Abstract
Industrial or urban wastewater treatment lagoons accumulate nutrients, which allows the proliferation of Cx. quinquefasciatus, vector of the etiologic agent of filariasis, and of Cx. saltanensis, vector of the protozoan causing malaria in chickens. The goal of this study was to evaluate the effectiveness and persistence of Aquabac® XT 1,200 UTI/mg, Teknar® 3,000 AAU/mg, and Vectobac® AS 1,200 UTI/mg; these products are liquid formulations whose active principles are crystals produced by Bacillus thuringiensis israelensis. Products were tested in two wastewater treatment lagoons of a meat cold-storage facility. The lagoons measured 1,419 mβ and 736 mβ, and received concentrations of 1 and 2 litres/hectare of each product, with three replicates each. Water pH, conductivity, oxygen and temperature were measured at each collection. Cx. quinquefasciatus and Cx. saltanensis were found in both lagoons. One litre/hectare concentration controlled 70 to 80% of the larvae in 24 and 48 hours, respectively; two litres/hectare concentration showed the best results, controlling 86 to 99% of the immature mosquitoes, with the highest efficiency observed at 48 hours. None of the products affected water quality according to the abiotic parameters examined. As a result, all products tested efficiently controlled these Culicidae under the local breeding conditions but had low persistence, with seventh-day larval indexes being similar to those recorded before application of the products. Weekly applications are recommended.
Introduction
The nutrients in decomposing matter found in effluent treatment lagoons promote physical and chemical changes in water. Such changes hinder the preservation of the aerobic aquatic fauna and enhance the breeding of mosquito larvae such as Cx. quinquefasciatus Say, 1823 and Cx. saltanensis Dyar, 1928 (O'Meara 2010).
Cx. quinquefasciatus is an urban and cosmopolitan mosquito, considered the main vector of the etiologic agent of filariasis in Brazil. The pathogen is transmitted in cities such as Manaus, Belém, Recife, Maceió, and Salvador, and other locations in the country (Deane 1951; Rachou 1956). Currently, only the metropolitan area of Recife, Pernambuco is considered an endemic area (Medeiros et al. 2003). According to the World Health Organization (WHO), 81 countries were endemic for the disease by the end of the year 2007, where 750 million people underwent treatment, and the aim has been to eradicate the disease (WHO 2008). The control of the arthropod vector is one of the strategies undertaken for such purpose.
Cx. saltanensis is an ornithophilic, neotropical mosquito (Lourenço-de-Oliveira & Heyden 1986) that can be infected with Plasmodium cathemerium, a sparrow homospory (Gabaldon et al. 1988). It has also been considered the primary vector of Plasmodium juxtanucleare (Lourenço-de-Oliveira & Castro 1991), etiologic agent of malaria in gallinaceous birds. Sibajev et al. (1993) described Crithidia ricardoi, a new species of Trypanosomatidae, where Cx. saltanensis is considered the primary host. Cx. saltanensis colonizes effluent treatment lagoons and has been the only mosquito species found in sanitary landfill effluent lagoons in Londrina, Paraná State (Zequi and Lopes 2007).
Both mosquito species are urban vectors, engage in disturbing behaviour during female blood meals, and cause allergies. They are also potential vectors for West Nile virus (WNV), which has been detected in horses in Mato Grosso do Sul (Pauvolid-Correa et al. 2011). Because they worsen the quality of life of people living near artificial breeding sites, integrated vector management or control programs are needed. One of the ecologically safest and most efficient biological control methods available is the bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) in effluent treatment lagoons or other aquatic environments. The efficiency of Bti has been reported for Culex spp. (Amalraj et al. 2000; Hallmon et al. 2000; Gunasekaran et al. 2002; Zequi and Lopes 2007; Bravo et al. 2011), although with a persistence of less than 30 days (Amalraj et al. 2000; Hallmon et al. 2000; Morais et al. 2007; Zequi and Lopes 2007). Under some conditions, persistent control of Aedes aegypti (Linnaeus, 1762) by Bti is more than 100 days (Mulla et al. 2004) or more than five months (Benjamin et al. 2005; Melo-Santos et al. 2009; Ritchie et al. 2010). The efficiency of Bti is inversely proportional to the increase in organic matter (Tetreau et al. 2012).
