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Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae)
Aquaculture 322-323 (2011) 249–254
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Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae) Jakeline G. França a,⁎, Maria J.T.R. Paiva b, Solange Carvalho a, Luciana Miashiro b, Julio V. Lombardi b,⁎ a b
Aquaculture Center, UNESP, Via de Acesso Prof. Paulo Donato Castellane, CEP 14884-900 Jaboticabal, SP, Brazil Fisheries Institute/APTA/SAA SP, Avenida Francisco Matarazzo, CEP 05001-900 São Paulo, SP, Brazil
a r t i c l e
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Article history: Received 18 May 2011 Received in revised form 29 September 2011 Accepted 4 October 2011 Available online 12 October 2011 Keywords: Potassium permanganate Ecotoxicity Oreochromis niloticus Ceriodaphnia dubia Pseudokirchneriella subcapitata
a b s t r a c t Potassium permanganate is a chemical compound widely used in aquaculture for the control and removal of parasites, and in the prevention of diseases caused by bacteria and fungi. However, this compound can be toxic to fish, being a strong oxidant. Moreover, there is no consistent information in the literature about its toxicity to non-target organisms. The purpose of this study was to evaluate the acute toxicity (LC50;96h) of potassium permanganate for tilapia, Oreochromis niloticus, and to determine its toxic effects on nontarget organisms using ecotoxicological assays performed with the microcrustacean Ceriodaphnia dubia and with the green microalgae Pseudokirchneriella subcapitata. The results showed that the concentration of 1.81 mg L− 1 of potassium permanganate caused acute toxic effect in tilapia fingerlings. The ecotoxicological assays demonstrated that concentrations above 0.12 mg L − 1 can cause chronic toxic effects on non-target organisms, indicating possible deleterious effects on the food chain of the aquatic ecosystem that may receive the discharge of effluents released by fish cultures treated with this chemotherapy. All toxic concentrations determined in this study were below those recommended in the literature for the use of this chemotherapy in fish cultures, demonstrating that this type of therapy should be more carefully considered in order to avoid damage to the treated fish and to the environment. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Currently, aquaculture faces the challenge of adapting to the concept of sustainability, since this activity has been the subject of social and scientific debate due its impacts on the environment (León-Santana and Hernández, 2008). Concern for the environment should be part of the production process, focusing on the development of systems that preserve the ecosystem in which they are placed, while maintaining a productive system of culture (Costa-Pierce, 2002). Thus, the development of this activity encourages speculation about the environmental aspects related to the stages of production and hence the impacts on natural ecosystems. According to Boyd (2003), considering the damage caused by aquaculture, the most common problem being water pollution caused by effluents from culture tanks. These effluents may contain high concentrations of nutrients, solids and other organic waste, which may cause serious impacts on the quality of the water bodies that receive them. Another important factor is related to the excessive and inappropriate use of chemicals in aquaculture, which can result in toxicity for ⁎ Corresponding authors. Tel.: + 55 1138717593; fax: + 55 1138717568. E-mail addresses: [email protected] (J.G. França), [email protected] (J.V. Lombardi). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.10.003
non-target organisms, the development of resistance to the compound by pathogens and the accumulation of waste (Holmstrom et al., 2003). Potassium permanganate is one of the most widely-used chemicals in aquaculture (Griffin et al., 2002). It is mainly used as a disinfectant in aquariums and tanks for the removal of parasites, such as: Ichthyophthirius multifiliis (Mitchell et al., 2008; Straus and Griffin, 2002), monogeneans (Pseudodactylogyrus anguillae and Pseudodactylogyrus bini) (Umeda et al., 2006), and also to control fungi and bacteria (Darwish et al., 2008, 2009; Schelenk et al., 2000). On the other hand, it is a product with a high degree of toxicity to fish because it is a strong oxidant, causing damage to delicate tissues like skin and gills, depending upon the concentration (Darwish et al., 2002). The regulatory agency for food and pharmaceutical products in the U.S. (FDA, 2007) establishes guidelines governing the standards of drugs and medicines in aquaculture, classifying some as “high regulatory priority drugs” and “low regulatory priority drugs”. Substances such as potassium permanganate and copper sulfate, known to be as potentially toxic to humans and other organisms (Kegley et al., 2010), are not included in any of the regulatory categories. Therefore, there is no pattern or limit for these drugs, and they can be used without restriction in aquaculture. According to the FDA (2007), the inclusion of these compounds into a regulatory class still depends on further studies.
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In this context, it is necessary to become familiar with and test products commonly used in fish cultures, such as potassium permanganate, to establish dose levels that do not cause deleterious effects on the treated fish. Also important to know are levels that do not cause damage to non-target organisms, i.e., representatives of different trophic levels of the aquatic ecosystem, which may be affected by the discharge of effluents from tanks with fish undergoing treatment. The purpose of this study was to determine the median lethal concentration LC50;96h of potassium permanganate for tilapia fingerlings (Oreochromis niloticus), in order to investigate the recommended concentrations of this compound as a chemotherapy in fish culture. Moreover, the additional intent of this study was to determine the effects of these concentrations on non-target organisms, performing standardized ecotoxicological assays for the microcrustacean cladoceran (Ceriodaphnia dubia) and the green microalgae (Pseudokirchneriella subcapitata).
The statistical analysis used in the acute toxicity test to determine the median lethal concentration (LC50;96h) was conducted using the Trimmed Spearman Karber method (Hamilton et al., 1977).
2.2. Ecotoxicological assays Ecotoxicological assays were performed with the same chemotherapy (potassium permanganate) in order to quantify its effect on non-target organisms (primary producers and primary consumers). Two organisms were selected that have been established as international standards for this type of assay (USEPA, 2002): the microcrustacean cladoceran (C. dubia) and the green microalgae (P. subcapitata).
2.3. Assays with the cladoceran C. dubia 2. Material and methods 2.1. Acute toxicity test with tilapia (O. niloticus) The experiment was conducted in the laboratory of Aquatic Toxicology, Institute of Fisheries - SP, São Paulo State, Brazil, under a controlled environment. The methodology for conducting the bioassay was standardized according to the recommendations made by APHA (2005). Fingerlings of tilapia, O. niloticus, were obtained from commercial fish culture, with average weight of 0.52 ± 0.10 g and mean total length of 3.35 cm ± 0.36 cm, and were acclimated for a period of 1 week in tanks with capacity of 250 L. During this period, fish were fed with a commercial ration containing 30% of crude protein, and were observed to evaluate possible signs of illness, stress, parasites, physical damage and mortality. For the test, fish were transferred to aquaria with artificial aeration, containing 5 L of solution. The density was two fish/L and 3 L of solution was replaced every 24 h. The total exposure period was 96 h, during which the fish were not fed (APHA, 2005). Dechlorined tap water was used in the acclimation and in the experiment, including the preparation and dilution of the tested concentrations. The chemical used was potassium permanganate (KMnO4) P.A., and the stock solution was prepared by diluting 1 g of potassium permanganate in 1000 mL of distilled water, resulting in a concentrated solution of 1000 mg L − 1 of potassium permanganate. This solution was prepared in sufficient quantities to provide the test concentrations. Immediately after adding the chemical, the solution was thoroughly mixed. The following physical and chemical aquatic parameters were monitored at the beginning and then every 24 h during the experiment: temperature (°C), dissolved oxygen (mg L − 1 and% of saturation), pH, electrical conductivity (μScm − 1), hardness and alkalinity (mg CaCO3 L − 1), and total ammonia (NH4 mg L − 1). To establish the potassium permanganate concentrations to be used in this study, a preliminary test was conducted, with concentrations based on minimum and maximum doses of this compound reported in the literature to treat fish diseases (Francis-Floyd and Klinger, 1997). The following concentrations were tested during 96 h, in triplicates: 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg L − 1 and a control group (with no addition of potassium permanganate). Based on the results obtained in the preliminary test, the following concentrations were established for the final test: 0.5, 1.0, 2.0, 4.0 and 6.0 mg L − 1 and a control group, whose assay was similarly conducted for 96 h, with three replicates for each treatment, totaling 18 aquaria. The occurrence of mortality was recorded for the periods of 24, 48, 72 and 96 h of exposure, with the removal of dead organisms each time.
