The compounds 2,6-diiodophenol (2a), 4-hydroxy-3,5-diiodoacetophenone (2b), 4-chloro-2,6-diiodophenol (2c) and 2,6-dichloro-4-iodophenol (2d) were prepared, purified and provided by Gallo et al. (2010) [9] (Scheme 1).

The iodination reaction of the phenolic compounds occurred selectively under mild reaction conditions, using water as solvent to reduce the environmental impact. Hydrogen peroxide (0.68 mL of a 30% (w/v) aqueous solution, 6 mmol) was added to a solution of the appropriate phenol (1a-d) (2 mmol) and iodine (0.76 g, 3 mmol) in distilled water (10 mL). The mixture was stirred at room temperature or at 50˚C for 24 h. an aqueous solution (10 mL) 10% (w/v) of sodium thiosulfate was then added to the mixture, which was extracted with dichloromethane or ethyl acetate (3 × 20 mL). The organic phase was dried using MgSO₄. After filtration, the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using a mixture of hexane/dichloromethane (1:1), unless indicated otherwise, yielding the desired product (2a-d).

Ae. aegyptieggs (Rockefeller strain by IBEX/Entomology/LAFICAVE/IOC/FIOCRUZ-RJ) were placed to hatch in an environmental chamber (BOD incubator) at a temperature of 27˚C ± 2˚C and relative humidity of 70% ± 5%. The larvae’s diet consisted of fish feed. Larvae were selected and allowed to grow to the adult stage. For blood supply of females used rats and as sugar food was available. The obtained Ae. aegypti eggswere used for both colony maintenance and toxicity bioassays [13] .

The larvicidal activity was monitored following the methodology described by the WHO-World Health Organization [14] . Ae. aegypti larvae (approximately 1000 individuals) were placed on a tray containing water and fed with fish feed (1g per liter of water) and 3^rd instar larvae were selected [15] .

Bioassays for establishing the baseline were performed using the compounds 2a, 2b, 2c and 2d. These compounds were weighed and sonicated in 1mL of dimethyl sulfoxide (DMSO) 0.5% (v/v), after which unchlorinated water was added up to a volume of 20 mL to produce solutions in concentrations of 1000 mg∙L⁻¹, 500 mg∙L⁻¹, 250 mg∙L⁻¹, 125 mg∙L⁻¹, 62.5 mg∙L⁻¹ and 31.25 mg∙L⁻¹ (ppm). A volume of 50 mL of solution and groups of ten 3^rd instar larvae were placed in plastic cups. After 24 hours of exposure, the number of dead larvae was counted, considering dead the ones that were immobile or did not respond to stimulation with a Pasteur pipette. The bioassay was performed in quadruplicate. The negative control was carried out concomitantly, using an aqueous solution of DMSO 0.5 (v/v), and the positive control was performed using Temephos^®. After establishing of baseline susceptibility other bioassay was performed in quadruplicate on three different days. The concentrations used now ranged from 120 mg∙L⁻¹ to 5 mg∙L⁻¹. All the experiments were accompanied with negative a control using an aqueous solution of DMSO 0.5 (v/v).

The eggs were separated and selected under a binocular stereomicroscope and placed in test tubes filled with unchlorinated water. The Ae. aegypti eggs were hatched and the ovicidal bioassays performed in a BOD incubator at a temperature of 27˚C ± 2˚C, relative humidity of 70% ± 5% and a 12:12 hours light-dark cycle. Two hundred Ae. aegypti eggs approximately 15 days old were used to avoid lowering the hatching rate [16] [17] . To check the incubation period and viability, the eggs were divided into five batches of 20 eggs each, which were kept in 20 mL test tubes, with 10 mL of water and fish food added to each tube. Daily bioassays were monitored using a binocular stereomicroscope to remove larvae, count hatched eggs and to prevent the adhesion of eggs to the tube walls.

The Probit analysis method was employed to obtain theLC₁₀, LC₅₀ and LC₉₀ values of the larval stages of Ae. aegypti and A. salina with 95% confidence interval P < 0.05 [18] .

