Sublethal effects of propiconazole on the metabolism of lambari Deuterodon iguape (Eigenmann 1907), a native species from Brazil

The objective of this study was to analyze the sublethal effects of propiconazole on Deuterodon iguape, a native fish common in Brazil, which has potential for aquaculture and use as a bioindicator. The hypothesis was to test whether D. iguape has a metabolism similar to Danio rerio so that its use in bioassays may be validated. Lethal concentration (LC50) and metabolic rates were studied in fish exposed to propiconazole. Specific oxygen consumption and ammonia excretion for D. iguape and D. rerio increased by 0.01 µg L−1 and then decreased as the propiconazole concentration increased. The decrease in the averages of specific oxygen consumption at the concentration of 0.1 µg L−1 represented a reduction in the metabolic rate compared to the control of 71% for D. iguape and 40% D. rerio. For the ammonia excretion, at the same concentration, there was a reduction of 68.7% and 45.4% for D. iguape and D. rerio, respectively. When comparing ammonia excretion of the two species for each concentration of propiconazole, there was a significant difference (p < 0.05) in relation to the control and for the highest concentration (0.1 µg L−1). As for specific oxygen consumption, there was a statistically significant difference only for the concentration of 0.1 µg L−1. D. iguape proved to be a good and useful bioindicator for ichthyologists or ecologists in studies of moderate pesticide contamination in freshwater aquatic environments, as its metabolic response was similar to D. rerio.


Introduction
Most water bodies in Brazil, and in the world, are contaminated by some type of chemical pollutant (Lopes et al. 2017). Agricultural pesticides are used excessively and beyond what is really necessary to control pests of agricultural crops. About 90% of what is applied is lost in the environment, and the biological response in terms of pest control is not reached (Barrera-Méndez et al. 2019). These losses occur due to factors such as application techniques, physical and chemical properties of pesticides, and environmental Abstract The objective of this study was to analyze the sublethal effects of propiconazole on Deuterodon iguape, a native fish common in Brazil, which has potential for aquaculture and use as a bioindicator. The hypothesis was to test whether D. iguape has a metabolism similar to Danio rerio so that its use in bioassays may be validated. Lethal concentration (LC50) and metabolic rates were studied in fish exposed to propiconazole. Specific oxygen consumption and ammonia excretion for D. iguape and D. rerio increased by 0.01 µg L −1 and then decreased as the propiconazole concentration increased. The decrease in the averages of specific oxygen consumption at the concentration of 0.1 µg L −1 represented a reduction in the metabolic rate compared to the control of 71% for D. iguape and 40% D. rerio. For the ammonia excretion, at the same concentration, there conditions (Ghormade et al. 2011). The impact of pesticides on the quality of groundwater has been a relevant and discussed subject worldwide (Lanchote et al. 2000). Less than 0.1% of the amount of pesticides applied to crops reaches the target organisms, while the other 99.9% has the potential to move to other environmental compartments, such as the surface and groundwater (Sabik et al. 2000).
Toxic compounds such as pesticides can affect aquatic organisms by compromising their behavioral, nutritional, and physiological status ( Van der Oost et al. 2003). For this reason, studies of the behavior and metabolism of fish and shrimp can assist in monitoring the environmental quality where these organisms are present. Using behavior and metabolism as a biomarker, it is possible to analyze the general physical state of these animals when in contact with certain toxic substances (Arias et al. 2007). Behavior analyses, specific oxygen consumption, and ammonia excretion can provide answers about the general health of aquatic organisms when under stress in the environment (Adams 1990).
An organism's metabolic rate is a useful and sensitive indication of its energy consumption. Therefore, in aerobic organisms, quantifying the rate of oxygen consumption can be directly associated with the amount of energy released from the oxidation of the food substrate. Based on the amount of oxygen consumed by an animal over a period of time, it is possible to calculate the energy spent during the same period to maintain its vital processes .
The evaluation of oxygen consumption and ammonia excretion in fish was used, for example, to study the toxic effects caused by: nanoparticles , ammonium chloride (Barbieri and Doi 2012), metallic trace elements (Barbieri 2007;Martinez et al. 2013;Ferrarini et al. 2016), and carbofurans (Campos-Garcia et al. 2016;Ruíz-Hidalgo et al. 2016).
