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Degradation and effects of the potential mosquito larvicides methazolamide and acetazolamide in sheepshead minnow (Cyprinodon variegatus)
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Ecotoxicology and Environmental Safety 64 (2006) 369–376 www.elsevier.com/locate/ecoenv
Degradation and effects of the potential mosquito larvicides methazolamide and acetazolamide in sheepshead minnow (Cyprinodon variegatus) Maria del Pilar Corenaa,, Peter van den Hurkb, He Zhongc, Catherine Brockc, Richard Moweryd, Jodie V. Johnsone, Paul J. Linsera a The Whitney Laboratory, University of Florida, St. Augustine, FL 32080, USA Department of Environmental Toxicology, Clemson University, Pendleton, SC, USA c Public Health Entomology Research and Education Center, Panama City, FL, USA d Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA e Chemistry Department, University of Florida, Gainesville, FL, USA
b
Received 20 September 2004; received in revised form 5 April 2005; accepted 14 May 2005 Available online 27 July 2005
Abstract To test for environmental persistence in order to determine the potential of carbonic anhydrase inhibitors as larvicides, the decomposition and degradation of samples containing methazolamide (MTZ) and acetazolamide (ACZ) in aqueous solution were monitored under different conditions. Additionally, nontarget species impact was assessed in an acute toxicity test using sheepshead minnow (Cyprinodon variegatus). The fish were exposed for 120 h to 103 and 104 M each compound in replicate seawater tanks. In the high-MTZ treatment, all fish died within 48 h, while mortality in the low-MTZ treatment was 27% at 120 h. In the high-ACZ treatment mortality reached 83% at 120 h. We observed no mortality for the lowest dose of ACZ. Tissue samples were collected from the fish to investigate absorption of the compounds. In the gills, MTZ concentrations were around 40 mg g1 and ACZ reached concentrations up to 80 mg g1. Liver concentrations were low for MTZ probably due to metabolism. r 2005 Elsevier Inc. All rights reserved. Keywords: Methazolamide; Acetazolamide; Fish; Mosquito larvae; Carbonic anhydrase inhibitors
1. Introduction Alpha carbonic anhydrases (CAs) are among the most conserved enzymes with regard to homology across species. However, sequence homology analysis between the CA isozymes present in mosquito larvae and those present in higher organisms indicates that subtle differences exist in the structure of the active site, which could make these isozymes suitable for larvicidal studies with specific inhibitors (Seron, 2004). Based on this new evidence, we have postulated that CA inhibitors tailored specifically to target particular larval CAs might have Corresponding author. Fax: +1 904 461 4008.
E-mail address: [email protected] (M. del Pilar Corena). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.05.015
potential as larvicides and still exhibit little environmental impact (Corena et al., 2004). This hypothesis is supported by similar research that recently emerged on the design of isozyme-specific and organ-selective CA inhibitors for therapeutic purposes (Supuran et al., 2003). Hundreds of CA inhibitors have been synthesized with only minor differences in their chemical structures. These compounds are regularly used for the treatment of glaucoma and as anticonvulsants, antihypertensives, and, lately, anticancer agents with minimum side effects and adverse reactions (Maren, 1967, 1976; Scozzafava et al., 2003). Furthermore, research has shown that high doses of these drugs can be administered to dogs, rats, and some species of invertebrates without lethal
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consequences (Maren, 1967; Henry et al., 2003). However, this is not the case for mosquito larvae. We have reported previously (Corena et al., 2004) that the sulfonamides methazolamide (MTZ) and acetazolamide (ACZ) have strong effects on the pH balance in the midgut of larval mosquitoes, with lethal effects for several species. Although the use of these inhibitors as larvicides has never been considered until now (primarily because of the high degree of homology among the different CAs in different organisms) the insecticidal activity of sulfonamides closely related to these two compounds has been documented previously (Wagner and Baer, 1965; Beesley and Peters, 1971; Beesley, 1973). The importance of developing new and environmentally safe larvicides becomes apparent as the spread of diseases such as malaria and dengue increases. West Nile virus and the sporadic appearance of foci of malaria in the southern United States are also evidence that the manifestation of these diseases is not limited to developing countries (CDC, 2004). Evidence also suggests the appearance of resistance to some of the available insecticides among mosquito populations worldwide, which exacerbates the problem. Current available mosquito control strategies include, among others, insecticide-impregnated bed nets, source reduction, chemical and biological larvicides, and some adulticides. Available larvicides include biological insecticides such as Bacillus thuringiensis (Bti) and Bacillus sphaericus (Bs) toxins, the chemicals methoprene and temephos, and oil films. Each of these compounds presents advantages and disadvantages with regard to stability, toxicity to nontarget species, and environmental effects (Pinkney et al., 1999). An insecticide must comply with a set of specific requirements before approval for mosquito control use. Among these requirements, it must have a relatively short life due to rapid chemical and biological degradation and this degradation must result in nontoxic compounds (Pinkney et al., 1999). Mosquito insecticides pose some risk to nontarget aquatic species and the aquatic ecosystem. As an example, temephos presents relatively low risk to birds and terrestrial species, but available information suggests that it may be toxic to some aquatic invertebrates (Pinkney et al., 1999). A fine balance between degradation, decomposition, and minimum effect on nontarget species must be achieved before a chemical compound is considered a potential insecticide. With regard to larvicides, the situation is multifaceted since a compound that degrades too quickly might not be as effective as a compound that remains in the water column throughout the developmental cycle of the larvae. Additionally, a compound that decomposes quickly might require several applications to the same site which increases the cost of mosquito control operations. On the other hand, a
chemical that persists in the environment might pose a more long-term risk to nontarget species. Therefore, it is important to determine not only the stability but also the toxicity to nontarget species of a particular chemical intended for larvicidal use. Effects that influence toxicity include, among others, the degree of exposure and stability of the compound and the physical distribution and transport of a particular compound relative to the organism being studied. CA is of special concern in fish toxicology investigations because it has a relatively high activity in gill tissue (VanGoor, 1948). Although the effects of CA inhibitors have been documented in several species of fish, the effect of sulfonamides such as MTZ and ACZ in sheepshead minnow (Cyprinodon variegatus) has not been documented. This species is easy to culture and maintain in the laboratory and is routinely used in fish acute toxicity tests as determined by the United States Environmental Protection Agency (US EPA, 1996). To determine the feasibility of MTZ and ACZ as environmentally acceptable larvicides, we studied their decomposition and degradation products in environmental samples and their toxic effects on sheepshead minnows.