The methodology for conducting assays with C. dubia followed the standard guidelines stipulated by the USEPA (2002). The assays were performed three times and each assay lasted seven days. To prevent stagnant conditions, the solution was exchanged every 24 h, starting at 48 h after the beginning of the assay. The highest concentration recommended in the literature (Francis-Floyd and Klinger, 1997) for direct use in the treatment of fish tanks (4.0 mg L − 1 potassium permanganate) was used for the highest concentration in this study. Five other concentrations were also tested, obtained by progressive dilution of the first. Thus, in addition to the control group, assays were conducted with the following concentrations: 0.12, 0.25, 0.50, 1.0, 2.0 and 4.0 mg L − 1 of potassium permanganate, which correspond respectively to the proportions of 3.1, 6.2, 12.5, 25.0, 50.0 and 100% of the parameter concentration (4.0 mg L − 1 potassium permanganate). All solutions were prepared in volumetric flasks and diluted with the same water used in the cultivation of the organisms (natural spring water, with quality certified by the Laboratory of Aquatic Ecotoxicology of the Institute of Fisheries). The culture vessels (plastic bottles of 20 mL) were filled with 15 mL of test-solution. Each vessel received the introduction of one organism (C. dubia neonates) 24 to 30 h old. The vessels were then placed in an incubator with an average temperature of 25.0 ± 0.7 °C, a photoperiod of 16 light hours: eight dark hours, and 1000 lx of illumination. Each assay was conducted with ten simultaneous replicates for each treatment. Daily, following 48 h, the organisms were transferred to another vessel containing the same concentration of solution. At this point the data on mortality and reproduction (number of neonates produced) were recorded. Food was provided to the organisms with each change of solution. Food was provided at 0.02 mL organism − 1 of the fermented ration having 2.50 g L − 1 of total solids content plus an additional 0.04 mL organism − 1 of a suspension of the microalgae P. subcapitata (Chlorophyceae), with the approximate ratio of 2.0 × 10 5 cells mL − 1 (USEPA, 2002). The control group exhibited a survival rate equal to or greater than 80%, with a minimum reproductive average of 12 neonates per genitor organism, as recommended in the literature (USEPA, 2002). The parameters for evaluating the results were: acute toxicity (survival rate of the parent organisms) and chronic toxicity (reproductive performance = number of produced neonates). Values of no observed effect concentration (NOEC) and observed effect concentration (OEC) were estimated using a statistically significant difference (P b 0.05) between a given concentration and the control group. For this calculation, the statistical package TOXSTAT 3.1 (Gulley et al., 1991) was used. Furthermore, the mean concentration inhibiting reproduction (IC50) was calculated by the linear interpolation method available in the program ICPin (Norberg-King, 1993).
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2.4. Assays with the green microalgae P. subcapitata The methodology for conducting the assays with P. subcapitata followed the recommendations of standardization stipulated by USEPA (2002). The assays were performed three times, at different periods, for 72 h each assay. The liquid medium “L. C. Oligo” ABNT (2005) was used for the cultivation of these organisms. Three days before the beginning of each set, a mass culture was prepared with 1 L of the culture medium “L. C. Oligo” and 50 mL of algal suspension. This mass culture was kept in a 2 L Erlenmeyer flask at a temperature of 25 °C, with constant illumination (4500 lx) and aeration. The culture was established to ensure that the algae were in the exponential growth phase. On the day of the assays, 100 mL of the mass culture was centrifuged for 15 min at 1500 rpm, the supernatant discarded and the sediment resuspended in 100 mL of culture medium. This procedure was repeated once more, and this new suspension was quantified (number of cells mL − 1) by counting in a Neubauer chamber, to determine the volume of inoculum to be used in the assays. This process of centrifugation and resuspension was performed to ensure a concentration 1of 1 × 10 5 cells in less than 1 mL of volume (USEPA, 2002). The calculation of the inoculum volume followed the formula: Vi = (Vf × Ci) / N. Where: Vi = volume of the inoculum (mL), Vf = volume of the final solution (mL), Ci = initial concentration of the vessel (cells mL− 1), N = number of cells of the suspension (cells mL− 1). The assays were run in triplicate (100 mL Erlenmeyer flasks) for each concentration, containing 50 mL of the sample and an inoculum with 1 × 10 5 cells of algae. The choice of the test-concentrations of potassium permanganate considered the same criteria previously mentioned for the assays with C. dubia. Following inoculation, the bottles were covered with plastic wrap and placed in an incubator for 72 h, under the conditions: temperature of 25 °C, constant illumination (4500 lx), and mechanical stirring of 175 rpm. After 72 h of exposure, algal counts (cells mL − 1) were determined using a Neubauer chamber. The validation of the assays followed the criteria of USEPA (2002) for toxicity assays with algae within 72 h of exposure. The assays were considered valid if, for the control group, the final algal biomass was at least 16 times greater than the initial biomass. The inhibition of algal growth at the end of the exposure period, measured by counting the number of microalgae (cells mL − 1) in a Neubauer chamber, was used to determine toxicity. The values of no observed effect concentration (NOEC) and observed effect concentration (OEC) were estimated according to the same criteria previously mentioned for the assays with C. dubia. 3. Results and discussion 3.1. Acute toxicity test for tilapia (O. niloticus) Concentrations of 4.0 and 6.0 mg L − 1 of potassium permanganate showed high toxicity, and after 24 h, the mortality observed in these concentrations were 70 and 100%, respectively. In the treatments with 1.0 and 2.0 mg L − 1 of potassium permanganate, high mortality rates were also recorded at the end of the exposure period: 43.3% and 33.3%, respectively. The mortality values for the control group were below 10%, which were in accordance with the acceptable limit for toxicity tests (APHA, 2005). Only under the concentration of 0.5 mg L − 1 was no toxicity observed, since only 3% of mortality occurred after 96 h, similar to the control group (Table 1). During the exposure period, the mortality of tilapia fingerlings was directly related to the concentration of potassium permanganate in the water, and this was higher in the first 24 h of exposure, with small changes after this period (Table 1). Similar results were observed by Silva et al. (2006), after the acute exposure (96 h) of Colossoma macropomum to potassium permanganate. Similarly, Marking
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Table 1 Cumulative average mortality (%) of tilapia (O. niloticus) over time in the acute toxicity test with potassium permanganate. Time (h) Treatments Potassium permanganate (mg L− 1)
24
Control 0.5 1.0 2.0 4.0 6.0
3.33 3.33 26.6 43.3 70.0 100
48
72
96
3.33 3.33 33.3 43.3 76.6 –
3.33 3.33 33.3 43.3 80.0 –
3.33 3.33 33.3 43.3 83.3 –
Mortality (%)
