Influence of Sediment on the Acute Toxicity of Diflubenzuron and p-Chloroaniline Mixtures to Tilapia: Isobolographic Analysis and Gill Enzyme Activity

Abstract

Several fish farmers have used the insecticide Diflubenzuron (DFB) to control ectoparasites that cause diseases in fish species. This compound can be degraded to produce the toxic metabolite p-chloroaniline (PCA) within the aquatic environment. Mixtures of these compounds may interact - such as through antagonism, addition, or synergism - and some methods are available for the study and classification of these chemical interactions. The toxic effects of DFB, PCA, and their mixtures in 75%, 50%, and 25% PCA were analyzed in tilapia fish (Oreochromis niloticus) after 96 h, both in the presence and absence of sediment. Catalase (CAT), acid phosphatase (AcP), and alkaline phosphatase (AlP), enzymes found in Tilapia gills, were used as biomarkers. The presence of sediment decreased the bioavailability of compounds in the water. An antagonistic relationship was observed in the mixture of 25% PCA, while a synergistic relationship occurred in the 75%-PCA mixture. The fish exhibited oxidative stress and possible damage to the gills with alterations in the activities of the CAT, AcP, and AlP enzymes. In conclusion, DFB and PCA should be utilized carefully to minimize risks to aquatic life and human health. The observations strongly support the use of branchial catalases and phosphatases in tilapia as biological biomarkers of water pollution. The data may contribute to the establishment of maximum allowable concentrations of DFB and PCA in Brazil and other countries, as well as aid in the diagnosis of aquatic environments contaminated with these pollutants.

Keywords:
Diflubenzuron; P-chloroaniline; Biomarkers; Catalase; Phosphatases; Sediment; Tilapia fish.

HIGHLIGHTS

Diflubenzuron and its metabolite affect gill enzymes activity

Sediment decreases the toxicity of diflubenzuron and p-chloroaniline mixture

Diflubenzuron and its metabolite have synergistic toxic effect

INTRODUCTION

Fish are a source of food, income, and livelihood for countless people worldwide. Fish farming has grown significantly over the past fifty years. In 2022, fisheries and aquaculture production reached an all-time high of 223.2 million tons [¹]. Reducing fish mortality caused by diseases from ectoparasites is crucial for increasing the development of fish production. For this purpose, chemicals such as insecticides have been frequently used [²-⁴].

Diflubenzuron (DFB) is an insecticide belonging to the benzoyl urea group. It inhibits chitin synthesis, thereby controlling the presence of ectoparasites [²].

In fish farming, DFB does not cause toxic effects on fish. However, several studies have shown that various physiological and biochemical factors can influence the health of these organisms [³,⁵,⁶]. Additionally, in the aquatic environment, DFB can undergo degradation or metabolism, producing several metabolites, including p-chloroaniline (PCA) [⁷]. PCA is a priority pollutant in environmental risk assessments because it is highly toxic to aquatic organisms [⁸]. Furthermore, PCA is considered potentially carcinogenic and mutagenic to humans [⁷].

Although many compounds have been studied in water toxicity mixtures, usually only one is considered [⁹]. The combinations of these compounds can undergo interactions, such as antagonism, addition, and synergism, the mechanisms of which are well documented [¹⁰]. The use of mathematical models can help predict the presence and intensity of these effects [¹¹-¹³].

Additionally, the presence of sediment must also be considered, as contaminant adsorption is not ruled out and can interfere with the bioavailability of the compounds. For instance, diflubenzuron can attach to residues in fish farms and may enter the food chain through resuspension [⁴].

Fish gills are in direct contact with water [¹⁴,¹⁵]; therefore, biochemical and morphological changes in these organs can be assessed as biomarkers of pollution in the aquatic environment [¹⁶,¹⁷]. In this context, changes in enzymatic activities of catalase (CAT), acid phosphatase (AcP) and alkaline phosphatase (AlP) in fish gills exposed to pesticides have been reported [¹⁸-²¹].

Even though these enzymes are widely used as biomarkers of environmental pollution [¹⁶], there are no references in the literature regarding the effects of DFB and PCA mixtures on their activities.

While the use and disposal of these compounds have both been studied, it is necessary to investigate the potential damage they cause to aquatic biota under different conditions, such as in the absence and presence of sediment.