2. Materials and methods 2.1. Reagents MTZ, ACZ, methanol, and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich Corp. (St. Louis, MO, USA) DMSO was used to prepare stocks; then the appropriate dilutions were prepared in water. Phosphate-buffered saline (10 PBS) was prepared by dissolving 80 g of NaCl, 2.0 g of KCl, 14.4 g of Na2PO4, and 2.4 g of KH2PO4 in 800 mL distilled H2O. The pH was adjusted to 7.4 with HCl and the volume was adjusted to 1 L with additional distilled water. The final solution was sterilized in an autoclave and the 1 solution was prepared by diluting accordingly. 2.2. Analysis of decomposition products The parent compounds MTZ and ACZ were analyzed by HPLC by dissolving them in 1500 mL H2O and 500 mL methanol to final concentrations of 6 mM for ACZ and 4 mM for MTZ. To determine the effect of light and temperature on the decomposition of these compounds, stock solutions were prepared in DMSO and 103, 104, 105, and 106 M dilutions were prepared in deionized water. Each sample was prepared in triplicates, kept in a (20-mL) glass vial, and stored at 25 and 37 1C, either exposed for 12 h to sunlight or maintained in the dark for comparison purposes.
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Triplicate samples were kept under these conditions for 1 week and then frozen and kept frozen (in the dark) at 0 1C until analyzed. Sample analysis were performed (directly without derivatization) via reverse-phase highperformance liquid chromatography/electrospray ionization mass spectrometry (HPLC/UV(+)ESI-MS). Data were acquired on a Finnigan (San Jose, CA) LCQ operated in electrospray ionization (ESI) mode. Typical operating parameters were nitrogen sheath and auxiliary gases of 60 and 5 (unitless), respectively, heated capillary temperature of 240 1C, and ion spray voltage of 3.5 kV. The mobile phase was introduced by a Beckman Instruments (Fullerton, CA) System Gold Model 126 pump. A binary HPLC mobile phase system was used. Chromatographic separation was achieved with a Phenomenex (Torrace, CA) LUNA C18 guard column connected to a Waters (Milford, MA) Symmetry Shield RP18 (2.1 mm i.d. 150 mm, 5-mm particles, 100-A˚ pore). Mobile phases used were A ¼ 0.5% acetic acid+5 mM ammonium formate in water and B ¼ 0.5% acetic acid in methanol. A typical gradient at 0.15 mL/min for the Waters column was 0 min (A:B ¼ 100:0), 30 min (A:B ¼ 5:95); Injector, LCQ’s Rheodyne, 25-mL injection loop. UV detector, Applied Biosystems Model 785A Programmable Absorbance Detector, wavelength ¼ 254 nm. 2.3. Analysis of degradation products To determine the degradation rate of these compounds under laboratory conditions in the presence of fish, samples obtained from tanks containing 4 L estuarine water and 10 fish (C. variegatus) per tank were obtained at 24, 48, 72, 96, and 120 h. Each tank per treatment replicate (n ¼ 3) containing 103 and 104 M ACZ and MTZ was sampled in duplicate for each compound. Individual samples were stored in the dark and kept frozen in GC glass vials (1 mL). Sample analysis was performed using an HPLC system equipped with a C18 column (3 mm silica, 150 mm 4.6 mm) using 95% water, 5% acetonitrile (both containing 0.1% trifluoroacetic acid) gradient to 10% water, 90% acetonitrile over 7 min using a flow of 2.5 mL/min. 2.4. Fish toxicity studies Adult sheepshead minnows were collected in Panama City, Florida at the Public Health Entomology Research and Education Center (PHEREC). Ten fish per treatment were tested at two concentrations for each compound in triplicates. Fish were maintained in aerated aquariums with 4 L seawater collected at the estuary at the same location and were allowed to acclimate for 1–2 h. Aliquots of MTZ and ACZ stock solutions were added to each tank to achieve final concentrations of 103 and 104 M. Controls were
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treated with 0.1% DMSO without inhibitor to account for the effect of the solvent. Mortality was assessed after 24, 48, 72, 96, and 120 h. Fish were fed daily according to protocols established at PHEREC, maintained at 25 1C, and kept under a 12-h light/12-h dark cycle. Dead animals were removed, dried, tagged, and kept at 85 1C in plastic Ziploc bags. 2.5. Distribution of ACZ and MTZ in C. variegatus tissue Gills, livers, and female gonads from individual dead C. variegatus used in this study were collected after thawing, weighed, and homogenized in 2 mL PBS after addition of 10 mL internal standard (1 mg/mL; ACZ as standard for MTZ and vice versa). Samples were acidified with 50 mL of HCl (50%) and stored overnight at 4 1C. Extraction and quantification of MTZ and ACZ were performed according to a modification of the methods described by Iyer and Taft (1998). Briefly, stock solutions of MTZ and ACZ were prepared by dissolving these compounds in 5 mM sodium hydroxide. An aliquot of 500 mL of homogenized C. variegatus tissue was analyzed using 10 mL of internal standard. To this, 2.5 mL of 50% ammonium sulfamate was added and the samples were vortexed for 30 s. Samples were placed in boiling water for 30 s and then quickly placed in cold water. Then 3 mL of ethylacetate was added and the samples were vortexed again for 2 min. The mixture was centrifuged at 3000g for 10 min. The organic and aqueous layers were allowed to separate, the organic layer was transferred to a tube containing 500 mL of glycine buffer (50 mM, pH 10.0), and the resultant mixture was vortexed and centrifuged for 5 min. The organic layer was discarded and traces of ethyl acetate were removed from the aqueous phase by extraction with 500 mL of ether. The ether layer was aspirated and the remaining ether was removed under N2 gas after vortexing for 2 min. The resulting solution was used to determine composition by injecting 50-mL aliquots. Samples were analyzed by HPLC on a Waters Symmetry C-18 column (150 4.6 mm, 5 mm) and a mobile phase consisting of phosphate buffer (20 mM, pH 2.6); acetonitrile (0–50%) over 9-min gradient. The mobile phase flow rate and detection wavelength were 2 ml min1 and 268 nm, respectively. Standard curves were obtained by adding exact amounts of compound stock solutions (in 50% acetonitrile) to the glycine backextraction buffer. 3. Results 3.1. Decomposition of ACZ and MTZ Analysis of decomposition products of ACZ and MTZ produced different results for these two
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compounds. ACZ decomposed yielding a detectable decomposition product, UV absorbent with a molecular weight (MW) of 181 Da. The loss of the acetyl group (non-UV absorbent) is revealed in the MS/MS as shown in Fig. 1. The abundance of this 181-Da compound increased with temperature and light as seen in Table 1, where samples exposed to light at 37 1C showed that as much as 1.53% of the 181-Da compound was produced in 7 days. No obvious UV absorbent decomposition products were detected in the MTZ samples.