and Bills (1975) reported that the toxicity of potassium permanganate for ten species of fish exhibited only small changes after 24 h of exposure. According to Francis-Floyd and Klinger (1997), potassium permanganate is an oxidizing agent capable of reacting indiscriminately with any organic material present in the water, including bacteria, fungi and other parasites, as well as with fish tissues such as gills and skin. Thus, substances that are easily oxidized, rapidly decrease the activity of potassium permanganate and this initial demand on the compound decreases its effective concentration (Marking and Bills, 1975). This can be easily observed in changes to the water color, from pink to brown, due to the formation of manganese dioxide (MnO2), a product of the reduction of potassium permanganate (Straus and Griffin, 2002). Therefore, in the first hours of the experiment, the small amount of organic matter present had little effect on potassium permanganate toxicity, which explains the high mortality rate in the initial period of exposure (24 h). In addition to mortality, signs of intoxication of the exposed fish were also observed in solutions with higher concentrations of potassium permanganate, especially in the first 24 h. The first phase was characterized by the stress of the fingerlings at the first contact with the compound, represented by hyperactivity, with continuous, rapid movement. In the second phase, movement became less continuous. However, the fish exhibited darkening of the skin, greater movement of the fins, increased opercular beat and the search for oxygen at the air–water interface. This searching behavior for oxygen, could have been caused by the interference of this chemical with the respiratory mechanism, by the formation of minute particles of salts of manganese oxide (MnO2) blocking the gills of the fish (Kori-Siakpere, 2008). In the third phase, the animals exhibited lethargic movement until death. The same changes in behavior were observed by Silva et al. (2006), in fingerlings of C. macropomum at concentrations above 6.5 mg L − 1 of potassium permanganate. Straus (2004) also reported increased opercular movements, lethargy and loss of balance, after exposure of the hybrid striped bass (Morone chrysops × Morone saxatilis) to several concentrations of potassium permanganate. The median lethal concentration (LC50;96h) of potassium permanganate estimated in this study for fingerlings of tilapia was 1.81 mg L − 1, with a confidence interval (95%) between 1.48 and 2.22 mg L − 1. Table 2 lists the levels of acute toxicity of potassium permanganate for different fish species. Results similar to this study were reported for M. saxatilis with LC50;96h estimated a 1.58 mg L − 1, and for the rainbow trout (Oncorhyncus mykiss), with LC50;96h ranging from 0.879 to 1.73 mg L − 1 (Table 2). However, the LC50;96h value estimated in this experiment indicates that the tilapia fingerlings exhibit greater sensitivity as compared to other species such as M. saxatilis, Clarias gariepinus, C. macropomum and Pimephales promelas (Table 2). The variations found in the LC50;96h values may be explained, at least in part, by differences in species, age or body size of the fish.
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Table 2 Values of LC50;96h of potassium permanganate to different fish species. Species
Size
LC50;96h (mg L− 1)
References
Oreochromis niloticus Poecilia reticulata Danio rerio Clarias gariepinus Colossoma macropomum Pimephales promelas Morone saxatilis Morone saxatilis Morone saxatilis Chanos chanos Tilapia nilotica Tilapia nilotica Oncorhyncus mykiss Morone saxatilis Morone saxatilis
0.52 g 0.3 g 0.3 g 6.2 g 59.1 g Fingerlings 9.0 g Fingerlings 1.0 g 3.0 a 5.0 g Fingerlings Juvenile 2 a 5 cm Fingerlings Fingerlings
1.81 1.43 1.25 3.02 8.60 4.74 2.97 0.90 1.58 1.47 2.90 3.30 1.73 5.00 2.50
Present study Dolezelova et al. (2009) Dolezelova et al. (2009) Kori-Siakpere (2008) Silva et al. (2006) Hobbs et al. (2006) Straus (2004) Reardon and Harrell (1994) Bills et al. (1993) Cruz and Tamse (1989) Dureza (1988) Dureza (1988) Marking and Bills (1975) Hughes (1971) Wellborn (1969)
Table 3 gives the means of the chemical and physical variables of water observed in assay, which were similar among the different concentrations, suggesting that these variables did not interfere with results obtained. The results of toxicity of potassium permanganate obtained in the present study are strictly related to the standard environmental conditions established for the assays run inside laboratory, and it is difficult to make comparisons with other studies carried out under different standards, especially concerning to water quality variables. Furthermore, it has to be emphasized that the toxicity of any chemical in the natural environment may be different from laboratory conditions, due to the strong influence of water variables over the toxicity expression. Marking and Bills (1975), observed that the negative effect of potassium permanganate on aquatic organisms may also be enhanced by the physical and chemical properties of the aquatic environment, such as: high pH (8.5 to 9.5), low temperatures (7 to 17 °C) and high concentrations of hardness (180 to 300 mg L − 1 CaCO3). However, according to the author, the difference is apparently related not only to the chemical characteristics and temperature of the water, but the oxygen demand of organic material. In laboratory water contain little or no oxidizable material, most or all of the potassium permanganate remained available to produced toxicosis. Hobbs et al. (2006) reported that the alkalinity was higher in the pond water than in the synthetic water, but the toxicity of potassium permanganate was greater in the synthetic water. This suggests that the potassium permanganate is usually less toxic in surface waters than in laboratory waters. According to Francis-Floyd and Klinger (1997), the potassium permanganate at concentrations of 1 and 2 mg L − 1 can be considered safe for fish under long-term treatment. On the other hand, in studies conducted by Darwish et al. (2008, 2009) with Ictalurus punctatus and Table 3 Mean values of physical and chemical variables of the water in the acute toxicity test with O. niloticus exposed to potassium permanganate. Physical and chemical variables pH Conductivity electrical (μScm− 1) Dissolved oxygen (mg L− 1) Dissolved oxygen (%) Temperature (°C) Alkalinity (mg CaCO3 L− 1) Hardness (mg CaCO3 L− 1) Ammonium (mg L− 1)
Potassium permanganate (mg L− 1) 0.0
0.5
1.0
2.0
4.0
6.0
7.53 94.90
7.53 88.67
7.52 90.17
7.48 93.54
7.49 92.01
7.45 107.30
7.71 90.10 23.20 22.67 27.72 0.042
7.67 89.79 23.19 22.67 25.74 0.034
7.72 90.20 23.10 22.67 27.72 0.030
7.80 90.82 23.09 22.67 27.72 0.016
7.92 92.60 23.11 20.61 31.68 0.025
7.83 91.60 23.20 41.22 39.60 0.003
by Mitchell et al. (2008) with hybrid sunshine bass (M. chrysops female × M. saxatilis male) treated with recommended doses of potassium permanganate, the compound was effective only in the early stages of infection by Flavobacterium columnare and Ichthyobodo necator, respectively. However, its therapeutic values were limited when the infection became advanced, increasing the mortality of the challenged fish. Although there are some recommendations in the literature for high concentrations of potassium permanganate (1.0 to 4.0 mg L − 1) for the treatment and prevention of diseases in fish (Francis-Floyd and Klinger, 1997), this management should be practiced with caution, because at certain concentrations and exposure periods, this substance can be toxic to many species of aquatic organisms, including target and non-target organisms. 