Appropriate biological formulations for effluent treatment lagoons still need to be tested to ensure greater success in controlling these mosquitoes in urban areas. The efficacy of entomopathogenic products depends on several environmental factors, including water quality at the breeding sites, nutrient availability, local climate conditions, number of larvae in the lagoon and solar radiation (Mulla et al. 1984; Consoli et al. 1995; Lacey 2007).
This study aimed to evaluate the efficiency and persistence of three aqueous suspensions containing B. thuringiensis israelensis and to simultaneously monitor water quality (pH, conductivity, and dissolved and saturated oxygen levels) in two wastewater treatment lagoons of a meat cold-storage facility in Jataizinho and Arapongas (Paraná, Brazil).
Material and methods
Procedures and tests.
Aqueous suspensions of Aquabac® XT 1,200 UTI/mg (lot F295), Teknar® 3,000 AAU/mg (lot A206673) and Vectobac® AS 1,200 UTI/mg (lot 69-149- N9) were used for the field trials. One urban effluent treatment lagoon was selected in each of the two municipalities studied, Jataizinho and Arapongas, both in the state of Paraná. Jataizinho lagoon measured 33 x 43 m (1,419 mβ) and received waste from a swine slaughterhouse. Arapongas lagoon was 33 x 23 m (736 mβ), with effluents from a cattle slaughterhouse. Product concentration was determined according to the length and width of each lagoon. Larval colonization site or the site to be treated was defined as the quadrant obtained one meter away from the water's edge and one meter deep, typically the area where larvae occur and feed.
The starting concentration patterns for product application at both sites were one and two liters per hectare following the manufacturers' recommendations for polluted water or high concentration of larvae. To simulate the actual field conditions, the time intervals for each repetition of the applications were defined according to the persistence of the product and initial larval recovery rates in each lagoon. Three applications of each concentration of bio-insecticides were applied every seven days in different ponds. Arapongas lagoon was used to test Vectobac 1 L/ha (March 10-31, 2004), Teknar 1 L/ha (March 31 to April 21, 2004), and Teknar 2 L/ha (April 28 to May 19, 2004). The following products were applied to the Jataizinho lagoon: Aquabac 1 L/ha (March 31 to April 21, 2004), Aquabac 2 L/ha (April 28 to May 19, 2004), and Vectobac 2 L/ha (September 22 to October 13, 2004). The bioinsecticides were applied with a multi-spray atomizing pump in each repetition when the levels of initial larval infestation in ponds were checked.
Measurement of Abiotic Factors.
Water pH, conductivity and dissolved and saturated oxygen were checked before each application of products and collection of immatures, using Gehaka CG 220, Gehaka PG 1400 and Oakton DO 300 instruments. The environmental temperature and relative humidity of the site were monitored using a thermo-hygrometer (Gehaka) at 10 m from the lagoon edge and three meters above the soil, in a shady area.
Specimen collection and procedures.
Larvae were collected before (pre-treatment sample) and one, two, five and seven days after bioinsecticide application (20 samples per product and concentration); physical and chemical water and environmental parameters were simultaneously measured. Larvae were collected at each corner of the pond using a nylon net (20 cm in diameter and 0.1 mm mesh) at a distance of one meter from the edge. Larvae collected were counted in the laboratory; 5% of the larvae collected at 4th instar of each site were mounted on a microscope slide with Hoyer's solution for species identification.
Statistical analysis.
The treatments for the control of Culicidae were considered: three products in two different concentrations and seven days of assessment. For data analysis by ANOVA was found not to homogeneity. To achieve this significance, data were transformed into square root (x +0.5). For the averages of variables (pH, conductivity and oxygen) of the treatments were compared by Tukey test at 5%. ANOVA and the Tukey test at 5% significance were performed, using the SPSS program (SPSS Inc. 2005).
Results and discussion
Larvae found in both lagoons belonged to the species Cx. quinquefasciatus and Cx. saltanensis. Similar larval density was found for the two species in the Jataizinho lagoon. Infestation rate of Cx. quinquefasciatus in the Arapongas lagoon was higher. Teknar (1 L/ha) controlled 70.1% of the larval population 24 h after application; less control was observed thereafter, declining to 45.9% 48 h after application. The same product applied at a concentration of 2 L/ha was more efficient 48 h after application, reaching 85.4% of control as compared to the initial level (Table 1). At the 2 L/ha concentration, control was efficient until the fifth day (Table 1). The different concentrations had similar initial impacts, whereas the higher product concentrations increased the residual activity of the product.