3.2. Degradation of ACZ and MTZ Degradation of these two sulfonamides was monitored in duplicate samples taken from each tank and analyzed by HPLC. Results obtained are presented in Table 2. ACZ appeared to be more stable to degradation by living organisms than MTZ. After 120 h, 100% of the initial ACZ remained in solution without obvious signs of degradation. In contrast, at 120 h posttreatment, only 53.5% of the initial MTZ remained in solution.
Fig. 1. Decomposition products of ACZ in aqueous solution after 7 days. (A) Loss of an acetyl group from ACZ (m/z 223) generates the m/z 181 [M+H+]+ ion. (B) (+) ESI mass spectrum is dominated by the m/z 223 [M+H+]+ ion (top) which undergoes collision-induced dissociation tandem mass spectrometry readily to form the m/z 181 daughter ion with loss of the acetyl group (bottom).
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3.3. Fish mortality studies To determine the acute mortality effects of these two sulfonamides, concentrations of 103 and 104 M were tested for both ACZ and MTZ. We observed higher fish mortality in the tanks containing MTZ than in those containing ACZ at the same concentration over the same interval of time (Fig. 2). Mortality in the tanks treated with 103 M MTZ reached 100% over 48 h. In contrast, treatment with ACZ at the same concentration reached only 6.7% mortality during the same interval of time. Treatment with 104 M MTZ resulted in 3% observed mortality after 72 h while no mortality was observed for those fish treated with ACZ at the same concentration. The percentage of dead fish in the tanks treated with 104 M MTZ increased up to 26.7% in 120 h. In contrast, in the tanks treated with 104 M ACZ we did not observe any mortality or signs of stress. There was no mortality or signs of stress observed in the DMSO controls that did not contain sulfonamides.
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to 80 mg/g in gill tissue over the first 48 h of exposure, with much lower levels in gonadal tissue (Fig. 3A). Liver samples were also analyzed, but the data were judged to be unreliable because the internal standard (MTZ) appeared to be metabolized during the extraction procedure. Once accumulation was saturated, no significant changes were apparent. Accumulation of MTZ is rapid and throughout the entire organism. This compound was detected (24 h after exposure) in the gills, liver, and gonads at levels close to 40 mg/g (Fig. 3B). After 48 h the levels in the gill remained the same, while levels in liver and gonads appeared to decrease. This may be due to induced enzymatic metabolism. However, no animals survived after 72 h, so declined accumulation may be due to the moribund status of the 48-h animals.
4. Discussion Direct assay of the decomposition and degradation of drugs in biological fluids has relied on HPLC, gas
3.4. Distribution of ACZ and MTZ in C. variegatus tissue Data from dead fish were collected by preparing, homogenizing, and analyzing tissue from each fish separately. Results were averaged for each particular tissue. Our observations showed that ACZ accumulated Table 1 Abundance of the 181-Da decomposition product from ACZ increased with temperature and light Sample ID
25 1C-dark 25 1C-light 37 1C-dark 37 1C-light
MW 181 compound
Acetazolamide (ACZ)
%UV
% MS
%UV
% MS
0.37 3.85 4.06 5.82
0.00 0.83 1.03 1.53
99.63 96.15 95.94 94.18
100.00 99.17 98.97 98.47
Results shown are the average of duplicate samples (standard deviation ¼ 0.08) (% UV ¼ percentage detected by UV detector, % MS ¼ percentage calculated by mass spectrometer).
Fig. 2. Average cumulative fish mortality over time after treatment with ACZ and MTZ. Results shown are the average of three tanks studied per each concentration, in duplicate.
Table 2 Degradation of ACZ and MTZ in estuarine water over time analyzed by HPLC Sample
ACZ MTZ
Initial concentration (mM)
1.0 1.0
Concentration remaining in solution (mM)
% Initial sample remaining in solution after 120 h
After 72 h
After 96 h
After 120 h
1.0 0.58
1.0 0.63a
1.0 0.54
Results shown are the average of two samples from each tank (n ¼ 3, standard deviation ¼ 0.02). a Value is higher than previous one due to evaporation.
100.0 53.5
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Fig. 3. Distribution of ACZ and MTZ in C. variegatus tissue over time as determined by HPLC. Results shown are the average of individual dead fish analysis. (A) ACZ distribution. (B) MTZ distribution (error bars indicate one standard deviation).
chromatography, or MS (Drummer, 1998). The methods available until now to determine MTZ and ACZ have been aimed at their quantitation in biological fluids such as urine and blood. Additionally, the available literature is focused on investigating the decomposition of these drugs in liquid or tablet formulations intended to be administered to humans either intravenously or orally. There are no reports on the analysis of the effect of these two drugs in the environment since these compounds have never been intended to be used in this manner. This study, for the first time, tested the effect of MTZ and ACZ on a nontarget species of fish. The concentrations of ACZ and MTZ were determined using modified HPLC methods (Hossie et al., 1980; Hartley and Lucock, 1986; Parasrampuria and Gupta, 1989). The effects of temperature and the presence or absence of light on the decomposition of these two chemicals were investigated. Degradation in estuarine water was also studied. Finally, their effect on sheepshead minnows was monitored and their distribution in the gills, gonads, and liver of this species was determined.