Propiconazole is a broad-spectrum fungicide used to control fungal diseases in agricultural crops (Satapute and Kaliwal 2018). It has an action interfering with the ergosterol biosynthesis and inhibiting steroid demethylation (Ouadah-Boussouf and Babin 2016). Compared to other fungicides, propiconazole is difficult to degrade in the environment and exhibits relatively high acute toxicity, which can contaminate soil, water, and indirectly fauna, flora Garrison et al. 2011), and mainly a wide range of aquatic organisms (Cobas et al. 2016). These compounds, even in low concentrations, affect the structure and function of natural communities, causing damage ranging from molecular levels to that of entire populations, proving that intensive agricultural practices are highly impactful to the environment and are directly related to the reduction of biodiversity (Barbieri and Ferreira 2011). Kronvang et al. (2003) detected up to 130 ng g −1 of propiconazole in the sediment of streams in a Danish plain. Kahle et al. (2008) found it in concentrations of up to 27 ng L −1 in effluents of lakes and wastewater treatment plants in Switzerland. Kreuger (1998) observed propiconazole at maximum concentrations of 20 µg L −1 in the water of agricultural basins in the south from Sweden. In Brazil, the impacts related to the chronic and environmental toxicity of the application of pesticides were ignored or considered irrelevant for many years (Barbieri and Ferreira 2011). In addition to the possible exposure of these fungicides to humans and wildlife through soil sediment and residual water, their stereoselective transformation forming new compounds is more harmful to flora and fauna and is also concerning (Garrison et al. 2011). Due to their high mobility, particles of < 2 µm can be important carriers of propiconazole, causing water pollution (Wu et al. 2003).
There is a risk of contamination in the food chain, which can affect humans through the consumption of contaminated fish. In the meantime, there is also the danger of contamination at sublethal levels which can affect the predator-prey relationship, eating habits, reproductive success, and the general metabolism of fish (Arias et al. 2007).
Banana farming is one of the most important agricultural activities in Brazil. The crop ranks second in volume of fruit produced, with approximately 6.75 million tons per year (Statista 2018), second only to oranges (Hanada et al. 2015). Due to phytosanitary problems, mainly leaf diseases such as the black sigatoka (Mycosphaerella fijiensis) and the yellow sigatoka (Mycospaerella musicola), the crop can display a low productivity. These diseases are mainly controlled with the use of propiconazole, which acts on a wide spectrum of diseases caused by ascomycetes, basidiomycetes, and deuteromycetes (Garrison et al. 2011).
In Brazil, the use of exotic species for toxicity tests is mainly due to the scarcity of studies on the biology and sensitivity of allochthon species that could be used as test organisms. Therefore, there is an urgent need for studies in order to find native species of different trophic levels that are considered important to the environment, and can be standardized as test organisms, since the exotic species used have no ecological relevance (Zagatto and Bertoletti 2010). In the present study, lambari D. iguape was chosen because it is a native species of the Atlantic forest, easily produced in captivity and has a wide distribution in this habitat (Henriques et al. 2018). According to Baun et al. (2000), there is not a single species of organism that represents the effects caused by a pollutant in a given ecosystem. Therefore, there is a need to use several species of test organisms in order to represent the different levels in the trophic chain, increasing, thus, the probability of more comprehensive and reliable responses, involving organisms of different sensitivities. The lambari Deuterodon iguape is a small-sized Characiform native to the Atlantic Forest watershed (Fonseca et al. 2017). It is an endemic species of small rivers and streams in the tropical and subtropical forest region. It also has wide market possibilities, since recent studies have discovered, in addition to selling for human consumption, its use as live bait in recreational fishing (Henriques et al. 2019).
The zebrafish, Danio rerio, is the model fish most used in bioassays on genetics, neurophysiology, and biomedicine (Amsterdam and Hopkins 2006;Teng et al. 2019). Other important aspect in zebrafish is the transparent embryo for an easy teratogenesis assessment (Keshari et al. 2016). It has several attributes that make it particularly efficient for experimental manipulation. It is a small, robust, and very prolific fish, which can be kept easily and at a low cost in the laboratory (Spence et al. 2008). The great advantage of its use as a model organism is that, as a vertebrate, it is more comparable to humans than model species of invertebrates, such as Drosophila melanogaster (Barbazuk et al. 2000), and is more susceptible to genetic and embryological manipulation than model species of mammals, such as rats, in which these procedures are more complicated and expensive (Spence et al. 2008).