Larvae exposed 2,6-diiodophenol, 4-hydroxy-3,5-diiodoacetophenone, 4-chloro-2,6-diiodophenol and 2,6-dich- loro-4-iodophenol presented little or no movement in the initial hours of contact with the product. In contrast, after 24 hours, the larvae of the control group intensive movements, and exuviae was visible in the container, indicating that ecdysis occurred.

Table 1 shows the toxicity bioassays results for the phenolic compounds 2,6-diiodophenol and 4-chloro-2,6- diiodophenol. Despite the toxicity presented in isolated experiments with 500 mg∙L⁻¹ and 1000 mg∙L⁻¹ of phenolic compounds. The compounds 4-hydroxy-3,5-diiodoacetophenone and 2,6-dichloro-4-iodophenol were not used in the toxicity bioassays because of their low solubility and their poor dispersion, which rendered them unsuitable for dilution. However, they were solubilized in Tween 80^®, as described in part 3.1.1.

The slopes of plotes can be used to analyze the toxicities of the compounds. Figure 1 shows that the Temephos^® has the highest toxicity. The values of slopes, which were determined by analyzing the mortality curve, demonstrate that there are differences and/or heterogeneities in the larvicidal behaviours of the substances compounds, explain about the linearity of the curves. The phenolic compound 2,6-diiodophenol shows different behaviour which is due to the low solubility of this component. These differences are probably due to the low solubility of 2,6-diiodophenol, which led to high standard deviations, as indicated by the differences in larval deaths in the toxicity bioassays of the Rockefeller strain, since the linearity of the straight line requires values below 7. It can be shown thatfor 2,6-diiodophenol, this value is the tolerance limit of linearity, since the solutions of this compound contained supernatant, indicating problems of solubility and heterogeneity in the solution.

Figure 1 shows a Log-Probit mortality graph of the Rockefeller strain of Ae. aegypti exposed to 2,6-diiodo- phenol, 4-chloro-2,6-diiodophenol, based on the concentrations used in the bioassay with 3^rd instar larvae of Ae. aegypti. The analysis of the curves indicated that the toxicity and larvicidal characteristic of 4-chloro-2,6-dii- odophenol is higher than those of 2,6-diiodophenol. These results are confirmed by the analysis of the LC₅₀ for toxic and larvicidal behaviour of 4-chloro-2,6-diiodophenol, with LC₅₀ of 11.53 mg∙L⁻¹, that is almost eight-fold lower than 2,6-diiodophenol, with LC₅₀ is 88.34 mg∙L⁻¹, indicating that the toxicity of the former compound is higher than that of the latter. The slope of the straight line in the Log-Probit graph indicates the toxicity of larvicidal substances. The steeper the slope of the straight line the higher the concentration of compound required

^aToxicity bioassays performed in quadruplicate on three different days. (−) Experiments not performed 2,6-diiodophenol and 4-chloro-2,6-diiodo- phenol.

to control the population and eliminate the mosquito larvae.

Temephos^®, the insecticide/larvicide organophosphate, which is applied in the concentration 1 mg∙L⁻¹ for control of Ae. aegypti, is normally used because of its low oral toxicity in mammals [19] . In the present study it was used as positive control. The results indicate the same confidence interval for both LC₁₀ and LC₅₀ which are in principle opposed to WHO (World Health Organization). The experimental results are different from the values of WHO (1992) considers that the Diagnostic Concentration (DC; 3 × LC₉₉) or 0.012 mg∙L⁻¹. The value of LC₁₀ is close to the value expected for the WHO-World Health Organization CD. This value suggests pesticide resistance (WHO Tech. Rep. Ser. 818, 63p, 1992) (see Table 1 and Table 2).

Moreover, the control larvae that were subjected to the same toxicity bioassay method, showed susceptibility to insecticide. The Log-Probit mortality graph of the Rockefeller population exposed to phenolic compounds expresses the ratio of mortality caused by commercially known insecticides versus potential larvicidal actives. Note that the more distant (to the right) from the Temephos^® curve, then higher the concentration of substances required to cause larval death.