Studies on the decomposition of ACZ and MTZ have focused primarily on the decomposition of ACZ contained as a base form in tablets or as a sodium salt in injections. Previous studies have shown that the decomposition of ACZ follows a first-order equation (Parasrampuria and Gupta, 1989). ACZ sodium injection is stable for only 1 week at 2–8 1C and stability increases when the base form is used in oral formulations. It has been reported that ACZ in this form retains at least 94% of potency for up to 60 days (Alexander et al., 1991; Parasrampuria and Gupta, 1989, 1990). In this study, we observed minor decomposition of ACZ in aqueous solutions in the absence of microorganisms or fish over a period of 7 days. We did not observe MTZ decomposition under the same conditions. With regard to the products observed, we detected a UV-absorbent compound with a MW of 181 Da. Degradation products for ACZ have been reported as acetic acid and 5-amino-1,3,4-thiadiazole-2-sulfonamide. The latter has a MW of 181 Da (Parasrampuria and Gupta, 1989). Although we did not characterize this compound, it is unlikely that the product of decomposition that we observed differs from 5-amino-1,3,4thiadiazole-2-sulfonamide. We can model the decomposition of ACZ in the absence of living organisms since it follows a first-order equation as reported by Parasrampuria and Gupta (1989). As a first-order equation, the decrease in the concentration of ACZ over time can be written as d½ACZ=dt ¼ k½ACZ,
(1)
where [ACZ] is concentration of ACZ remaining in solution at a given time t and k is the rate constant. Although the accuracy of [ACZ] determined in this study is based on the assumption that the 40 mg/g of ACZ found in gill tissue is a negligible amount compared to the volume of the water added to the tanks, this concentration can be used to estimate the percentage of ACZ that would remain in the tank after a given period of time. Integration of Eq. (1) yields lnð½A=½A0 ¼ kt þ C,
(2)
where C (integration constant) can be evaluated using boundary conditions (when t ¼ 0, [A] ¼ [A]0, and [A]0 is the original starting ACZ concentration). Substituting the values obtained in our study in this equation, we obtained a rate constant (k) of 0.0022 per day. When trying to predict the amount of ACZ that would remain in water in a given period of time, one could use Eq. (2) and substitute the value of 0.0022 for the rate constant. For example, during 3 months (12 weeks or 84 days) of summer the fraction of active ACZ in solution would be 0.83 which corresponds to 83% of the original ACZ. In conclusion we hypothesize that, after 12 weeks, approximately 83% of ACZ would
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remain in aqueous solution (in the absence of living organisms) under the conditions presented in this study, such as light exposure and a water temperature of 25–37 1C (77–98.6 1F). This observation would favor the use of ACZ over MTZ as a potential larvicide. The obvious advantages to a compound that remains active in water for long periods of time with regard to larvicidal activity are the elimination of repeated applications of this chemical in sites containing mosquito larvae at all stages with the associated reduction in costs. With regard to the disadvantages, a chemical that lasts in its active form for long periods of time in the environment could have potential impact on some nontarget species. Our studies with C. variegatus showed that ACZ at concentrations lower than 104 M is not lethal for this species. The sheepshead minnow, perhaps the most commonly tested estuarine fish, was considered one of the most sensitive fish species in toxicological studies using pesticides (Clark et al., 1985). Our results demonstrated that C. variegatus is less sensitive to ACZ than to MTZ. Therefore, we speculate that in species less sensitive than C. variegatus ACZ will have minimum effects with regard to toxicology. However, it will be necessary in the future to determine the LC50 for this compound to determine the lowest dose necessary to have an impact on mosquito larvae without resultant mortality in nontarget species. With regard to the temperatures tested, a range between 77 and 98.6 1F was chosen to include the temperatures encountered in the water during the summer months in the eastern coast of the Gulf of Mexico around Panama City (average of 86 1F) where the fish were collected (NODC, 2005). This range was extended to include body temperature or 37 1C (98.6 1F) to be able to compare the results presented in this study with those presented in the literature. In the presence of living organisms, MTZ (a methylated tautomer of ACZ) appeared to be more readily degraded or absorbed than ACZ with only 50% of this compound remaining in solution after exposure to water containing microorganisms and fish during a period of 5 days. These results were unexpected as our results of decomposition showed that MTZ was very resistant to decomposition by heat or light. This compound would completely degrade according to our results in the presence of fish and microorganisms in 10 days if decomposition follows a first-order equation as that of ACZ. The advantages of using MTZ as a larvicide in this respect would be obvious since this compound would degrade quickly in the environment, minimizing the exposure and accumulation in aquatic organisms. However, the rapid degradation of this compound in the environment would require repeated applications to observe efficient larval control. Overall, when comparing these two inhibitors under the conditions presented in this study, ACZ appears to be a more suitable
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compound to use due to its low toxicity to sheepshead minnows and its persistence in the environment. With regard to fish mortality, the presence of CA has been documented in several species (Carter et al., 1976; Dimberg et al., 1981; Desforges et al., 2001). It is not surprising that we found these two inhibitors associated with fish tissue such as the gills, gonads, and liver. CA inhibitors, when administered intravenously, appear to have an effect on fish respiration and renal physiology (Maren et al., 1992; Henry et al., 1995; Peterson et al., 1997). This is in agreement with our observations of fish mortality at the highest concentrations of these compounds. With regard to metabolism of ACZ and MTZ, it has been reported that these two compounds are very resistant to both chemical and enzymatic hydrolysis. Extreme conditions are normally required for the chemical cleavage of sulfonamides to liberate the corresponding amines (Kim et al., 1990; Greene and Wuts, 1991). However, nonenzymatic conjugation of MTZ to glutathione has been described (Conroy et al., 1984) and the nonenzymatic sulfonamide cleavage of MTZ has been reported in in vivo studies (Maren et al., 1977; Kishida et al., 1990). Recently the involvement of glutathione-S-transferase in sulfonamide cleavage was demonstrated (Koeplinger et al., 1999). Preliminary results from experiments with mummichog (Fundulus heteroclitus), another estuarine fish species, indicate that ACZ is not metabolized, while MTZ is rapidly conjugated to glutathione in a reaction catalyzed by glutathione-S-transferase (P. van den Hurk, unpublished results). A similar pathway could explain the results that we observed in C. variegatus liver. However, future experiments will be needed to test this hypothesis. We conclude that there is potential in the use of CA inhibitors as mosquito larvicides. The primary target would be the mosquito larval midgut which exhibits an extremely alkaline pH (10–11). Therefore, one possibility would be to encapsulate these inhibitors in pHsensitive formulations designed to release the compound only at these extremely high pH values. The use of controlled-release formulations of mosquito larvicides would reduce the frequency of application and losses due to degradation of the insecticide compared with conventional formulations. The use of mosquito CAspecific inhibitors will likely reduce the mortality in nontarget species associated with these compounds. Additionally, the design of mosquito CA-specific inhibitors and slow-release formulations or the use of similar CA inhibitors chemically modified to specifically bind the mosquito enzyme at high pH would decrease the toxicity on nontarget species or the problems associated with the stability of these compounds in water. This study is the first attempt to identify the potential environmental problems associated with the use of CA inhibitors as larvicides. Further testing is
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required to determine the impact of ACZ and MTZ and other CA inhibitors on different nontarget species before consideration of these compounds as potential larvicidal formulations.
