exposed larvae were returned to the lab and placed in petri dishes for subsequent long-term mortal ity monitoring. The results, Table 2, show that those larvae exposed to field application did not survive as well as those that remained in the lab. Interpretation of these results relative to abate toxicity is 1 imited, however, because of the absence of field control samples. All larvae died within a day of the molt into the second zoeal stage at day 6. September 6, & 7, 1990 Field Control and Abate Application Studies During September, only the mangrove tree crab, Aratus, was found to be gravid and subsequently released larvae on 9/5/90. A field control test was performed on 9/6/90, followed with a field appl ication test.on 9/7/90. In addition to the crab and mosquito larvae, 14 day old snook fry also were placed in the field trays for observation. A fi e 1 d control study was performed on 9/6, wi th fi e 1 d test sites about 3 hours before high tide. Due to a limited high tide, the tidal water did not reach the test site until one hour after the initial placement, thus changes in sal inity and D. O. placed an excessive stress on both the snook and aratus larvae. The Aratus were replaced at 1:00, after the tidal water had entered the test site, and were monitored again at 4:00, after the high tide had receded. The high mortality, Table 4, reflect the affect of exposing crab and fish larvae to adverse environmental conditions. Once the high tide had entered the test site, the crab larvae exhibited excellent survival. We did not have enough snook fry for repl acement. The effects of abate application on Aratus and Aedes larvae were studied on 9/7/90. These data, Table 5, show 12 of 15 Uca larvae alive after 2 and 4 hours at the control site with 20 out of 30 surviving at the test site, indicating possible effects of abate on the Aratus larvae. Omitting tray #1 due to the impact of heavy organic flock, however, a different view of the situation emerges with 11 out of 15 surviving at 2 and 6 hours at the test site. These results show the importance of careful monitoring and control of environmental conditions and the need for a greater number of larvae to enhance statistical interpretation of the data. field conditions appears to be 70 to 80% The normal mortality of Aratus under over the first 4 hours to tidal flux. The Aratus in-field larvae survival tests show no apparent increase in mortality due to abate. 7

11 Table 4. Survival of crab (Aratus), snook (Centropomus) and mosquito (Aedes) larvae during and following exposure to a saltwater environment: 9/6/90. No abate applied. (gy Time Number of live larvae 0 9/6/90 Aratus CentroQomus Aedes 12:00 noon : Re-stock Aratus from exposed population 2: : Table 5. Number of larvae in each field cone. Test Control Site Test Site Aratus Aedes Aratus Aedes Aratus Aedes T-3 T-3 T -1 T-2 T -1 T-2 A 11.(;. A 11.(;. A 11.(;. A 11.(;. A 11.(; (;. 1: :00 (Accl. ) :30 Abate application 4: ' : ' 0 1 Heavy organic flock inside mesh cones interfering with 1 arva 1 movement 8

12 Abate concentrations in water are given in Table 6. Abate concentrations in filter paper and adult Uca are given in Tabl e 7. These data show a sl ight amount of abate drifting to the control area (filter paper data) but more was detectable in the control site water. An add it i ona 1 important observat i on is the difference between abate concentrations in surface versus subsurface water. Higher abate concentrations at the surface imply a greater exposure and impac on those organisms (mosquito larvae) in contact with the surface, whereas these organisms not in contact with the surface (crab larvae) would experience a much lower exposure level. C. Problems 1. Organic flock and suspended particles introduced into some larval cones during monitoring activities. 2. Lack of proper food to sustain growth of Uca and Aratus past several zoeal larval stages. 3. Difficulty in seeing and counting large numbers of crab larvae (30 or more per site) needed to provide statistical evaluati6n and interpretation of data. 4. Difficulty in maintaining consistent environmental conditions between control and test sites and between application episodes. 9

13 Table 6. Application 2 September Abate Concentrations in Ti da 1 Pool Water Adjacent to Larval Trays. Time Test Site Surface Sub-Surface ("gil) ("gil) Control Site Surface Sub-Surface ("gil ) ("gil ) Spike1 110% 109% Pre App ndz nd nd' nd IHr ± nd nd 2Hr ± ' 4Hr 4.60 ± ± 2.29 nd nd 24Hr 0.76 ± 0.07 nd I z 3 Standard recovery nd = Not detected Mean ± standard deviation (n=2) = Sample not taken ]0

14 Table 7. Abate Appl ication 2, September 7, Abate Application Impacting Filter Paper and in Adult Uca. Test Site Control Site Uca g/g Fi Hers g/m2 Filters g/m2 open canopy open canopy Pre Appl. nd' nd 2. 1 Hr Hr. 0.8 ± 3 24 Hr ± 2 I NO _ Not detected 2 = Sample not collected Mean ± standard deviation (n=3) 11

15 IV. FUTURE STUDIES A. Construct boardwalks to reduce stirring up mud that impacts larvae held in trays. B. Develop proper feeding and handling techniques for rearing Uca and Aratus larvae through all zoeal stages into the megalops stage. C. Complete investigations of the sublethal effects of Temephos and Altosid on the development of crab larvae after exposure to larvicide applications in the saltmarsh. V. ITEMIZED TOTAL HOUR REPORT: Hours charged to project for the pay periods 14 to 20 from June 28, 1990 to October 3, Conner, Troy F. Davis, Andrea S. Evens, Terrence J. Harris, Carmen C Hartsell, M. Lanny Hasbrouck, Patricia Henry, Michael S. Martino, Jason C. Pierce, Richard H. Total Hours