The legislation establishing the maximum permissible limits for chemicals in bodies of water in Brazil reports a small number of agrochemicals, although many compounds are currently in use. Diflubenzuron, among many other agrochemicals, is not included in the list of this legislation. Therefore, it is necessary to collect data on its adverse effects in order to implement public policies regarding the establishment of maximum permissible levels in bodies of water [⁶].

According to the Food and Agriculture Organization (FAO), the tilapia (Oreochromis niloticus) is one of the main cultivated species around the world. Due to its relevant commercial interest, this fish is bred within the most diverse production systems [²²]. Moreover, this species is ecologically significant since it has been found in natural ecosystems, like rivers and reservoirs, across several regions of Brazil, including the Northeastern, Southeastern, and Amazonian biomes [²³,²⁴].

Tilapia have been employed as test organisms in order to assess the risk of chemicals used in their production system [²⁵,²⁶], as well as that of pesticides (or other chemicals) that might contaminate aquatic ecosystems [²⁷,²⁸,²⁹].

In a previous study we reported on the acute effects of DFB and PCA mixtures in tilapia, in both the presence and absence of soil, and the alteration of enzymatic activity in hepatic tissue [³⁰]. This research was aimed at evaluating the joint acute action of DFB and PCA using a mathematical model combined with an isobolographic analysis. Additionally, this study focused on assessing the activity of tilapia gill enzymes after exposure to sublethal doses of the mixtures, again in the presence and absence of sediment.

MATERIAL AND METHODS

Test organisms

Adult and juvenile tilapia (Oreochromis niloticus) were obtained from a local fish farm (Piscicultura Polettini, Mogi Mirim, São Paulo State, Brazil). The fish were acclimated to the laboratory conditions at the Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), for one month, in 180-liter plastic tanks containing dechlorinated water. The tanks were maintained under constant aeration, a natural light-dark cycle, and a temperature of 28 ± 2 °C. The physical and chemical parameters of the water were monitored and remained stable throughout the acclimation period: a pH of 7.4 ± 0.4; a dissolved oxygen level of 8 ± 2 mg O₂.L^-1; a conductivity level of 160 ± 5 µS.cm^-1 and a hardness of 50 ± 2 mg CaCO₃. L^-1

The animals were fed with TetraMin^® Plus (Tetra Holding US Inc.) commercial fish food during the acclimation period. All experiments involving the fish were approved by the Ethics Committee on the Use of Animals at the State University of Campinas (CEUA/UNICAMP) legislation, under registration Nº 2756-1 and 3641-1 (Law Nº 11794/2008).

Acute toxicity tests

The acute toxicity bioassays were conducted using the commercial formulation of Diflubenzuron (Dimilin^®, Chemtura, Brazil) and p-chloroaniline (Sigma-Aldrich, technical grade, purity > 99%). The chemicals were tested both individually and in mixtures with PCA proportions of 75%, 50%, and 25%. The test solutions were prepared using carbon-filtered water.

The renewal of the test solutions and the analysis of the compounds in the solution were not performed during the assay. This action is supported by the stability of the compounds under the physicochemical conditions and the durability of the tests [³¹].

The test concentrations of diflubenzuron, p-chloroaniline, and their mixtures were 0.0 (control group), 0.1, 1.0, 10.0, and 100.0 mg.L^-1 [³²]. The tests were conducted in the presence and absence of artificial sediment which was prepared by mixing sand (70%), kaolin (20%), and peat (10%) [³³].

Ten juvenile Tilapia (length and average weight of 3 cm and 6 g respectively) were exposed to test solutions in 10 L glass aquaria (five fish per aquarium and two replicates per concentration) with continuous aeration and a controlled temperature (28 ± 2 °C) in the absence or presence of 900 g sediment [³⁴]. The fish were not fed during the test. The water’s physical and chemical parameters were monitored and remained consistent with those of the acclimation period. The number of individual deaths was recorded daily and at the end of 96 hours. The lethal concentration that affected 50% of the population (LC50-96h) was determined.

Evaluation of the DFB and PCA mixtures

The procedure for a joint mixture action was based on a method proposed by Marking [¹³]. The technique involves calculating the additive toxicity index of a given mixture by calculating the toxicity of the individual compound using the following equation:

$$\mathrm{S}=\frac{\mathrm{Am}}{\mathrm{Ai}}+\frac{\mathrm{Bm}}{\mathrm{Bi}}$$

Where S = the sum of individual compound toxicity; A and B correspond to the chemicals (in this case, DFB and PCA); and i and m correspond to the LC50-96h values for the isolated compound and mixture, respectively.