3.2. Ecotoxicological assays with C. dubia In the assays with the microcrustacean cladoceran C. dubia, acute toxicity at the concentration of 0.25 mg L − 1 of potassium permanganate was observed, with total mortality (100% of the organisms) during the first 24 h of exposure. At the lowest concentration tested (0.12 mg L − 1), high mortality (70% of test-organisms) was observed only in Assay I (Table 4). The evaluation of chronic toxicity (inhibition of reproduction of C. dubia) showed statistical similarity between the number of neonates produced in the control group and the concentration 0.12 mg L − 1 of potassium permanganate in the three assays (Fig. 1), indicating the absence of chronic toxicity at this concentration. All other concentrations of potassium permanganate were highly toxic to C. dubia. These concentrations are below what is considered safe for the use in aquaculture, both for the treatment of fish diseases: 1.0 to 4.0 mg L − 1 (Francis-Floyd and Klinger, 1997); and for the use as algaecide: between 2.0 and 4.0 mg L − 1 (Dorzab and Barkoh, 2005; Smith, 2005). This compound may affect the biodiversity of aquatic systems if used as proposed in the literature. In the present study, the inhibition concentration percentage (ICp50) is the concentration that inhibits 50% of the reproduction of organisms. In the study performed by Markle et al. (2000) using Raphidocelis subcapitata for evaluating of effluent toxicity, the authors concluded that the ICp50 constitutes a much better tool to evaluate the sensitivity of species, when compared to results obtained in assays using the No observed effect concentration (NOEC) and the Observed effect concentration (OEC) as response values. The ICp50;168h for the cladoceran C. dubia was determined to be 0.17 mg L − 1, and the confidence interval (95%) was between 0.15 and 0.18 mg L − 1 for potassium permanganate, within the range defined for the values of OEC and NOEC (Fig. 1). Information on the toxicity of potassium permanganate to nontarget species is very limited. However, the toxic potential of this compound was also recorded by Hobbs et al. (2006), in toxicity tests conducted with five aquatic species from different trophic levels. In these tests, the authors found that the median lethal concentration LC50;96h for potassium permanganate was 0.058 mg L − 1 for C. dubia, 0.053 mg L − 1 for Dapnhia magna, 2.13 mg L − 1 for Pimephales promelas, 4.74 mg L − 1 for Hyalela azteca and 4.43 mg L − 1 for Chironomus tentans. According to these authors, most of these values are below Table 4 Cumulative mortality of C. dubia after seven days of exposure to different concentrations of potassium permanganate. Potassium permanganate (mg L− 1) Control
0.12
0.25
0.5
1.0
2.0
4.0
70 20 10
100 100 100
100 100 100
100 100 100
100 100 100
100 100 100
Mortality (%) Assay I Assay II Assay III
0 0 0
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diversity and abundance of species from other levels of the trophic web. 4. Conclusion This study demonstrated that concentrations of potassium permanganate (1.0 to 4.0 mg L − 1) usually recommended for the treatment of fish diseases or to control algae in tanks of fish culture, can be toxic to tilapia (O. niloticus), as well as to the aquatic microorganisms standardized for ecotoxicological assays: cladocera (C. dubia) and chlorophycea (P. subcapitata). Acknowledgments Fig. 1. Graphical representation of the toxicity of potassium permanganate to C. dubia at different concentrations, for seven days of exposure. Average of three assays. * significant difference (P b 0.05). NOEC = no observed effect concentration, OEC = observed effect concentration.
The authors would like to thank the São Paulo Research Foundation (FAPESP) for financial support (Process 0756481-6R). References
what is recommended for use in fish treatment (at least 2.0 mg L − 1), corroborating the results found in this study. These results suggest a significant environmental risk if the effluent from fish tanks in treatment are released directly into a water body. 3.3. Ecotoxicological assays with P. subcapitata Fig. 2 shows the results of algal growth inhibition obtained in the assays with potassium permanganate. There is a significant difference between the control and the tested concentrations, except for the concentration of 0.12 mg L − 1. Therefore, the concentrations of 0.25 and 0.12 mg L − 1 correspond to the values of observed effect concentration (OEC) and no observed effect concentration (NOEC), respectively. In this study, the ICP50;72h for the green microalgae P. subcapitata was estimated at 0.54 mg L − 1, with a confidence interval (95%) between 0.28 and 0.68 mg L − 1 of potassium permanganate. However, the concentrations recommended for the use of this compound, both for the treatment of fish diseases, which is 1.0 to 4.0 mg L − 1 (Francis-Floyd and Klinger, 1997), and for the control of algae in tanks, which is 2.0 to 4.0 mg L − 1 (Dorzab and Barkoh, 2005; Smith, 2005), seem to be highly toxic to P. subcapitata. Fig. 2 shows that such concentrations cause increased inhibition of algal growth, exhibiting a typical dose–response relationship, i.e., the higher the product concentration in water, the lower the growth rate of the algae P. subcapitata. Due to their niche as primary producers, microalgae are ecologically important organisms, and highly sensitive indicators of pollutants discharged into the water (Paixão et al., 2008). Damage to the population structure of these organisms can cause changes in
Fig. 2. Growth rate of the microalgae P. subcapitata exposed to different concentrations of potassium permanganate, for 72 h. Average of three assays. * significant difference (P b 0.05). NOEC = no observed effect concentration, OEC = observed effect concentration.
American Public Health Association (APHA), American Water Works Association (AWWA), Water Environment Federation (WEF), 2005. Standard Methods for the Examination of Water and Wastewater 21th ed. American Public Health Association, Washington, D.C. Associação Brasileira de Normas e Técnicas (ABNT), 2005. NBR 12548: Ecotoxicologia aquática - Toxicidade crônica - Método de ensaio com algas (Chlorophyceae). Associação Brasileira de Normas e Técnicas, Rio de Janeiro. Bills, T.D., Marking, L.L., Howe, G.E., 1993. Sensitivity of Juvenile Striped Bass to Chemicals Used in Aquaculture, Resource Publication 192. U.S. Department of Interior, Fish and Wildlife Service, Washington, D.C., USA. Boyd, C.E., 2003. Guidelines for aquaculture effluent management at farm-level. Aquaculture 226, 101–112. Costa-Pierce, B.A., 2002. Ecology as the paradigm for the future of aquaculture. In: Costa-Pierce, B.A. (Ed.), Ecological Aquaculture: The Evolution of the Blue Revolution. Blackwell Science, Malden, Massachusetts, pp. 339–372. Cruz, E.R., Tamse, C.T., 1989. Acute toxicity of potassium permangante to milkfish fingerlings, Chanos chanos. Bulletin of Environmental Contamination and Toxicology 43, 785–788. Darwish, A.M., Griffin, D.L., Straus, D.L., Mitchell, A.J., 2002. Histological and hematological evaluation of potassium permanganate exposure in channel catfish. Journal of Aquatic Animal Health 14, 134–144. Darwish, A.M., Mitchell, A.J., Hobbs, M.S., 2008. In vitro and in vivo evaluation of potassium permanganate treatment efficacy for the control of acute experimental infection of Flavobacterium columnare in channel catfish. North American Journal of Aquaculture 70, 314–322. Darwish, A.M., Mitchell, A.J., Straus, D.L., 2009. Evaluation of potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus. Journal of Fish Diseases 32, 193–199. Dolezelova, P., Macova, S., Plhalova, L., Pistekova, V., Svobodova, Z., Bedanova, I., Voslarova, E., 2009. Comparison of the sensitivity of different fish species to medical substances. Neuroendocrinology Letters 30, 248–252. Dorzab, T., Barkoh, A., 2005. Toxicity of copper sulfate and potassium permanganate to rainbow trout and golden alga Prymnesium parvum. In: Barkoh, A., Fries, L.T. (Eds.), Management of Prymnesium parvum at Texas State Fish Hatcheries: Management Data Series N° 236. Dureza, L.A., 1988. Toxicity and lesions in the gills of Tilapia nilotica fry and fingerlings exposed to formalin, furanace, potassium permanganate and malachite green, Doctoral Dissertation, Auburn University. Food, Drug Administration (FDA), 2007. Enforcement Priorities for Drug Use in Aquaculture, Center for Veterinary Medicine Program Policies and Procedures Manual 1240.4200. U.S. Food and Drug Administration. Francis-Floyd, R., Klinger, R., 1997. Use of potassium permanganate to control external infections of ornamental fish. University of Florida Institute of Food and Agricultural Sciences Extension Fact Sheet FA-37. Griffin, B.R., Davis, K.B., Darwish, A., Straus, D.L., 2002. Effect of exposure to potassium permanganate on stress indicators in channel catfish Ictalurus punctatus. Journal of the World Aquaculture Society 33, 1–9. Gulley, D.D., Boelter, A.M., Bergman, H.L., 1991. TOXSTAT 3.3: Fish Physiology and Toxicology Laboratory. Dept. Zool. and Physiol., Univ. WY, Laramie, Wyoming. Hamilton, M.A., Russo, R.C., Thurston, R.V., 1977. Trimmed Spearman–Karber method for estimating median lethal concentrations in toxicity bioassays. Environmental Science & Technology 11, 714–719. Hobbs, M.S., Grippo, R.S., Farris, J.L., Griffin, B.R., Harding, L.L., 2006. Comparative acute toxicity of potassium permanganate to nontarget aquatic organisms. Environmental Toxicology and Chemistry 25, 3046–3052. Holmstrom, K., Graslund, S., Wahlstrom, A., Poungshompoo, S., Bengtsson, B.E., Kautsky, N., 2003. Antibiotic use in shrimp farming and implications for environmental impacts and human health. International Journal of Food Science & Technology 38, 255–266. Hughes, J.S., 1971. Tolerance of striped bass, Morone saxatilis (Walbaum), larvae and fingerlings to nine chemicals used in pond culture. Proceedings of the 24th Annual