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Ecotoxicology and Environmental Safety 64 (2006) 369–376 www.elsevier.com/locate/ecoenv
Degradation and effects of the potential mosquito larvicides methazolamide and acetazolamide in sheepshead minnow (Cyprinodon variegatus) Maria del Pilar Corenaa,, Peter van den Hurkb, He Zhongc, Catherine Brockc, Richard Moweryd, Jodie V. Johnsone, Paul J. Linsera a The Whitney Laboratory, University of Florida, St. Augustine, FL 32080, USA Department of Environmental Toxicology, Clemson University, Pendleton, SC, USA c Public Health Entomology Research and Education Center, Panama City, FL, USA d Department of Chemistry and Biochemistry, Baylor University, Waco, TX, USA e Chemistry Department, University of Florida, Gainesville, FL, USA
b
Received 20 September 2004; received in revised form 5 April 2005; accepted 14 May 2005 Available online 27 July 2005
Abstract To test for environmental persistence in order to determine the potential of carbonic anhydrase inhibitors as larvicides, the decomposition and degradation of samples containing methazolamide (MTZ) and acetazolamide (ACZ) in aqueous solution were monitored under different conditions. Additionally, nontarget species impact was assessed in an acute toxicity test using sheepshead minnow (Cyprinodon variegatus). The fish were exposed for 120 h to 103 and 104 M each compound in replicate seawater tanks. In the high-MTZ treatment, all fish died within 48 h, while mortality in the low-MTZ treatment was 27% at 120 h. In the high-ACZ treatment mortality reached 83% at 120 h. We observed no mortality for the lowest dose of ACZ. Tissue samples were collected from the fish to investigate absorption of the compounds. In the gills, MTZ concentrations were around 40 mg g1 and ACZ reached concentrations up to 80 mg g1. Liver concentrations were low for MTZ probably due to metabolism. r 2005 Elsevier Inc. All rights reserved. Keywords: Methazolamide; Acetazolamide; Fish; Mosquito larvae; Carbonic anhydrase inhibitors
1. Introduction Alpha carbonic anhydrases (CAs) are among the most conserved enzymes with regard to homology across species. However, sequence homology analysis between the CA isozymes present in mosquito larvae and those present in higher organisms indicates that subtle differences exist in the structure of the active site, which could make these isozymes suitable for larvicidal studies with specific inhibitors (Seron, 2004). Based on this new evidence, we have postulated that CA inhibitors tailored specifically to target particular larval CAs might have Corresponding author. Fax: +1 904 461 4008.
E-mail address: [email protected] (M. del Pilar Corena). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.05.015
potential as larvicides and still exhibit little environmental impact (Corena et al., 2004). This hypothesis is supported by similar research that recently emerged on the design of isozyme-specific and organ-selective CA inhibitors for therapeutic purposes (Supuran et al., 2003). Hundreds of CA inhibitors have been synthesized with only minor differences in their chemical structures. These compounds are regularly used for the treatment of glaucoma and as anticonvulsants, antihypertensives, and, lately, anticancer agents with minimum side effects and adverse reactions (Maren, 1967, 1976; Scozzafava et al., 2003). Furthermore, research has shown that high doses of these drugs can be administered to dogs, rats, and some species of invertebrates without lethal
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consequences (Maren, 1967; Henry et al., 2003). However, this is not the case for mosquito larvae. We have reported previously (Corena et al., 2004) that the sulfonamides methazolamide (MTZ) and acetazolamide (ACZ) have strong effects on the pH balance in the midgut of larval mosquitoes, with lethal effects for several species. Although the use of these inhibitors as larvicides has never been considered until now (primarily because of the high degree of homology among the different CAs in different organisms) the insecticidal activity of sulfonamides closely related to these two compounds has been documented previously (Wagner and Baer, 1965; Beesley and Peters, 1971; Beesley, 1973). The importance of developing new and environmentally safe larvicides becomes apparent as the spread of diseases such as malaria and dengue increases. West Nile virus and the sporadic appearance of foci of malaria in the southern United States are also evidence that the manifestation of these diseases is not limited to developing countries (CDC, 2004). Evidence also suggests the appearance of resistance to some of the available insecticides among mosquito populations worldwide, which exacerbates the problem. Current available mosquito control strategies include, among others, insecticide-impregnated bed nets, source reduction, chemical and biological larvicides, and some adulticides. Available larvicides include biological insecticides such as Bacillus thuringiensis (Bti) and Bacillus sphaericus (Bs) toxins, the chemicals methoprene and temephos, and oil films. Each of these compounds presents advantages and disadvantages with regard to stability, toxicity to nontarget species, and environmental effects (Pinkney et al., 1999). An insecticide must comply with a set of specific requirements before approval for mosquito control use. Among these requirements, it must have a relatively short life due to rapid chemical and biological degradation and this degradation must result in nontoxic compounds (Pinkney et al., 1999). Mosquito insecticides pose some risk to nontarget aquatic species and the aquatic ecosystem. As an example, temephos presents relatively low risk to birds and terrestrial species, but available information suggests that it may be toxic to some aquatic invertebrates (Pinkney et al., 1999). A fine balance between degradation, decomposition, and minimum effect on nontarget species must be achieved before a chemical compound is considered a potential insecticide. With regard to larvicides, the situation is multifaceted since a compound that degrades too quickly might not be as effective as a compound that remains in the water column throughout the developmental cycle of the larvae. Additionally, a compound that decomposes quickly might require several applications to the same site which increases the cost of mosquito control operations. On the other hand, a
chemical that persists in the environment might pose a more long-term risk to nontarget species. Therefore, it is important to determine not only the stability but also the toxicity to nontarget species of a particular chemical intended for larvicidal use. Effects that influence toxicity include, among others, the degree of exposure and stability of the compound and the physical distribution and transport of a particular compound relative to the organism being studied. CA is of special concern in fish toxicology investigations because it has a relatively high activity in gill tissue (VanGoor, 1948). Although the effects of CA inhibitors have been documented in several species of fish, the effect of sulfonamides such as MTZ and ACZ in sheepshead minnow (Cyprinodon variegatus) has not been documented. This species is easy to culture and maintain in the laboratory and is routinely used in fish acute toxicity tests as determined by the United States Environmental Protection Agency (US EPA, 1996). To determine the feasibility of MTZ and ACZ as environmentally acceptable larvicides, we studied their decomposition and degradation products in environmental samples and their toxic effects on sheepshead minnows.