The hypothesis tested in this study was that the Atlantic forest lambari D. iguape has a metabolism similar to zebrafish D. rerio so that its use in bioassays can also be validated and mainly be used as a bioindicator species of aquatic environment conditions. This study aimed to verify the toxicity and sublethal effects of propiconazole on the Atlantic Forest lambari D. iguape and zebrafish D. rerio, through the evaluation of its metabolic rate.
In the laboratory, a total of 120 fish of each species were kept for 5 days in independent 500-L tanks, with constant aeration and daily water change (20%) to acclimate them to the conditions of the laboratory. The freshwater used for maintenance, passed through three 2-µm filters, two 1-µm filters, and one 0.5-µm filter arranged sequentially. The fish were fed with extruded commercial feed, 2.0 mm with 36% crude protein (CP), 7% ether extract (EE), and 4% crude fiber (CF), placed in the tanks, in the proportion of 2% of the live weight. Feeding was suspended 24 h before the experiments.

Tested substance
In this study, the main element, propiconazole, , (100 ng μL −1 in methanol, PESTANAL®, analytical standard-Empirical Formula C 15 H 17 Cl 2 N 3 O 2 ) ( Fig. 1), used as an active ingredient in the composition of the commercial pesticide formula TILT, one of the most used pesticides by Brazilian banana growers.
Determining the LC50 for fish subjected to propiconazole The experiment for determining the average lethal concentration, which represents the concentration calculated to cause 50% mortality in a tested population over a given period (LC50) (Rand and Petrocelli 1985), was short term, up to 96 h, with partial renewal (semi-static) and with 12 h of light cycle. D. iguape and D. rerio were placed in 20-L aquariums at 25 °C. For each treatment, 20 fish of each species (5 for each replica) were exposed to propiconazole concentrations of 0.1; 0.5; 1; and 2.5 µg L −1 , in addition to a control treatment (without propiconazole). Mortality was recorded every 2 h until the first 12 h and later at 24, 36, 48, 72, and 96 h. For each treatment, the percentage of survival was plotted against the exposure period (Barbieri 2007).
To obtain the propiconazole desired concentration, the necessary volume of the main substance (1 mg propiconazole mL −1 ) was calculated for volume aquarium and set with the help of a micropipette at the end of the acclimation. Water chemical analysis to confirm exposure concentrations of propiconazole was performed using an HPLC system (1200 series, Agilent Technologies, CA, USA) coupled to a 6130 quadrupole mass spectrometer with a G1978B multimode ion source [electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)].
The acute lethal effects of low concentrations of propiconazole in both species were analyzed by determining the average lethal concentration (LC50). The LC50 was calculated using the trimmed Spearman-Karber statistical method (with Abbott correction), proposed by Hamilton et al. (1978).
The pH, dissolved oxygen, and the concentration of ammonia, nitrite, and nitrate were monitored at the beginning and at the end of each test, to verify the water quality conditions of the experiment. For this measurement, pHmeters, ATAGO-S/Mill refractometer, Winkler's method for dissolved oxygen (Winkler 1888), and Koroleff's (1970) colorimetric method for ammonia were used.

Routine metabolism
The two species of fish studied were similar in size, D. iguape (3.0 ± 0.32 cm) and D. rerio (2.6 ± 0.21 cm), but regardless, we examined both the consumption of oxygen and the excretion of specific ammonia, that is, per unit weight. Consumption and excretion were divided by the animal's weight, specific consumption = consumption g −1 and specific excretion = excretion g −1 .
Both fish species acclimated to a temperature of 25 °C were exposed to concentrations of 0.0, 0.01, 0.05, and 0.1 µ L −1 of propiconazole for a period of 2 h. Five fish of each species for each concentration were subjected to measurement of oxygen consumption and ammonia excretion in each of the four concentrations with three replicates (Barbieri and Ferreira 2011).