Gunasekaran et al. (2004) controlled more than 80% of Cx. quinquefasciatus in sewage using Teknar HP-D (1,200 IU/mg) at the 2 L/ha concentration twice a week, at three-day intervals. Mulla et al. (2003), using high doses of Bti, failed to extend control of Culex. These results show that there are many biotic and abiotic factors that affect the action of Bti. According to Lacey (2007), the efficiency of Bti can be affected by temperature, solar radiation, turbidity, presence of vegetation, mosquito species, strategies and rates of food ingestion by the larvae, among other factors.
High conductivity levels and low concentrations of dissolved and saturated oxygen were recorded for the lagoon water. However, none of the abiotic parameters evaluated was affected by Teknar at the 1 L/ha concentration. High colonization of breeding sites in alkaline water with pH 7 to 9 for Cx. quinquefasciatus (Tables 2 to 3) had been previously reported by Fernández et al. (1986). They also suggested that low oxygen indexes at breeding sites with high amounts of organic matter are associated with the presence of protozoans with high reduction potential such as Metopus sp., an indica- tor of polluted water, this being the appropriate breeding site for the mosquito.
Vectobac at the 1 L/ha concentration controlled 82.4 and 85.6% of the lagoon larvae 24 and 48 h after application, respectively, but the larval count returned to initial levels 5 days after application of this product (Table 2). The 2 L/ha concentration controlled 98.1% of the initial larval population 24 h after application and 99.3% at 48 h, but there was an increase in the number of larvae on the fifth day (Table 2). Hallmon et al. (2000) controlled Cx. quinquefasciatus with Vectobac AS, two to three days after application in plastic containers, with repeated applications every ten days. Zequi and Lopes (2007) found that Vectobac at 2 L/ ha effectively controlled Cx. saltanensis in slurry lagoons for up to 15 days, with 100% larval mortality 24 h after application, and recommended biweekly applications for similar conditions. Amalraj et al. (2000) tested Vectobac AS at 1.2 and 2.4 L/ha concentrations at smaller breeding sites, obtaining 80% control of larvae of Cx. quinquefasciatus for 1.8 days in septic cesspools. Conductivity was the only chemical parameter to decrease on the seventh day after Vectobac application at the 1 and 2 L/ha concentrations. Such a decrease might not have been related to the bioinsecticide application because higher product concentration did not produce significant changes or the application of 1L/ha was sufficient to maximally alter conductivity (Table 2). A fluctuation in the physical and chemical parameters (pH and conductivity) between the fifth and seventh day was observed with 2 L/ha concentration but without statistical difference (Table 1). Such changes are typical of lagoons with continuous effluent influx and higher or lower needs for organic matter, depending on the intensity of activities of the waste producer.
Efficacy of Teknar (1 L/ha concentration) against Cx. quinquefasciatus and Cx. saltanensis applied to the effluent treatment lagoon of a cattle slaughterhouse (Arapongas, Paraná), with three replicates performed (March 31 to April 21, 2004; April 28 to May 19, 2004), and measurements of abiotic factors.
Original data; results were square root (x +0.5) transformed for determination of larval mortality rate.
Same letters on same row indicate no difference according to the Tukey test at 5% significance level.
Coefficient of variation.
Aquabac XT controlled 76.5 and 71.5% of the larvae at a concentration of 1 L/ha one and two days after application, respectively (Table 3). At 2 L/ha concentration, 95.4% control was observed 24 h after application and 97.7%, 48 h after application (Table 3); the initial larval counts were reached again on the seventh day. Aquabac XT seems to have caused changes in pH and oxygen (saturated and dissolved) only at the 2L/ha concentration (Table 3), but these parameters returned to almost baseline levels after 7 days.