2. Materials and methods 2.1. Reagents MTZ, ACZ, methanol, and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich Corp. (St. Louis, MO, USA) DMSO was used to prepare stocks; then the appropriate dilutions were prepared in water. Phosphate-buffered saline (10 PBS) was prepared by dissolving 80 g of NaCl, 2.0 g of KCl, 14.4 g of Na2PO4, and 2.4 g of KH2PO4 in 800 mL distilled H2O. The pH was adjusted to 7.4 with HCl and the volume was adjusted to 1 L with additional distilled water. The final solution was sterilized in an autoclave and the 1 solution was prepared by diluting accordingly. 2.2. Analysis of decomposition products The parent compounds MTZ and ACZ were analyzed by HPLC by dissolving them in 1500 mL H2O and 500 mL methanol to final concentrations of 6 mM for ACZ and 4 mM for MTZ. To determine the effect of light and temperature on the decomposition of these compounds, stock solutions were prepared in DMSO and 103, 104, 105, and 106 M dilutions were prepared in deionized water. Each sample was prepared in triplicates, kept in a (20-mL) glass vial, and stored at 25 and 37 1C, either exposed for 12 h to sunlight or maintained in the dark for comparison purposes.
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Triplicate samples were kept under these conditions for 1 week and then frozen and kept frozen (in the dark) at 0 1C until analyzed. Sample analysis were performed (directly without derivatization) via reverse-phase highperformance liquid chromatography/electrospray ionization mass spectrometry (HPLC/UV(+)ESI-MS). Data were acquired on a Finnigan (San Jose, CA) LCQ operated in electrospray ionization (ESI) mode. Typical operating parameters were nitrogen sheath and auxiliary gases of 60 and 5 (unitless), respectively, heated capillary temperature of 240 1C, and ion spray voltage of 3.5 kV. The mobile phase was introduced by a Beckman Instruments (Fullerton, CA) System Gold Model 126 pump. A binary HPLC mobile phase system was used. Chromatographic separation was achieved with a Phenomenex (Torrace, CA) LUNA C18 guard column connected to a Waters (Milford, MA) Symmetry Shield RP18 (2.1 mm i.d. 150 mm, 5-mm particles, 100-A˚ pore). Mobile phases used were A ¼ 0.5% acetic acid+5 mM ammonium formate in water and B ¼ 0.5% acetic acid in methanol. A typical gradient at 0.15 mL/min for the Waters column was 0 min (A:B ¼ 100:0), 30 min (A:B ¼ 5:95); Injector, LCQ’s Rheodyne, 25-mL injection loop. UV detector, Applied Biosystems Model 785A Programmable Absorbance Detector, wavelength ¼ 254 nm. 2.3. Analysis of degradation products To determine the degradation rate of these compounds under laboratory conditions in the presence of fish, samples obtained from tanks containing 4 L estuarine water and 10 fish (C. variegatus) per tank were obtained at 24, 48, 72, 96, and 120 h. Each tank per treatment replicate (n ¼ 3) containing 103 and 104 M ACZ and MTZ was sampled in duplicate for each compound. Individual samples were stored in the dark and kept frozen in GC glass vials (1 mL). Sample analysis was performed using an HPLC system equipped with a C18 column (3 mm silica, 150 mm 4.6 mm) using 95% water, 5% acetonitrile (both containing 0.1% trifluoroacetic acid) gradient to 10% water, 90% acetonitrile over 7 min using a flow of 2.5 mL/min. 2.4. Fish toxicity studies Adult sheepshead minnows were collected in Panama City, Florida at the Public Health Entomology Research and Education Center (PHEREC). Ten fish per treatment were tested at two concentrations for each compound in triplicates. Fish were maintained in aerated aquariums with 4 L seawater collected at the estuary at the same location and were allowed to acclimate for 1–2 h. Aliquots of MTZ and ACZ stock solutions were added to each tank to achieve final concentrations of 103 and 104 M. Controls were
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treated with 0.1% DMSO without inhibitor to account for the effect of the solvent. Mortality was assessed after 24, 48, 72, 96, and 120 h. Fish were fed daily according to protocols established at PHEREC, maintained at 25 1C, and kept under a 12-h light/12-h dark cycle. Dead animals were removed, dried, tagged, and kept at 85 1C in plastic Ziploc bags. 2.5. Distribution of ACZ and MTZ in C. variegatus tissue Gills, livers, and female gonads from individual dead C. variegatus used in this study were collected after thawing, weighed, and homogenized in 2 mL PBS after addition of 10 mL internal standard (1 mg/mL; ACZ as standard for MTZ and vice versa). Samples were acidified with 50 mL of HCl (50%) and stored overnight at 4 1C. Extraction and quantification of MTZ and ACZ were performed according to a modification of the methods described by Iyer and Taft (1998). Briefly, stock solutions of MTZ and ACZ were prepared by dissolving these compounds in 5 mM sodium hydroxide. An aliquot of 500 mL of homogenized C. variegatus tissue was analyzed using 10 mL of internal standard. To this, 2.5 mL of 50% ammonium sulfamate was added and the samples were vortexed for 30 s. Samples were placed in boiling water for 30 s and then quickly placed in cold water. Then 3 mL of ethylacetate was added and the samples were vortexed again for 2 min. The mixture was centrifuged at 3000g for 10 min. The organic and aqueous layers were allowed to separate, the organic layer was transferred to a tube containing 500 mL of glycine buffer (50 mM, pH 10.0), and the resultant mixture was vortexed and centrifuged for 5 min. The organic layer was discarded and traces of ethyl acetate were removed from the aqueous phase by extraction with 500 mL of ether. The ether layer was aspirated and the remaining ether was removed under N2 gas after vortexing for 2 min. The resulting solution was used to determine composition by injecting 50-mL aliquots. Samples were analyzed by HPLC on a Waters Symmetry C-18 column (150 4.6 mm, 5 mm) and a mobile phase consisting of phosphate buffer (20 mM, pH 2.6); acetonitrile (0–50%) over 9-min gradient. The mobile phase flow rate and detection wavelength were 2 ml min1 and 268 nm, respectively. Standard curves were obtained by adding exact amounts of compound stock solutions (in 50% acetonitrile) to the glycine backextraction buffer. 3. Results 3.1. Decomposition of ACZ and MTZ Analysis of decomposition products of ACZ and MTZ produced different results for these two
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compounds. ACZ decomposed yielding a detectable decomposition product, UV absorbent with a molecular weight (MW) of 181 Da. The loss of the acetyl group (non-UV absorbent) is revealed in the MS/MS as shown in Fig. 1. The abundance of this 181-Da compound increased with temperature and light as seen in Table 1, where samples exposed to light at 37 1C showed that as much as 1.53% of the 181-Da compound was produced in 7 days. No obvious UV absorbent decomposition products were detected in the MTZ samples.