Before the beginning of the experiments, the animals were kept in respirometers with continuous water circulation for at least 60 min, to alleviate the stress resulting from handling. Then, the water supply was suspended, and the respirometers were closed so that the fish consumed the oxygen present in a known volume of water, for a period of 1 h (Barbieri and Doi 2012). The respirometers were protected by a shield to isolate the animals from possible movements in the laboratory. The difference between oxygen concentrations, determined at the beginning and at the end of confinement, represented the animal's consumption during the period; the same was applied for ammonia. To minimize the effect of the lack of oxygen on metabolism, the duration of the experiments was regulated in such a way that the oxygen concentration at the end of the experiments was always greater than 70% of its initial concentration  (Barbieri and Ferreira 2011). Dissolved oxygen was determined using the Winkler method (1888) and ammonia concentration using the Nessler method (standard methods for the examination of water and wastewater).

Statistical analysis
Behavioral data were assessed according to species considering ammonia excretion and specific oxygen consumption in relation to the different concentrations of propiconazole tested. In addition, the species were compared separately according to ammonia excretion values and specific oxygen consumption for each propiconazole concentration. Initially, normality was analyzed based on the Shapiro-Wilk test and the homogeneity of variances by the Levene test. For the species model as a function of ammonia excretion and specific oxygen consumption, for each concentration, the differences were analyzed based on the Student's T test. As for the model in which each species was analyzed separately in relation to the different concentrations, the data were submitted to analysis of variance (ANOVA) and to the post-hoc Tukey test. For both experiments, the differences were considered significant when p < 0.05.

Results
During the entire experiment, the average temperature was 24.7 ± 0.6 °C. The water parameters pH, ammonium, nitrite, and nitrate did not significantly differ among the experimental units (p < 0.05) and continued within the range considered as acceptable for tropical fish species.
The acute toxicity of propiconazole to D. iguape and D. rerio exposed to different concentrations of this pesticide for periods of up to 96 h, expressed as LC50, is shown in Table 1. These results showed that propiconazole produced higher toxicities in both species of fish.

Mortality
The percent mortality of D. iguape exposed to propiconazole at each 24-h interval is shown in Table 1. No deaths of control animals were observed. The higher the concentration of pesticide the fish was exposed to, the higher the mortality observed. After being exposed to propiconazole, death was first observed at a concentration of 0.05 µg L −1 in the first 72 h. Mortality rates of 100% were observed after a 24-h exposure period at concentrations of 1.0 µg L −1 and were also 100% after 96 h at a concentration of 0.1 µg L −1 .
Only 26.66% average mortality was observed during the first 24 h at 0.05 µg L −1 , while 100% mortality rates during the first 48 h. Zebrafish exposed to propiconazole at each 24-h interval is shown in Table 1. No deaths of control animals were observed. Mortality rates of 100% were observed after a 24-h exposure period at concentrations of 1.0 µg L −1 and were also 100% after 96 h at a concentration of 0.05 µg L −1 . Only 33.33% average mortality was observed during the first 24 h at 0.10 µg L −1 , while 100% mortality rates during the first 72 h.

Routine metabolism
The specific oxygen consumption for D. iguape acclimated to a temperature of 25 °C, initially increased at a concentration of 0.01 µg L −1 and then decreased due to the increase in the concentration of propiconazole (Fig. 2). At the concentration of 0.1 µg L −1 , the specific oxygen consumption in relation to the exposure time decreases significantly in comparison with the control (Fig. 2). The decrease in the averages of specific oxygen consumption at a concentration of 0.1 µg L −1 represented a 71% decrease in the metabolic rate compared to the control.
There was a significant increase in the averages of specific oxygen consumption at a concentration of 0.01 µg L −1 . Using the ANOVA statistical test (Tukey, p < 0.05), it was found that the average of specific oxygen consumption at a concentration of 0.05 µg L −1 was not significantly different in relation to the time of exposure.
The specific oxygen consumption for D. rerio acclimated at a temperature of 25 °C also increases in the concentration of 0.01 µg L −1 and then decreases due to the increase in the concentration of propiconazole (Fig. 2). There was a significant increase in the averages of specific oxygen consumption at a concentration of 0.01 µg L −1 . For 0.05 and 0.1 µg L −1 concentrations, there were significant decreases. The decrease in the averages of specific oxygen consumption at a concentration of 0.1 µg L −1 represented a 40% decrease in the metabolic rate compared to the control. Using the ANOVA statistical test (Tukey, p < 0.05), it is found that the averages of specific oxygen consumption at concentrations 0.05 and 0.1 µg L −1 were significantly different in relation to the time of exposure (Fig. 2).