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Schelenk, D., Colley, W.C., Alfy, A.E., Kirby, R., Griffin, B.R., 2000. Effects of the oxidant potassium permanganate on the expression of gill metallothionein mRNA and its relationship to sublethal whole animal endpoints in channel catfish. Toxicological Sciences 54, 177–182. Silva, A.L.F., Chagas, E.C., Gomes, L.C., Araujo, L.D., Silva, C.R., Brandão, F.R., 2006. Toxicity and sublethal effects of potassium permanganate in Tambaqui (Colossoma macropomum). Journal of the World Aquaculture Society 37, 318–321. Smith, D.G., 2005. Efficacy of potassium permanganate to reduce Prymnesium parvum ichthyotoxicity. In: Barkoh, A., Fries, L.T. (Eds.), Management of Prymnesium parvum at Texas State Fish Hatcheries: Management Data Series No. 236. Straus, D.L., 2004. Comparison of the acute toxicity of potassium permanganate to Hybrid Striped Bass in well water and diluted well water. Journal of the World Aquaculture Society 35, 55–60. Straus, D.L., Griffin, B.R., 2002. Efficacy of potassium permanganate in treating Ichthyophthiriasis in channel catfish. Journal of Aquatic Animal Health 14, 145–148. Umeda, N., Nibe, H., Hara, T., Hirazawa, N., 2006. Effects of various treatments on hatching of eggs and viability of oncomiracidia of the monogenean Pseudodactylogyrus anguillae and Pseudodactylogyrus bini. Aquaculture 253, 148–153. United States Environmental Protection Agency (USEPA), 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, fourth ed. EPA. Wellborn, T.L.J., 1969. The toxicity of nine therapeutic and herbicidal compounds to striped bass. The Progressive Fish Culturist 31, 27–32.
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Toxicity of the therapeutic potassium permanganate to tilapia Oreochromis niloticus and to non-target organisms Ceriodaphnia dubia (microcrustacean cladocera) and Pseudokirchneriella subcapitata (green microalgae) Jakeline G. França a,⁎, Maria J.T.R. Paiva b, Solange Carvalho a, Luciana Miashiro b, Julio V. Lombardi b,⁎ a b
Aquaculture Center, UNESP, Via de Acesso Prof. Paulo Donato Castellane, CEP 14884-900 Jaboticabal, SP, Brazil Fisheries Institute/APTA/SAA SP, Avenida Francisco Matarazzo, CEP 05001-900 São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history: Received 18 May 2011 Received in revised form 29 September 2011 Accepted 4 October 2011 Available online 12 October 2011 Keywords: Potassium permanganate Ecotoxicity Oreochromis niloticus Ceriodaphnia dubia Pseudokirchneriella subcapitata
a b s t r a c t Potassium permanganate is a chemical compound widely used in aquaculture for the control and removal of parasites, and in the prevention of diseases caused by bacteria and fungi. However, this compound can be toxic to fish, being a strong oxidant. Moreover, there is no consistent information in the literature about its toxicity to non-target organisms. The purpose of this study was to evaluate the acute toxicity (LC50;96h) of potassium permanganate for tilapia, Oreochromis niloticus, and to determine its toxic effects on nontarget organisms using ecotoxicological assays performed with the microcrustacean Ceriodaphnia dubia and with the green microalgae Pseudokirchneriella subcapitata. The results showed that the concentration of 1.81 mg L− 1 of potassium permanganate caused acute toxic effect in tilapia fingerlings. The ecotoxicological assays demonstrated that concentrations above 0.12 mg L − 1 can cause chronic toxic effects on non-target organisms, indicating possible deleterious effects on the food chain of the aquatic ecosystem that may receive the discharge of effluents released by fish cultures treated with this chemotherapy. All toxic concentrations determined in this study were below those recommended in the literature for the use of this chemotherapy in fish cultures, demonstrating that this type of therapy should be more carefully considered in order to avoid damage to the treated fish and to the environment. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Currently, aquaculture faces the challenge of adapting to the concept of sustainability, since this activity has been the subject of social and scientific debate due its impacts on the environment (León-Santana and Hernández, 2008). Concern for the environment should be part of the production process, focusing on the development of systems that preserve the ecosystem in which they are placed, while maintaining a productive system of culture (Costa-Pierce, 2002). Thus, the development of this activity encourages speculation about the environmental aspects related to the stages of production and hence the impacts on natural ecosystems. According to Boyd (2003), considering the damage caused by aquaculture, the most common problem being water pollution caused by effluents from culture tanks. These effluents may contain high concentrations of nutrients, solids and other organic waste, which may cause serious impacts on the quality of the water bodies that receive them. Another important factor is related to the excessive and inappropriate use of chemicals in aquaculture, which can result in toxicity for ⁎ Corresponding authors. Tel.: + 55 1138717593; fax: + 55 1138717568. E-mail addresses: [email protected] (J.G. França), [email protected] (J.V. Lombardi). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.10.003
non-target organisms, the development of resistance to the compound by pathogens and the accumulation of waste (Holmstrom et al., 2003). Potassium permanganate is one of the most widely-used chemicals in aquaculture (Griffin et al., 2002). It is mainly used as a disinfectant in aquariums and tanks for the removal of parasites, such as: Ichthyophthirius multifiliis (Mitchell et al., 2008; Straus and Griffin, 2002), monogeneans (Pseudodactylogyrus anguillae and Pseudodactylogyrus bini) (Umeda et al., 2006), and also to control fungi and bacteria (Darwish et al., 2008, 2009; Schelenk et al., 2000). On the other hand, it is a product with a high degree of toxicity to fish because it is a strong oxidant, causing damage to delicate tissues like skin and gills, depending upon the concentration (Darwish et al., 2002). The regulatory agency for food and pharmaceutical products in the U.S. (FDA, 2007) establishes guidelines governing the standards of drugs and medicines in aquaculture, classifying some as “high regulatory priority drugs” and “low regulatory priority drugs”. Substances such as potassium permanganate and copper sulfate, known to be as potentially toxic to humans and other organisms (Kegley et al., 2010), are not included in any of the regulatory categories. Therefore, there is no pattern or limit for these drugs, and they can be used without restriction in aquaculture. According to the FDA (2007), the inclusion of these compounds into a regulatory class still depends on further studies.