The Additive Index (AI) was calculated from these equations:

$$AI=\frac{1}{S}-1\text{when}S⩽1$$

$$AI=S(-1)=1\text{when}S⩾1$$

If the value of AI is 0, a simple additive effect is diagnosed; for AI < 0, the result is antagonistic, and for AI > 0, the product is synergistic [³⁵].

After obtaining the AI value, the Magnification Factor (MF) was calculated to demonstrate how much more or less toxic (in times) the mixture is compared to the isolated compounds. The MF value was calculated by adding 1 to the AI value in the case of synergism. In the case of antagonism, the MF value was obtained by the reciprocal of “1+AI” being AI in absolute [¹³].

The isobolograms were used to display any toxicological interactions (synergism, additivity, or antagonism). This procedure was applied to all the tested binary mixtures of DFB and PCA without mathematical derivation. These graphs were structured by plotting the concentrations of each pollutant, either alone or in combination, that induced a 50% lethality in fish (LC50) [³⁵,³⁶]. A point above the solid line indicates an antagonistic effect, while a point below the line indicates synergism. A point exactly on the line denotes an additive effect.

Test for enzymatic evaluation

For the enzymatic analysis, ten adult fish were exposed to sublethal doses of the compounds and their mixtures, both in the presence and absence of sediment, for 96 hours. The sublethal doses were calculated based on the LC50 values obtained from the acute toxicity test (LC₅₀/10, LC₅₀/50, and LC₅₀/100).

The procedures for this test were the same as those described in the acute toxicity tests. After 96 hours, the animals were anesthetized with benzocaine (0.1 g.L^-1) and sacrificed by spinal cord section to remove the gills. The organs were divided into equal portions, weighed, and homogenized in the appropriate buffer for each enzyme at a ratio of 1:4 (weight/volume).

The homogenate was centrifuged at 10,000 x g for 20 minutes at 4 °C. The supernatant was collected and stored at -80 °C before being used to determine the total protein and enzyme activities.

Enzyme activities

The supernatant of gill tissue was used to quantify the CAT, AcP, and AlP enzymes. The CAT activity was determined using hydrogen peroxide (H₂O₂) as a substrate, and the decrease in absorbance at 240 nm was measured [³⁰]. The reaction mixture contained a 50 mM phosphate buffer (pH 7), 30 mM of H₂O₂, and an enzyme extract. The specific activity of catalase was expressed as nmoles of H₂O₂.min^-1.mg^-1.

AcP and AlP activity was determined by incubating 10 µL of the supernatant with 10 mM p-nitrophenyl phosphate (pNPP) as a substrate and 100 mM acetate buffer at pH 5 (for AcP) or 250 mM glycine buffer at pH 9.4, plus 20 mM MgCl₂ (for AlP), at 37 °C for 40 minutes [³⁰, ³⁶]. The reaction was stopped by adding 1 mL of 1 M sodium hydroxide. The product of these reactions p-nitrophenol (p-NP) was measured at 405 nm. The specific activities of AcP and AlP were expressed as nmoles of pNP produced per minute per milligram (nmoles pNP.min^-1.mg^-1). All the enzymatic analyses were performed in triplicate. The absorbance values were read using an EON BioTek microplate spectrophotometer.

Protein concentration

The protein concentrations in the samples were determined in triplicate using the Lowry method [³⁷], with bovine serum albumin as the standard.

Statistical analysis

The LC50-96h values were calculated using probit analysis (Statgraphics Plus v. 5.1 software) and were considered statistically different when there was no overlap of the 95% confidence intervals. GraphPad Prism v. 5.1 software was used for other statistical analyses, and the data were presented as mean ± SD and analyzed by a one-way analysis of variance (ANOVA). The Dunnett test was used to verify differences between tested concentrations and the control. Differences were considered significant for P < 0.05 or P < 0.01.

RESULTS

Acute toxicity

The results of the acute toxicity bioassays are summarized in Table 1. LC50-96h values greater than 100 mg.L-1 were obtained for DFB and the mixture containing 75% of this compound, in both the presence and absence of sediment. PCA showed higher toxicity toward the fish when tested alone and in the combinations with 50% and 75% of this compound. However, in the presence of sediment, the median lethal concentration for PCA alone and in the mixtures of 50% and 75% PCA increased.