3.2. Degradation of ACZ and MTZ Degradation of these two sulfonamides was monitored in duplicate samples taken from each tank and analyzed by HPLC. Results obtained are presented in Table 2. ACZ appeared to be more stable to degradation by living organisms than MTZ. After 120 h, 100% of the initial ACZ remained in solution without obvious signs of degradation. In contrast, at 120 h posttreatment, only 53.5% of the initial MTZ remained in solution.
Fig. 1. Decomposition products of ACZ in aqueous solution after 7 days. (A) Loss of an acetyl group from ACZ (m/z 223) generates the m/z 181 [M+H+]+ ion. (B) (+) ESI mass spectrum is dominated by the m/z 223 [M+H+]+ ion (top) which undergoes collision-induced dissociation tandem mass spectrometry readily to form the m/z 181 daughter ion with loss of the acetyl group (bottom).
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3.3. Fish mortality studies To determine the acute mortality effects of these two sulfonamides, concentrations of 103 and 104 M were tested for both ACZ and MTZ. We observed higher fish mortality in the tanks containing MTZ than in those containing ACZ at the same concentration over the same interval of time (Fig. 2). Mortality in the tanks treated with 103 M MTZ reached 100% over 48 h. In contrast, treatment with ACZ at the same concentration reached only 6.7% mortality during the same interval of time. Treatment with 104 M MTZ resulted in 3% observed mortality after 72 h while no mortality was observed for those fish treated with ACZ at the same concentration. The percentage of dead fish in the tanks treated with 104 M MTZ increased up to 26.7% in 120 h. In contrast, in the tanks treated with 104 M ACZ we did not observe any mortality or signs of stress. There was no mortality or signs of stress observed in the DMSO controls that did not contain sulfonamides.
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to 80 mg/g in gill tissue over the first 48 h of exposure, with much lower levels in gonadal tissue (Fig. 3A). Liver samples were also analyzed, but the data were judged to be unreliable because the internal standard (MTZ) appeared to be metabolized during the extraction procedure. Once accumulation was saturated, no significant changes were apparent. Accumulation of MTZ is rapid and throughout the entire organism. This compound was detected (24 h after exposure) in the gills, liver, and gonads at levels close to 40 mg/g (Fig. 3B). After 48 h the levels in the gill remained the same, while levels in liver and gonads appeared to decrease. This may be due to induced enzymatic metabolism. However, no animals survived after 72 h, so declined accumulation may be due to the moribund status of the 48-h animals.
4. Discussion Direct assay of the decomposition and degradation of drugs in biological fluids has relied on HPLC, gas
3.4. Distribution of ACZ and MTZ in C. variegatus tissue Data from dead fish were collected by preparing, homogenizing, and analyzing tissue from each fish separately. Results were averaged for each particular tissue. Our observations showed that ACZ accumulated Table 1 Abundance of the 181-Da decomposition product from ACZ increased with temperature and light Sample ID
25 1C-dark 25 1C-light 37 1C-dark 37 1C-light
MW 181 compound
Acetazolamide (ACZ)
%UV
% MS
%UV
% MS
0.37 3.85 4.06 5.82
0.00 0.83 1.03 1.53
99.63 96.15 95.94 94.18
100.00 99.17 98.97 98.47
Results shown are the average of duplicate samples (standard deviation ¼ 0.08) (% UV ¼ percentage detected by UV detector, % MS ¼ percentage calculated by mass spectrometer).
Fig. 2. Average cumulative fish mortality over time after treatment with ACZ and MTZ. Results shown are the average of three tanks studied per each concentration, in duplicate.
Table 2 Degradation of ACZ and MTZ in estuarine water over time analyzed by HPLC Sample
ACZ MTZ
Initial concentration (mM)
1.0 1.0
Concentration remaining in solution (mM)
% Initial sample remaining in solution after 120 h
After 72 h
After 96 h
After 120 h
1.0 0.58
1.0 0.63a
1.0 0.54
Results shown are the average of two samples from each tank (n ¼ 3, standard deviation ¼ 0.02). a Value is higher than previous one due to evaporation.
100.0 53.5
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Fig. 3. Distribution of ACZ and MTZ in C. variegatus tissue over time as determined by HPLC. Results shown are the average of individual dead fish analysis. (A) ACZ distribution. (B) MTZ distribution (error bars indicate one standard deviation).
chromatography, or MS (Drummer, 1998). The methods available until now to determine MTZ and ACZ have been aimed at their quantitation in biological fluids such as urine and blood. Additionally, the available literature is focused on investigating the decomposition of these drugs in liquid or tablet formulations intended to be administered to humans either intravenously or orally. There are no reports on the analysis of the effect of these two drugs in the environment since these compounds have never been intended to be used in this manner. This study, for the first time, tested the effect of MTZ and ACZ on a nontarget species of fish. The concentrations of ACZ and MTZ were determined using modified HPLC methods (Hossie et al., 1980; Hartley and Lucock, 1986; Parasrampuria and Gupta, 1989). The effects of temperature and the presence or absence of light on the decomposition of these two chemicals were investigated. Degradation in estuarine water was also studied. Finally, their effect on sheepshead minnows was monitored and their distribution in the gills, gonads, and liver of this species was determined.