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In this context, it is necessary to become familiar with and test products commonly used in fish cultures, such as potassium permanganate, to establish dose levels that do not cause deleterious effects on the treated fish. Also important to know are levels that do not cause damage to non-target organisms, i.e., representatives of different trophic levels of the aquatic ecosystem, which may be affected by the discharge of effluents from tanks with fish undergoing treatment. The purpose of this study was to determine the median lethal concentration LC50;96h of potassium permanganate for tilapia fingerlings (Oreochromis niloticus), in order to investigate the recommended concentrations of this compound as a chemotherapy in fish culture. Moreover, the additional intent of this study was to determine the effects of these concentrations on non-target organisms, performing standardized ecotoxicological assays for the microcrustacean cladoceran (Ceriodaphnia dubia) and the green microalgae (Pseudokirchneriella subcapitata).
The statistical analysis used in the acute toxicity test to determine the median lethal concentration (LC50;96h) was conducted using the Trimmed Spearman Karber method (Hamilton et al., 1977).
2.2. Ecotoxicological assays Ecotoxicological assays were performed with the same chemotherapy (potassium permanganate) in order to quantify its effect on non-target organisms (primary producers and primary consumers). Two organisms were selected that have been established as international standards for this type of assay (USEPA, 2002): the microcrustacean cladoceran (C. dubia) and the green microalgae (P. subcapitata).
2.3. Assays with the cladoceran C. dubia 2. Material and methods 2.1. Acute toxicity test with tilapia (O. niloticus) The experiment was conducted in the laboratory of Aquatic Toxicology, Institute of Fisheries - SP, São Paulo State, Brazil, under a controlled environment. The methodology for conducting the bioassay was standardized according to the recommendations made by APHA (2005). Fingerlings of tilapia, O. niloticus, were obtained from commercial fish culture, with average weight of 0.52 ± 0.10 g and mean total length of 3.35 cm ± 0.36 cm, and were acclimated for a period of 1 week in tanks with capacity of 250 L. During this period, fish were fed with a commercial ration containing 30% of crude protein, and were observed to evaluate possible signs of illness, stress, parasites, physical damage and mortality. For the test, fish were transferred to aquaria with artificial aeration, containing 5 L of solution. The density was two fish/L and 3 L of solution was replaced every 24 h. The total exposure period was 96 h, during which the fish were not fed (APHA, 2005). Dechlorined tap water was used in the acclimation and in the experiment, including the preparation and dilution of the tested concentrations. The chemical used was potassium permanganate (KMnO4) P.A., and the stock solution was prepared by diluting 1 g of potassium permanganate in 1000 mL of distilled water, resulting in a concentrated solution of 1000 mg L − 1 of potassium permanganate. This solution was prepared in sufficient quantities to provide the test concentrations. Immediately after adding the chemical, the solution was thoroughly mixed. The following physical and chemical aquatic parameters were monitored at the beginning and then every 24 h during the experiment: temperature (°C), dissolved oxygen (mg L − 1 and% of saturation), pH, electrical conductivity (μScm − 1), hardness and alkalinity (mg CaCO3 L − 1), and total ammonia (NH4 mg L − 1). To establish the potassium permanganate concentrations to be used in this study, a preliminary test was conducted, with concentrations based on minimum and maximum doses of this compound reported in the literature to treat fish diseases (Francis-Floyd and Klinger, 1997). The following concentrations were tested during 96 h, in triplicates: 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg L − 1 and a control group (with no addition of potassium permanganate). Based on the results obtained in the preliminary test, the following concentrations were established for the final test: 0.5, 1.0, 2.0, 4.0 and 6.0 mg L − 1 and a control group, whose assay was similarly conducted for 96 h, with three replicates for each treatment, totaling 18 aquaria. The occurrence of mortality was recorded for the periods of 24, 48, 72 and 96 h of exposure, with the removal of dead organisms each time.
The methodology for conducting assays with C. dubia followed the standard guidelines stipulated by the USEPA (2002). The assays were performed three times and each assay lasted seven days. To prevent stagnant conditions, the solution was exchanged every 24 h, starting at 48 h after the beginning of the assay. The highest concentration recommended in the literature (Francis-Floyd and Klinger, 1997) for direct use in the treatment of fish tanks (4.0 mg L − 1 potassium permanganate) was used for the highest concentration in this study. Five other concentrations were also tested, obtained by progressive dilution of the first. Thus, in addition to the control group, assays were conducted with the following concentrations: 0.12, 0.25, 0.50, 1.0, 2.0 and 4.0 mg L − 1 of potassium permanganate, which correspond respectively to the proportions of 3.1, 6.2, 12.5, 25.0, 50.0 and 100% of the parameter concentration (4.0 mg L − 1 potassium permanganate). All solutions were prepared in volumetric flasks and diluted with the same water used in the cultivation of the organisms (natural spring water, with quality certified by the Laboratory of Aquatic Ecotoxicology of the Institute of Fisheries). The culture vessels (plastic bottles of 20 mL) were filled with 15 mL of test-solution. Each vessel received the introduction of one organism (C. dubia neonates) 24 to 30 h old. The vessels were then placed in an incubator with an average temperature of 25.0 ± 0.7 °C, a photoperiod of 16 light hours: eight dark hours, and 1000 lx of illumination. Each assay was conducted with ten simultaneous replicates for each treatment. Daily, following 48 h, the organisms were transferred to another vessel containing the same concentration of solution. At this point the data on mortality and reproduction (number of neonates produced) were recorded. Food was provided to the organisms with each change of solution. Food was provided at 0.02 mL organism − 1 of the fermented ration having 2.50 g L − 1 of total solids content plus an additional 0.04 mL organism − 1 of a suspension of the microalgae P. subcapitata (Chlorophyceae), with the approximate ratio of 2.0 × 10 5 cells mL − 1 (USEPA, 2002). The control group exhibited a survival rate equal to or greater than 80%, with a minimum reproductive average of 12 neonates per genitor organism, as recommended in the literature (USEPA, 2002). The parameters for evaluating the results were: acute toxicity (survival rate of the parent organisms) and chronic toxicity (reproductive performance = number of produced neonates). Values of no observed effect concentration (NOEC) and observed effect concentration (OEC) were estimated using a statistically significant difference (P b 0.05) between a given concentration and the control group. For this calculation, the statistical package TOXSTAT 3.1 (Gulley et al., 1991) was used. Furthermore, the mean concentration inhibiting reproduction (IC50) was calculated by the linear interpolation method available in the program ICPin (Norberg-King, 1993).