Table 1
Median lethal concentration (LC50-96h), in mg.L^-1, of DFB, PCA, and their binary mixtures in Tilapia fish.

Conditions	DFB	PCA	75%PCA	50%PCA	25%PCA
Without sediment	> 100	25 (16.7-29.56)	15 (10.7-21.3)	40 (29.15-42.22)	> 100
With sediment	> 100	37 (31.7-42.9)	25 (23.8-27.2)	45 (43.9-49.8)	> 100

Evaluation of DFB and PCA joint action

According to Marking’s Additive Index (AI), the mixtures that exhibited antagonistic toxicity without sediment were 50% PCA (AI = -0.033) and 25% PCA (AI = -0.77) (Table 2). Therefore, the toxic effects of these mixtures were 0.96 and 0.57 times lower respectively, when compared to the toxicities displayed by the individual compounds.

Table 2
The Additive Index (AI) and Magnification Factors (MF) were calculated after exposure of Tilapia to different proportions of DFB and PCA mixtures.

Conditions	75% PCA			50% PCA		25% PCA
	AI		MF (times)	AI	MF (times)	AI	MF (times)
Without sediment		+1,02 (2.33 to 0.03)1	2.0**	-0,033 (-0.47 to 0.57)	0.96	-0,77 (-0.59 to -1.24)	0.57*
With sediment	+0.69 (1.01 to 0.41)		1.7**	+0,19 (0.37 to -0.03)	1.2	-0,42 (-0.33 to -0.53)	0.70*

• ¹
  Range of AI in parenthesis;
• ^*
  Significant antagonism;
• ^**
  Significant synergism

However, only the 25% PCA mixture range did not overlap with zero, so it was considered to exhibit an actual antagonist effect (Table 2). In the presence of sediment, the mixture with 25% PCA still showed significant antagonistic interaction with DFB (AI = -0.42), with toxicity being 0.7 times lower. Nevertheless, in the presence of sediment, synergistic interactions between the compounds slightly increased as the PCA concentration in the mixtures was augmented 1.2 times for 50% PCA and 1.7 times for 75% PCA. An actual significant synergistic effect was detected for this last mixture (Table 2).

The meaning of AI values is demonstrated by the points in the isobolograms shown in Figure 1. This graphical representation displays the LC50-96h value, indicating the compound concentration needed to achieve this effect. The presence or absence of the antagonistic interactions involving sediment was observed at low PCA concentrations (25%) within the mixture. Synergic interactions occurred at higher PCA concentrations (75%) under the same conditions. In the absence of sediment, no interaction was observed (additive effect) for the 50% DFB + 50% PCA mixture; however, a slight synergism was observed in the presence of sediment.

Figure 1
Isobolographic analysis for mixtures of DFB and PCA: without sediment (A); with sediment (B). The additive line is the zero-interaction isobole constructed from LC50-96h values for each compound alone. LC50 values are represented by 75% PCA (▲), 50% PCA ( ● ), and 25% PCA ( ■ )

Enzymatic activities

A notable increase in CAT activity was observed for the lowest PCA concentration tested, exceeding 2,000%. However, 2 mg.L^-1 of DFB alone (Figure 2B) and in the 25% PCA mixture (Figure 2E) caused decreases of 35% and 21% in CAT activity, respectively. A reduction of 43% was observed for the 0.8 mg.L^-1 concentration of the 50% mixture of each compound (Figure 2D). CAT activities decreased by 80% at 0.45 mg.L^-1 of the 50% PCA mixture in the presence of sediment (Figure 2D). A similar reduction (70%) was observed for 1 mg.L^-1 of the 25% PCA mixture (Figure 2E).

Figure 2
Effect of PCA, DFB, and their mixtures on the catalase (CAT) activities of tilapia gills (n = 10) after 96 hours of exposure, in the presence and absence of sediment. The numbers on the control bars (zero concentration) indicating 100% activity correspond to nmol of H₂O₂ decomposed per minute per mg of protein. *Significantly different from the control at P<0.05 and ** at P<0.01. Data are expressed as the mean (±standard deviation)

In the absence of sediment, AcP showed a significant increase of approximately 125% in activity at 25:75 DFB/PCA mixture (Figure 3C) with significant increases observed at all tested concentrations.