The excretion of ammonia for D. iguape acclimated to a temperature of 25 °C initially increases and then decreases as the concentration of propiconazole increased (Fig. 3). The decrease in the averages of ammonia excretion at a concentration of 0.1 µg L −1 represented a 68.7% decrease in the metabolic rate compared to the control.
Using the ANOVA statistical test (Tukey, p < 0.05), it is found that the mean of ammonia excretion at a concentration of 0.1 µg L −1 was significantly different in relation to the control (Fig. 3).
The results of ammonia excretion obtained from exposure to propiconazole showed a decrease in excretion rates for D. rerio. Using the ANOVA statistical test (Tukey, p < 0.05), it is found that the mean of ammonia excretion at a concentration of 0.1 µg L −1 was significantly different in relation to the control (Fig. 3). The decrease in the averages of ammonia excretion at a concentration of 0.1 µg L −1 represented a decrease in the metabolic rate of around 45.4% in relation to the control.
When comparing the two species for each concentration of propiconazole tested, it is observed that for the excretion of ammonia, there was a significant difference (p < 0.05) in the controls and for the highest concentration applied (0.1 µg L −1 ). As for the specific oxygen consumption, there is only a statistically significant difference for the concentration of 0.1 µg L −1 ( Table 2).

Discussion
Fish are excellent biological models to be used in the environmental monitoring of polluted and unpolluted aquatic environments (Damato and Barbieri, 2012). In addition, they can be found in most aquatic environments, playing an important ecological role in food chains (Cort and Ghisi 2014;Barbieri et al. 2019).
Fish of the Astyanax and Deuterodon genera, popularly known as D. iguape, have excellent potential as a bioindicator because they are very common, small, omnivorous specimens with considerable economic value and are beginning to be used in several studies for biomonitoring and bioassays in Brazil (Cort and Ghisi 2014).
In the present study, it was observed that D. iguape was a good biological model, responding well as a bioindicator, corroborating with other studies that also used D iguape to study lethal and sublethal effects of pesticides (Erbe et al. 2010;    -Krawczyk et al. 2015;Galvan et al. 2016) and effects of other pollutants such as gasoline (Galvan et al. 2016) and carbofuran ). Therefore, due to its availability and mainly its sensitivity to small changes in the aquatic environment that resulted in measurable changes, this fish can be used in Brazil as a biological model in biomonitoring studies and bioassays with pesticides. According to Arias et al. (2007), in recent years, aquatic biota is constantly exposed to a large number of toxic substances released daily in open environments, without proper treatment, from different sources of emission. Among the pollutants present in the water are fungicides, such as propiconazole. These fungicides can cause mortality and alterations in the metabolism of fish, as observed in the results obtained in this study with tests carried out with D. iguape and zebrafish D. rerio, examining toxicity (LC50), specific oxygen consumption, and ammonia excretion. It is common for fish and other aquatic organisms to be subject to receiving water contaminated by pesticides because they are close to vegetable cultivation fields treated with these substances (Hernández-Moreno et al. 2011;Cobas et al. 2016).
The toxicity tests with D. rerio are recommended because it is an organism used worldwide as a standard to establish LC50-96 h for pesticides and other pollutants. The propiconazole LC50 was compared between D. rerio and D. iguape in order to recognize possible differences in sensitivity between species. According to the LC50-96 h value calculated in this study, the acute toxicity of propiconazole for D. iguape was similar to that observed for D. rerio, with values of 0.05 (0.04-0.06) and 0.03 (0.02-0.04) µg L −1 , respectively. In this case, we found no significant difference in sensitivity between D. iguape and D. rerio. However, the toxicity of propiconazole was approximately three times greater for D. rerio than for D. iguape, when the exposure period was 24 h. This result leads to the supposition that D. iguape is more tolerant to the fungicide in the first 24 h of exposure.
The toxicity of propiconazole to fish has not been well documented, especially for D. iguape and D. rerio. For example, Hemalatha et al. (2016) obtained the 96-h LC50 of propiconazole for the fish Labeo rohita at 8.9 µL L −1 , a toxicity value much higher than those recorded in the present study where the LC50 for D. iguape and D. rerio were 0.05 µg L −1 and 0.03 µg L −1 , respectively. Wilfriel (2005) recorded 96-h LC50 values of propiconazole for various fish between 5.3 and 6.8 mg L −1 (Oncorhynchus mykiss 5.3 mg L −1 , Cyprinus carpio 6.8 mg L −1 , and Lepomis macrochiurus 6.4 mg L −1 ). Hernández-Moreno et al. (2011) argue that there is a great variability in the results of LC50 found for different species, and even within the same species, therefore, comparisons of results should be interpreted with caution to avoid erroneous conclusions possibly due to the applied test, the testing conditions, and stage of life of the exposed organisms, among other factors. However, the fact is that concentrations found in the environment of 12.90 mg L −1 can be very harmful to fish (Teng et al. 2019).