All field trials were conducted at 19.2 to 29.4 °C water temperatures, environmental temperatures between 11.1 and 38 °C, and 25 to 99% relative humidity (Table 4). Such fluctuations are common in northern Paraná State during the summer (Fritzsons et al. 2008), when most of the reproductive activity of Culicidae occurs by colonization of the breeding sites, assuring a high egg hatching rate and constantly high quantity of larvae. Egg rafts were collected at every sampling period, indicating the presence of adults near the lagoons, assuring the immediate recolonization of breeding sites (data not shown). Camargo et al. (1994) reported 93% eclosion from egg rafts at 27 °C for Cx. quinquefasciatus. Vianna et al. (1996) showed that hatching of eggs of Cx. quinquefasciatus was not significantly affected by temperature under natural conditions, reaching 90% eclosion during a large part of the year. Zequi and Lopes (2012) found a 97.48% egg hatching rate for Cx. saltanensis, with emergence occurring between 12.29 and 13.12 days for males and females, respectively, at 27 °C and relative humidity of 80 ± 5% in the laboratory. These data on the reproductive ecology of the species and the limits of temperature changes in the environment suggest that temperature does not affect the reproductive behaviour of the species. Therefore, the population changes observed were directly related to the pathogenic action of the bioinsecticide, where high temperature reduces the time between egg hatching and the emergence of adults, thereby facilitating a potential rapid recolonization of the site. High temperature and excess of nutrients significantly increase larval density at breeding sites. Such conditions indirectly affect the impact of Bti because high temperatures and ultraviolet rays affect crystal persistence in the environment (Lacey 2007). According to Tetreau et al. (2013), ultraviolet rays increase the sensitivity of mosquitos to Bti, thereby increasing its efficiency for short-term periods. At low temperatures, food intake and screening by the larvae occur at lower rates, requiring higher product concentration to achieve better results (Becker et al. 1992).
Efficacy of Vectobac AS (1 and 2 L/ha concentrations) against Cx. quinquefasciatus and Cx. saltanensis applied to the effluent treatment lagoons of a cattle (Arapongas, Paraná) and a swine slaughterhouse (Jataizinho, Paraná), with three replicates performed (March 10-31, 2004; Sep- tember 22 to October 13, 2004), and measurements of abiotic factors.
Primary data; results were square root (x +0.5) transformed for determination of larval mortality rate.
Same letters on same row indicate no difference by the Tukey test at 5% significance level.
Coefficient of variation.
Efficacy of Aquabac XT (1 and 2 L/ha concentrations) against Cx. quinquefasciatus and Cx. saltanensis applied to the effluent treatment lagoon of a swine slaughterhouse (Jataizinho, Paraná) with three replicates performed (March 31 to April 21, 2004; April 28 to May 19, 2004), and measurements of abiotic parameters.
Primary data; results were square root (x + 0.5) transformed for determination of larval mortality rate.
Same letters on same row indicate no difference by the Tukey test at 5% significance level.
Coefficient of variation.
Environmental and water temperature and relative humidity in lagoons where formulations containing Bacillus thuringiensis israelensis were applied, with three replicates for each concentration.
Larval density was high in both lagoons (Tables 1 to 3). This might have hindered the persistence of Bti action. Becker et al. (1992) argue that Bti efficacy decreases linearly as larval density increases. Insecticide efficiency also decreases with higher solar intensity and presence of other competitors for filtration, such as the micro-crustacean Daphnia. Nayar et al. (1999) also tested the effect of larval densities and light intensity on Bti efficiency, with results similar to those obtained by Becker et al. (1992) and to our results in this study. Morais et al. (2007) found a high infestation by Cx. quinquefasciatus in the Pinheiros River in the city of São Paulo, even after applications of adulticides, organophosphorus larvicides, and B. sphaericus. They related such a high infestation to the breeding site's potential for mosquito development, such as its large amount of nutrients. This same phenomenon was observed in lagoons where the products were applied, probably competing directly with Bti crystals during the larval filtering process during feeding. The low levels of dissolved oxygen found in the lagoons can account for the very few aquatic predators found, allowing for the proliferation of Culicidae (Juliano 2009).
Conclusion
Biological products containing Bti, such as those tested in our trials are an ecological alternative to chemical insecticides for controlling Culicidae in effluent treatment lagoons. None of the products has high persistence rates and must be applied weekly at the 2 L/ha concentration for efficient control and environmental safety. Because of the limited persistence of biological products in this environment and the high-pressure colonization by Culicidae, depending on the context, Bti does not exhibit the same efficiency for different places. Therefore, the control strategy must be adapted to each location to be treated, because effluent treatment lagoons are sites conducive to the breeding of Culicidae and are difficult to control, thereby requiring constant monitoring.
Footnotes
Acknowledgements
This work was supported by Capes (Coordination for the Improvement of Higher Education Personnel - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Dr. A. Ley- va helped with English editing of the manuscript.