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2.4. Assays with the green microalgae P. subcapitata The methodology for conducting the assays with P. subcapitata followed the recommendations of standardization stipulated by USEPA (2002). The assays were performed three times, at different periods, for 72 h each assay. The liquid medium “L. C. Oligo” ABNT (2005) was used for the cultivation of these organisms. Three days before the beginning of each set, a mass culture was prepared with 1 L of the culture medium “L. C. Oligo” and 50 mL of algal suspension. This mass culture was kept in a 2 L Erlenmeyer flask at a temperature of 25 °C, with constant illumination (4500 lx) and aeration. The culture was established to ensure that the algae were in the exponential growth phase. On the day of the assays, 100 mL of the mass culture was centrifuged for 15 min at 1500 rpm, the supernatant discarded and the sediment resuspended in 100 mL of culture medium. This procedure was repeated once more, and this new suspension was quantified (number of cells mL − 1) by counting in a Neubauer chamber, to determine the volume of inoculum to be used in the assays. This process of centrifugation and resuspension was performed to ensure a concentration 1of 1 × 10 5 cells in less than 1 mL of volume (USEPA, 2002). The calculation of the inoculum volume followed the formula: Vi = (Vf × Ci) / N. Where: Vi = volume of the inoculum (mL), Vf = volume of the final solution (mL), Ci = initial concentration of the vessel (cells mL− 1), N = number of cells of the suspension (cells mL− 1). The assays were run in triplicate (100 mL Erlenmeyer flasks) for each concentration, containing 50 mL of the sample and an inoculum with 1 × 10 5 cells of algae. The choice of the test-concentrations of potassium permanganate considered the same criteria previously mentioned for the assays with C. dubia. Following inoculation, the bottles were covered with plastic wrap and placed in an incubator for 72 h, under the conditions: temperature of 25 °C, constant illumination (4500 lx), and mechanical stirring of 175 rpm. After 72 h of exposure, algal counts (cells mL − 1) were determined using a Neubauer chamber. The validation of the assays followed the criteria of USEPA (2002) for toxicity assays with algae within 72 h of exposure. The assays were considered valid if, for the control group, the final algal biomass was at least 16 times greater than the initial biomass. The inhibition of algal growth at the end of the exposure period, measured by counting the number of microalgae (cells mL − 1) in a Neubauer chamber, was used to determine toxicity. The values of no observed effect concentration (NOEC) and observed effect concentration (OEC) were estimated according to the same criteria previously mentioned for the assays with C. dubia. 3. Results and discussion 3.1. Acute toxicity test for tilapia (O. niloticus) Concentrations of 4.0 and 6.0 mg L − 1 of potassium permanganate showed high toxicity, and after 24 h, the mortality observed in these concentrations were 70 and 100%, respectively. In the treatments with 1.0 and 2.0 mg L − 1 of potassium permanganate, high mortality rates were also recorded at the end of the exposure period: 43.3% and 33.3%, respectively. The mortality values for the control group were below 10%, which were in accordance with the acceptable limit for toxicity tests (APHA, 2005). Only under the concentration of 0.5 mg L − 1 was no toxicity observed, since only 3% of mortality occurred after 96 h, similar to the control group (Table 1). During the exposure period, the mortality of tilapia fingerlings was directly related to the concentration of potassium permanganate in the water, and this was higher in the first 24 h of exposure, with small changes after this period (Table 1). Similar results were observed by Silva et al. (2006), after the acute exposure (96 h) of Colossoma macropomum to potassium permanganate. Similarly, Marking
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Table 1 Cumulative average mortality (%) of tilapia (O. niloticus) over time in the acute toxicity test with potassium permanganate. Time (h) Treatments Potassium permanganate (mg L− 1)
24
Control 0.5 1.0 2.0 4.0 6.0
3.33 3.33 26.6 43.3 70.0 100
48
72
96
3.33 3.33 33.3 43.3 76.6 –
3.33 3.33 33.3 43.3 80.0 –
3.33 3.33 33.3 43.3 83.3 –
Mortality (%)
and Bills (1975) reported that the toxicity of potassium permanganate for ten species of fish exhibited only small changes after 24 h of exposure. According to Francis-Floyd and Klinger (1997), potassium permanganate is an oxidizing agent capable of reacting indiscriminately with any organic material present in the water, including bacteria, fungi and other parasites, as well as with fish tissues such as gills and skin. Thus, substances that are easily oxidized, rapidly decrease the activity of potassium permanganate and this initial demand on the compound decreases its effective concentration (Marking and Bills, 1975). This can be easily observed in changes to the water color, from pink to brown, due to the formation of manganese dioxide (MnO2), a product of the reduction of potassium permanganate (Straus and Griffin, 2002). Therefore, in the first hours of the experiment, the small amount of organic matter present had little effect on potassium permanganate toxicity, which explains the high mortality rate in the initial period of exposure (24 h). In addition to mortality, signs of intoxication of the exposed fish were also observed in solutions with higher concentrations of potassium permanganate, especially in the first 24 h. The first phase was characterized by the stress of the fingerlings at the first contact with the compound, represented by hyperactivity, with continuous, rapid movement. In the second phase, movement became less continuous. However, the fish exhibited darkening of the skin, greater movement of the fins, increased opercular beat and the search for oxygen at the air–water interface. This searching behavior for oxygen, could have been caused by the interference of this chemical with the respiratory mechanism, by the formation of minute particles of salts of manganese oxide (MnO2) blocking the gills of the fish (Kori-Siakpere, 2008). In the third phase, the animals exhibited lethargic movement until death. The same changes in behavior were observed by Silva et al. (2006), in fingerlings of C. macropomum at concentrations above 6.5 mg L − 1 of potassium permanganate. Straus (2004) also reported increased opercular movements, lethargy and loss of balance, after exposure of the hybrid striped bass (Morone chrysops × Morone saxatilis) to several concentrations of potassium permanganate. The median lethal concentration (LC50;96h) of potassium permanganate estimated in this study for fingerlings of tilapia was 1.81 mg L − 1, with a confidence interval (95%) between 1.48 and 2.22 mg L − 1. Table 2 lists the levels of acute toxicity of potassium permanganate for different fish species. Results similar to this study were reported for M. saxatilis with LC50;96h estimated a 1.58 mg L − 1, and for the rainbow trout (Oncorhyncus mykiss), with LC50;96h ranging from 0.879 to 1.73 mg L − 1 (Table 2). However, the LC50;96h value estimated in this experiment indicates that the tilapia fingerlings exhibit greater sensitivity as compared to other species such as M. saxatilis, Clarias gariepinus, C. macropomum and Pimephales promelas (Table 2). The variations found in the LC50;96h values may be explained, at least in part, by differences in species, age or body size of the fish.