In the tests carried out with lambari and zebrafish, propiconazole, when used alone, demonstrated a significant effect in reducing the specific consumption of oxygen and the excretion of ammonia in the highest concentrations tested.
The decrease in specific oxygen consumption is closely associated with a decrease in metabolism , observed during the experiments through the low consumption of individuals exposed to higher concentrations of propiconazole compared to those exposed to lower concentrations.
This study demonstrates the action of propiconazole as a potentially toxic substance for the metabolic functions of D. iguape and D. rerio fish. It was observed that the specific oxygen consumption and ammonia excretion decreased at the highest concentration (0.1 µg L −1 ) for both fish. This physiological response to the presence of xenobiotics is directly associated with changes in metabolism and occurs due to the fish's attempt to maintain its homeostasis . Atypical situations can stimulate protein synthesis not directly related to growth, such as stress and thermal shock, toxicity to metals, toxicity to pesticides, deprivation of nutrients, and metabolic disorders, among others (Mommsent, 1998;Damato and Barbieri 2012).
According to Rand and Petrocelli (1985), fish can absorb pesticides directly from the water, and the gills are the main absorbing organ. The decrease in specific oxygen consumption is closely associated with a decrease in metabolism, a fact observed during experiments carried out through the low mobility of individuals exposed to higher concentrations of pesticides (Campos-Garcia et al. 2016). According to Vargas et al. (1991), xenobiotics affect the breathing processes of organisms by inducing them to use other sources of energy, which can be used for detoxification reactions and stabilization of metabolic patterns, which may explain the reduction in specific oxygen consumption to the extent where the concentration of propiconazole was increased.
The exposure of Nile tilapia Oreochromis niloticus to Folidol 600, an organophosphate insecticide whose active ingredient is methyl parathion, widely used in aquaculture tanks to eliminate predator insect larvae, promoted not only a significant reduction in oxygen consumption and an increase in ammonia excretion, but also an increase in hematological parameters (hematocrit and hemoglobin concentration) and an inhibition of cholinesterase activity (AChE, BChE, and PChE), as the concentration and exposure time increased. Lesions of the branchial tissue as well as the loss of ability to maintain homeostasis were identified as possible causes of these alterations. Both at low and high concentrations of the pesticide, cholinesterase activity was inhibited, which may reflect the fish's attempt to compensate to stay alive (Barbieri and Ferreira 2011). Campos-Garcia et al. (2016), in studies conducted with tilapia (O. niloticus), obtained an increase in specific oxygen consumption in individuals subjected to high concentration of carbofuran carbonate, which resulted in an increase in the metabolic rates of fish. A similar result was recorded by Barbieri et al. (2019) studying the effects of carbofuran on lambari Astyanax ribeirae.
In the tests for ammonia excretion performed with D. iguape and D. rerio, there was a statistically significant decrease in relation to the control when subjected to the presence of the fungicide, propiconazole, at the highest concentrations, demonstrating changes in the excretion of both fish. Barbieri and Ferreira (2011) identified changes in the excretion of ammonia in toxicity studies carried out with tilapia, O. niloticus, exposed to different concentrations of Folidol 600, which were similar to the results obtained in this study for D. iguape and zebrafish exposed to propiconazole. Areechon and Plumb (1990) and Heath et al. (1993) proposed that this response probably occurs due to a possible lesion in the branchial tissue, resulting in internal hypoxia and stimulation of erythropoiesis.
According to Mommsen (1998), atypical situations can stimulate protein synthesis that are not directly related to growth, such as stress from heat shock, nutrient deprivation, metabolic disorders, metal toxicity, viral infection, and others.
In freshwater fish, the final residues of protein metabolism are excreted mainly in the form of ammonia, and the mechanisms of this excretion are through gills and kidneys, and even in some fish species, the skin can perform this function (Bombardelli and Hayashi 2005). The results of ammonia excretion obtained from exposure to propiconazole showed a decrease in excretion rates; this fact suggests a decrease in protein metabolism as a mechanism to maintain the energy balance of fish submitted to propiconazole.