The analysis of mortality of simple organisms such as the marine microcrustacean Artemia Salina Leach enables a comprehensive evaluation of toxicity and is considered a preliminary bioassay in the study of assets with potential biological activity [21] .

The first step in this assessment was the cultivation of A. salina to ensure a high hatching rate of cysts and attain a survival rate of at least 95% under the conditions of the bioassay. With adequate lighting, temperature and oxygenation, it was possible to reproduce the optimum environmental conditions for A. salina, obtaining high hatching and survival rates. Brine Shrimp Lethality Test (BSL) was held from eggs of A. salina Miramar-high hatching in a sea salt solution (38 g∙L⁻¹) were used for the toxicity test, while quinidine sulfate served as the positive control, and DMSO 0.5 (v/v) as the negative control. Initially, the dilutions were made with unchlorinated tap water, but after 24 hours the mortality rate was 100%. Therefore, the dilutions were made with water in which the eggs had hatched, thus ensuring the viability of 100% of the Artemia Salina nauplii population for the analysis.

Dolabella (1997) evaluated solvents that are toxic and non-toxic to A. salina and found that water and Tween 80^® were non-toxic (100% survival), DMSO was moderately toxic, and ethane followed by methanol were highly toxic, causing 100% mortality at a concentration of 5% (v/v) [22] . Table 3 shows the lethal concentrations to A. salineexposed to phenolic compoundssolubilized in Tween 80^®.

Figure 3 shows a Log-Probit mortality graph for theA. salinaexposed to 2,6-diiodophenol, 4-chloro-2,6-dii- odophenol, 4-hydroxy-3,5-diiodoacetophenone and2,6-dichloro-4-iodophenolsolubilized with 0.5% Tween 80^®. The values of the angular coefficient “slope” for the phenolic compounds 2,6-diiodophenol, 4-hydroxy-3,5-dii- odoacetophenone and 2,6-dichloro-4-iodophenol weresimilar to those found for Ae. aegypti larvae, except for4-chloro-2,6-diiodophenol.

A comparison of larvicidal activity and toxicity indicated that all the compounds under study are toxic to A. salinaand Ae. aegypti. The Lethal Concentration (LC) of the compound 2,6-diiodophenol which caused a mortality rate of 10% of the population of A. salina was 56.97 mg∙L⁻¹, while the lethal concentration of this compound that resulted in 10% mortality of the population of Ae. aegypti larvae was 54.12 mg∙L⁻¹ (LC₁₀) and most of the LCs values were lower for A. salina (LC₁₀, LC₅₀ and LC₉₀) than Ae. aegypti larvae.

Analysis of Table 2 and Table 3 reveals that lower concentrations of all the phenolic compounds are needed

^a*oxicity bioassays were performed in quadruplicate on three different days.

to kill A. salina than to kill Ae. aegypti larvae, clearly indicating that these phenolic compounds have higher larvicidal activity against A. salina than Ae. aegypti larvae. These results demonstrate the importance of using this crustacean for the detection of bioactive substances. Studies involving new actives of different origins with potential biological behaviour are increasing, so the analysis of bioindicators of lethality is imperative. The bioassay with A. salina proved to be an excellent indicator of toxicity, and is a simple and low cost method of analysis.

The phenolic compounds 2,6-diiodophenol (2a) and 4-chloro-2,6-diiodophenol (2c) have different toxicity and larvicidal behaviour. The compound 4-chloro-2,6-diiodophenol (2c) showed the highest toxicity for larvae of Ae. aegypti and A. salina, exhibiting higher toxicity than 2,6-diiodophenol. However, because the solubility of compounds 2,6-diiodophenol and 4-chloro-2,6-diiodophenol is low, these compounds had to be dissolved with Tween 80^® (surfactant) to reduce the problems of homogeneity in toxicity bioassays. The use of Tween 80^® augmented the larvicidal action of the phenolic compounds. LCs, in general, have been reduced.