Studies on the decomposition of ACZ and MTZ have focused primarily on the decomposition of ACZ contained as a base form in tablets or as a sodium salt in injections. Previous studies have shown that the decomposition of ACZ follows a first-order equation (Parasrampuria and Gupta, 1989). ACZ sodium injection is stable for only 1 week at 2–8 1C and stability increases when the base form is used in oral formulations. It has been reported that ACZ in this form retains at least 94% of potency for up to 60 days (Alexander et al., 1991; Parasrampuria and Gupta, 1989, 1990). In this study, we observed minor decomposition of ACZ in aqueous solutions in the absence of microorganisms or fish over a period of 7 days. We did not observe MTZ decomposition under the same conditions. With regard to the products observed, we detected a UV-absorbent compound with a MW of 181 Da. Degradation products for ACZ have been reported as acetic acid and 5-amino-1,3,4-thiadiazole-2-sulfonamide. The latter has a MW of 181 Da (Parasrampuria and Gupta, 1989). Although we did not characterize this compound, it is unlikely that the product of decomposition that we observed differs from 5-amino-1,3,4thiadiazole-2-sulfonamide. We can model the decomposition of ACZ in the absence of living organisms since it follows a first-order equation as reported by Parasrampuria and Gupta (1989). As a first-order equation, the decrease in the concentration of ACZ over time can be written as d½ACZ=dt ¼ k½ACZ,
(1)
where [ACZ] is concentration of ACZ remaining in solution at a given time t and k is the rate constant. Although the accuracy of [ACZ] determined in this study is based on the assumption that the 40 mg/g of ACZ found in gill tissue is a negligible amount compared to the volume of the water added to the tanks, this concentration can be used to estimate the percentage of ACZ that would remain in the tank after a given period of time. Integration of Eq. (1) yields lnð½A=½A0 ¼ kt þ C,
(2)
where C (integration constant) can be evaluated using boundary conditions (when t ¼ 0, [A] ¼ [A]0, and [A]0 is the original starting ACZ concentration). Substituting the values obtained in our study in this equation, we obtained a rate constant (k) of 0.0022 per day. When trying to predict the amount of ACZ that would remain in water in a given period of time, one could use Eq. (2) and substitute the value of 0.0022 for the rate constant. For example, during 3 months (12 weeks or 84 days) of summer the fraction of active ACZ in solution would be 0.83 which corresponds to 83% of the original ACZ. In conclusion we hypothesize that, after 12 weeks, approximately 83% of ACZ would
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remain in aqueous solution (in the absence of living organisms) under the conditions presented in this study, such as light exposure and a water temperature of 25–37 1C (77–98.6 1F). This observation would favor the use of ACZ over MTZ as a potential larvicide. The obvious advantages to a compound that remains active in water for long periods of time with regard to larvicidal activity are the elimination of repeated applications of this chemical in sites containing mosquito larvae at all stages with the associated reduction in costs. With regard to the disadvantages, a chemical that lasts in its active form for long periods of time in the environment could have potential impact on some nontarget species. Our studies with C. variegatus showed that ACZ at concentrations lower than 104 M is not lethal for this species. The sheepshead minnow, perhaps the most commonly tested estuarine fish, was considered one of the most sensitive fish species in toxicological studies using pesticides (Clark et al., 1985). Our results demonstrated that C. variegatus is less sensitive to ACZ than to MTZ. Therefore, we speculate that in species less sensitive than C. variegatus ACZ will have minimum effects with regard to toxicology. However, it will be necessary in the future to determine the LC50 for this compound to determine the lowest dose necessary to have an impact on mosquito larvae without resultant mortality in nontarget species. With regard to the temperatures tested, a range between 77 and 98.6 1F was chosen to include the temperatures encountered in the water during the summer months in the eastern coast of the Gulf of Mexico around Panama City (average of 86 1F) where the fish were collected (NODC, 2005). This range was extended to include body temperature or 37 1C (98.6 1F) to be able to compare the results presented in this study with those presented in the literature. In the presence of living organisms, MTZ (a methylated tautomer of ACZ) appeared to be more readily degraded or absorbed than ACZ with only 50% of this compound remaining in solution after exposure to water containing microorganisms and fish during a period of 5 days. These results were unexpected as our results of decomposition showed that MTZ was very resistant to decomposition by heat or light. This compound would completely degrade according to our results in the presence of fish and microorganisms in 10 days if decomposition follows a first-order equation as that of ACZ. The advantages of using MTZ as a larvicide in this respect would be obvious since this compound would degrade quickly in the environment, minimizing the exposure and accumulation in aquatic organisms. However, the rapid degradation of this compound in the environment would require repeated applications to observe efficient larval control. Overall, when comparing these two inhibitors under the conditions presented in this study, ACZ appears to be a more suitable
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compound to use due to its low toxicity to sheepshead minnows and its persistence in the environment. With regard to fish mortality, the presence of CA has been documented in several species (Carter et al., 1976; Dimberg et al., 1981; Desforges et al., 2001). It is not surprising that we found these two inhibitors associated with fish tissue such as the gills, gonads, and liver. CA inhibitors, when administered intravenously, appear to have an effect on fish respiration and renal physiology (Maren et al., 1992; Henry et al., 1995; Peterson et al., 1997). This is in agreement with our observations of fish mortality at the highest concentrations of these compounds. With regard to metabolism of ACZ and MTZ, it has been reported that these two compounds are very resistant to both chemical and enzymatic hydrolysis. Extreme conditions are normally required for the chemical cleavage of sulfonamides to liberate the corresponding amines (Kim et al., 1990; Greene and Wuts, 1991). However, nonenzymatic conjugation of MTZ to glutathione has been described (Conroy et al., 1984) and the nonenzymatic sulfonamide cleavage of MTZ has been reported in in vivo studies (Maren et al., 1977; Kishida et al., 1990). Recently the involvement of glutathione-S-transferase in sulfonamide cleavage was demonstrated (Koeplinger et al., 1999). Preliminary results from experiments with mummichog (Fundulus heteroclitus), another estuarine fish species, indicate that ACZ is not metabolized, while MTZ is rapidly conjugated to glutathione in a reaction catalyzed by glutathione-S-transferase (P. van den Hurk, unpublished results). A similar pathway could explain the results that we observed in C. variegatus liver. However, future experiments will be needed to test this hypothesis. We conclude that there is potential in the use of CA inhibitors as mosquito larvicides. The primary target would be the mosquito larval midgut which exhibits an extremely alkaline pH (10–11). Therefore, one possibility would be to encapsulate these inhibitors in pHsensitive formulations designed to release the compound only at these extremely high pH values. The use of controlled-release formulations of mosquito larvicides would reduce the frequency of application and losses due to degradation of the insecticide compared with conventional formulations. The use of mosquito CAspecific inhibitors will likely reduce the mortality in nontarget species associated with these compounds. Additionally, the design of mosquito CA-specific inhibitors and slow-release formulations or the use of similar CA inhibitors chemically modified to specifically bind the mosquito enzyme at high pH would decrease the toxicity on nontarget species or the problems associated with the stability of these compounds in water. This study is the first attempt to identify the potential environmental problems associated with the use of CA inhibitors as larvicides. Further testing is
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required to determine the impact of ACZ and MTZ and other CA inhibitors on different nontarget species before consideration of these compounds as potential larvicidal formulations.
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