The fish Geophagus brasiliensis exposed to the herbicide 2,4-D (dichlorophenoxyacetic acid), a pesticide commonly used in pastures and in the control of aquatic macrophytes, showed a significant reduction in the consumption of dissolved oxygen in the water and an increase in ammonia excretion, probably related to the accumulation and metabolism of the contaminant in their lysosomes as well as other metabolic changes (Barbieri, 2008). Perhaps the same may have occurred with D. iguape and D. rerio, which had altered oxygen consumption and ammonia excretion, with a statistical difference between them only at the highest concentration (0.1 µg L −1 ). This result may indicate a difference in sensitivity between the two species, although in relation to the control, the species at this concentration were statistically different.
Gills play an important role in transporting gases for respiration and in regulating the osmotic and ionic balance of fish (Tabassum et al. 2016). The gills are composed of a single layer of vulnerable delicate tissues that can be easily damaged by any xenobiotic dissolved or suspended in the media in which the organism lives (Guzmán-Guillén et al. 2015). The changes in oxygen consumption as well as in the ammonia excretion recorded in the present study may be related to the histopathological lesions observed by Tabassum et al. (2016), such as edema, hypertrophy of epithelial necrosis, bulging binge fusion of gill lamellae, and other degenerative changes in the vascularization of the gills that occur when fish are exposed to xenobiotics (Melo et al. 2015). In summary, propiconazole proved to be toxic to D. iguape and D. rerio causing significant changes in metabolism and toxicity in both species of fish.
In view of the increase and the diverse uses of pesticides in Brazil, their ecological effects must always be studied and evaluated. The risks of toxicity of pesticides to fish are fundamental mainly because they are consumed by human populations. The data obtained here from acute toxicity can be useful to evaluate the negative short-term results on D. iguape, a fish commonly used to feed riverside communities. These results suggest that metabolic responses can be used as potential biomarkers to monitor residual pesticides present in aquatic environments and provide useful parameters to assess the physiological effects in fish. This implies that the results can serve as parameters for taking necessary precautions in the application of pesticides to avoid problems related to the life cycle of fish and other aquatic organisms. Moreover, these results will be able to provide indices for further studies considering the environmental risk assessment. However, the application of these findings needs to be complemented with more detailed histological studies before they can be established as special biomarkers for monitoring the aquatic environment.

Conclusion
Deuterodon iguape and Danio rerio responded well to exposure of varying propiconazole concentrations, as observed through changes in oxygen consumption and ammonia excretion.
In environmental monitoring, fish are excellent biological models for comparing polluted and unpolluted areas. In addition, these animals can be found in almost any aquatic environment, playing an important ecological role in the food chain. D. iguape was found to be a good biological model, and that specific oxygen consumption and specific ammonia excretion were good physiological biomarkers for exposure to propiconazole.
Much remains to be studied about the mechanisms of interactions between propiconazole and other environmental factors such as pH, temperature, and hardness in the aquatic environment, and how fish are affected when they come into contact with this pesticide. Therefore, further studies are needed to better understand the potential environmental risks of exposure to propiconazole and especially the toxicokinetic and toxicodynamic characteristics of this xenobiotic in the environment.
Authors' contributions MBH analyzed and interpreted data, was a major contributor in writing the manuscript, and was the author who submitted the manuscript. KFOR analyzed and interpreted data and was a contributor in writing the manuscript. LCB performed the statistical analysis and was a contributor in writing the manuscript. EB analyzed and interpreted data and was an important contributor in writing the manuscript. All authors read and approved the final manuscript.
Funding This study was financially supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)-São Paulo Research Foundation (process 2018/19747-2) and the National Council for Scientific and Technological Development (CNPq, Brazil, for the productivity research grant, process no. 302705/2020-1).

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability (software application or custom code) Not applicable in this section.

Declarations
Ethics approval and consent to participate This study followed the ethical principles for animal experimentation adopted by the Brazilian School of Animal Experimentation (COBEA) and received authorization (no.14/2018) from the Ethics Committee on Animal Experimentation of the Fisheries Institute, São Paulo, Brazil.
Consent for publication Not applicable in this section.