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Table 2 Values of LC50;96h of potassium permanganate to different fish species. Species
Size
LC50;96h (mg L− 1)
References
Oreochromis niloticus Poecilia reticulata Danio rerio Clarias gariepinus Colossoma macropomum Pimephales promelas Morone saxatilis Morone saxatilis Morone saxatilis Chanos chanos Tilapia nilotica Tilapia nilotica Oncorhyncus mykiss Morone saxatilis Morone saxatilis
0.52 g 0.3 g 0.3 g 6.2 g 59.1 g Fingerlings 9.0 g Fingerlings 1.0 g 3.0 a 5.0 g Fingerlings Juvenile 2 a 5 cm Fingerlings Fingerlings
1.81 1.43 1.25 3.02 8.60 4.74 2.97 0.90 1.58 1.47 2.90 3.30 1.73 5.00 2.50
Present study Dolezelova et al. (2009) Dolezelova et al. (2009) Kori-Siakpere (2008) Silva et al. (2006) Hobbs et al. (2006) Straus (2004) Reardon and Harrell (1994) Bills et al. (1993) Cruz and Tamse (1989) Dureza (1988) Dureza (1988) Marking and Bills (1975) Hughes (1971) Wellborn (1969)
Table 3 gives the means of the chemical and physical variables of water observed in assay, which were similar among the different concentrations, suggesting that these variables did not interfere with results obtained. The results of toxicity of potassium permanganate obtained in the present study are strictly related to the standard environmental conditions established for the assays run inside laboratory, and it is difficult to make comparisons with other studies carried out under different standards, especially concerning to water quality variables. Furthermore, it has to be emphasized that the toxicity of any chemical in the natural environment may be different from laboratory conditions, due to the strong influence of water variables over the toxicity expression. Marking and Bills (1975), observed that the negative effect of potassium permanganate on aquatic organisms may also be enhanced by the physical and chemical properties of the aquatic environment, such as: high pH (8.5 to 9.5), low temperatures (7 to 17 °C) and high concentrations of hardness (180 to 300 mg L − 1 CaCO3). However, according to the author, the difference is apparently related not only to the chemical characteristics and temperature of the water, but the oxygen demand of organic material. In laboratory water contain little or no oxidizable material, most or all of the potassium permanganate remained available to produced toxicosis. Hobbs et al. (2006) reported that the alkalinity was higher in the pond water than in the synthetic water, but the toxicity of potassium permanganate was greater in the synthetic water. This suggests that the potassium permanganate is usually less toxic in surface waters than in laboratory waters. According to Francis-Floyd and Klinger (1997), the potassium permanganate at concentrations of 1 and 2 mg L − 1 can be considered safe for fish under long-term treatment. On the other hand, in studies conducted by Darwish et al. (2008, 2009) with Ictalurus punctatus and Table 3 Mean values of physical and chemical variables of the water in the acute toxicity test with O. niloticus exposed to potassium permanganate. Physical and chemical variables pH Conductivity electrical (μScm− 1) Dissolved oxygen (mg L− 1) Dissolved oxygen (%) Temperature (°C) Alkalinity (mg CaCO3 L− 1) Hardness (mg CaCO3 L− 1) Ammonium (mg L− 1)
Potassium permanganate (mg L− 1) 0.0
0.5
1.0
2.0
4.0
6.0
7.53 94.90
7.53 88.67
7.52 90.17
7.48 93.54
7.49 92.01
7.45 107.30
7.71 90.10 23.20 22.67 27.72 0.042
7.67 89.79 23.19 22.67 25.74 0.034
7.72 90.20 23.10 22.67 27.72 0.030
7.80 90.82 23.09 22.67 27.72 0.016
7.92 92.60 23.11 20.61 31.68 0.025
7.83 91.60 23.20 41.22 39.60 0.003
by Mitchell et al. (2008) with hybrid sunshine bass (M. chrysops female × M. saxatilis male) treated with recommended doses of potassium permanganate, the compound was effective only in the early stages of infection by Flavobacterium columnare and Ichthyobodo necator, respectively. However, its therapeutic values were limited when the infection became advanced, increasing the mortality of the challenged fish. Although there are some recommendations in the literature for high concentrations of potassium permanganate (1.0 to 4.0 mg L − 1) for the treatment and prevention of diseases in fish (Francis-Floyd and Klinger, 1997), this management should be practiced with caution, because at certain concentrations and exposure periods, this substance can be toxic to many species of aquatic organisms, including target and non-target organisms. 3.2. Ecotoxicological assays with C. dubia In the assays with the microcrustacean cladoceran C. dubia, acute toxicity at the concentration of 0.25 mg L − 1 of potassium permanganate was observed, with total mortality (100% of the organisms) during the first 24 h of exposure. At the lowest concentration tested (0.12 mg L − 1), high mortality (70% of test-organisms) was observed only in Assay I (Table 4). The evaluation of chronic toxicity (inhibition of reproduction of C. dubia) showed statistical similarity between the number of neonates produced in the control group and the concentration 0.12 mg L − 1 of potassium permanganate in the three assays (Fig. 1), indicating the absence of chronic toxicity at this concentration. All other concentrations of potassium permanganate were highly toxic to C. dubia. These concentrations are below what is considered safe for the use in aquaculture, both for the treatment of fish diseases: 1.0 to 4.0 mg L − 1 (Francis-Floyd and Klinger, 1997); and for the use as algaecide: between 2.0 and 4.0 mg L − 1 (Dorzab and Barkoh, 2005; Smith, 2005). This compound may affect the biodiversity of aquatic systems if used as proposed in the literature. In the present study, the inhibition concentration percentage (ICp50) is the concentration that inhibits 50% of the reproduction of organisms. In the study performed by Markle et al. (2000) using Raphidocelis subcapitata for evaluating of effluent toxicity, the authors concluded that the ICp50 constitutes a much better tool to evaluate the sensitivity of species, when compared to results obtained in assays using the No observed effect concentration (NOEC) and the Observed effect concentration (OEC) as response values. The ICp50;168h for the cladoceran C. dubia was determined to be 0.17 mg L − 1, and the confidence interval (95%) was between 0.15 and 0.18 mg L − 1 for potassium permanganate, within the range defined for the values of OEC and NOEC (Fig. 1). Information on the toxicity of potassium permanganate to nontarget species is very limited. However, the toxic potential of this compound was also recorded by Hobbs et al. (2006), in toxicity tests conducted with five aquatic species from different trophic levels. In these tests, the authors found that the median lethal concentration LC50;96h for potassium permanganate was 0.058 mg L − 1 for C. dubia, 0.053 mg L − 1 for Dapnhia magna, 2.13 mg L − 1 for Pimephales promelas, 4.74 mg L − 1 for Hyalela azteca and 4.43 mg L − 1 for Chironomus tentans. According to these authors, most of these values are below Table 4 Cumulative mortality of C. dubia after seven days of exposure to different concentrations of potassium permanganate. Potassium permanganate (mg L− 1) Control
0.12
0.25
0.5
1.0
2.0
4.0
70 20 10
100 100 100
100 100 100
100 100 100
100 100 100
100 100 100
Mortality (%) Assay I Assay II Assay III
0 0 0
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diversity and abundance of species from other levels of the trophic web. 4. Conclusion This study demonstrated that concentrations of potassium permanganate (1.0 to 4.0 mg L − 1) usually recommended for the treatment of fish diseases or to control algae in tanks of fish culture, can be toxic to tilapia (O. niloticus), as well as to the aquatic microorganisms standardized for ecotoxicological assays: cladocera (C. dubia) and chlorophycea (P. subcapitata). Acknowledgments Fig. 1. Graphical representation of the toxicity of potassium permanganate to C. dubia at different concentrations, for seven days of exposure. Average of three assays. * significant difference (P b 0.05). NOEC = no observed effect concentration, OEC = observed effect concentration.
The authors would like to thank the São Paulo Research Foundation (FAPESP) for financial support (Process 0756481-6R). References
what is recommended for use in fish treatment (at least 2.0 mg L − 1), corroborating the results found in this study. These results suggest a significant environmental risk if the effluent from fish tanks in treatment are released directly into a water body. 3.3. Ecotoxicological assays with P. subcapitata Fig. 2 shows the results of algal growth inhibition obtained in the assays with potassium permanganate. There is a significant difference between the control and the tested concentrations, except for the concentration of 0.12 mg L − 1. Therefore, the concentrations of 0.25 and 0.12 mg L − 1 correspond to the values of observed effect concentration (OEC) and no observed effect concentration (NOEC), respectively. In this study, the ICP50;72h for the green microalgae P. subcapitata was estimated at 0.54 mg L − 1, with a confidence interval (95%) between 0.28 and 0.68 mg L − 1 of potassium permanganate. However, the concentrations recommended for the use of this compound, both for the treatment of fish diseases, which is 1.0 to 4.0 mg L − 1 (Francis-Floyd and Klinger, 1997), and for the control of algae in tanks, which is 2.0 to 4.0 mg L − 1 (Dorzab and Barkoh, 2005; Smith, 2005), seem to be highly toxic to P. subcapitata. Fig. 2 shows that such concentrations cause increased inhibition of algal growth, exhibiting a typical dose–response relationship, i.e., the higher the product concentration in water, the lower the growth rate of the algae P. subcapitata. Due to their niche as primary producers, microalgae are ecologically important organisms, and highly sensitive indicators of pollutants discharged into the water (Paixão et al., 2008). Damage to the population structure of these organisms can cause changes in
Fig. 2. Growth rate of the microalgae P. subcapitata exposed to different concentrations of potassium permanganate, for 72 h. Average of three assays. * significant difference (P b 0.05). NOEC = no observed effect concentration, OEC = observed effect concentration.
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