Figure 3
Effect PCA, DFB, and their mixtures on acid phosphatase (AcP) and alkaline phosphatase (AlP) activities in the gills of tilapia (n = 10) after 96 hours of exposure in the presence and absence of sediment. The numbers on the control bars (zero concentration), representing 100% activity, correspond to nmol of p-nitrophenol produced per minute per mg of protein. *Significantly different from the control at P<0.05 and ** P<0.01. Data are expressed as the mean (±standard deviation)

In the case of AlP, the most significant change was observed in the absence of sediment, where activity increased by approximately 170% at a concentration of 0.4 mg L^-1 containing 50% PCA (Figure 3D). However, decreases in AlP activity were also noted. The greatest reduction (over 50%) occurred in the DFB (Figure 3B) and 75% PCA (Figure 3C) treatments conducted without sediment.

DISCUSSION

The presence of sediment in the acute toxicity test decreased the bioavailability of DFB and PCA compounds in water. In aquatic environments, xenobiotics may partition between the aqueous phase and the sedimented particles. This partition is influenced by the physical and chemical properties of the water, the sediment type present, and the molecular nature of the xenobiotics [³⁸]. Additionally, the low water solubility and high partition coefficients for DFB and PCA contribute to their association with sediment particles [⁵]. Bellemjid and coauthors [³⁹] and Medeiros and coauthors [³⁴] demonstrated the efficiency of sediment in reducing the toxicity and bioavailability of the pesticide carbendazim, and of the insecticide teflubenzuron, respectively.

The presence of sediment did not alter the interactions between DFB and PCA in the mixtures (Figure 1). Exposure to a combination of 75% DFB and 25% PCA elicited a less severe response in the fish compared to that of the pesticides applied individually, indicating an antagonistic effect. However, the antagonistic interaction between DFB and PCA did not mitigate the toxic effects of this mixture in the aquatic environment. For instance, DFB in the aquatic environment can undergo degradation or metabolism, potentially leading to the formation of additional PCA. As observed in Table 2, the mixture predominantly composed of PCA (75%) exhibited a synergistic effect. Interactions involving various xenobiotics, which pose significant risks to many aquatic organisms, have been reported [⁹,³⁶,⁴⁰]. However, there remains a deficit in the scientific literature regarding the environmental impact of the DFB and PCA mixture on aquatic organisms.

The isobologram was used as a graphical representation of the synergism and antagonism of compounds without mathematical derivation. The visual representation illustrates LC50-96h and the compound concentrations required to achieve this effect. The observed interactions between DFB and PCA in the mixtures suggest that Marking's Additive Index (AI) is suitable for predicting the combined toxicity of these compounds. This index was applied to provide a prognostic assessment of the toxic effects of compound mixtures on the environment [³⁶,⁴¹].

Additionally, the results indicated that sublethal concentrations of DFB, PCA, and their mixtures can alter key enzymes used as markers in fish metabolism. In fish species, the gills are the first organs to come into direct contact with aquatic xenobiotics [⁴²].

The measurement of antioxidant enzyme activities can express the oxidative damage that xenobiotics induce in aquatic ecosystems. In this context, the antioxidant enzyme CAT can protect organisms against hydrogen peroxide (H2O2) generated in many situations of stress [⁴³].

The CAT enzyme has been widely utilized as a biomarker of environmental pollution due to its sensitivity to various xenobiotics. Lombardero and coauthors [⁴⁴] used gill CAT as biomarkers in order to biomonitori pesticide pollution in a shallow lake. Carvalho and coauthors [⁴⁵] observed a decline in catalase (CAT) activity in the gills of Tilapia exposed to samples of water from the Monjolinho River (São Carlos, SP, Brazil), which were contaminated with metals. CAT activity was statistically higher in fish gills from a river contaminated with mercury, confirming the capacity of the metal interference with redox equilibrium [⁴⁶].

Numerous pesticides can stimulate the development of antioxidative defenses. In this context, catalase (CAT) is one of the most extensively studied antioxidant enzymes [⁴⁷].

In this work, generally, CAT activity after 96 hours of Tilapia exposure to the compounds (both individually and in mixtures) showed a significant increase in the absence of sediment. The results are consistent with CAT activation, which occurs to counteract oxidative stress in gill cells due to excessive H₂O₂ production [⁴⁰]. Several authors have reported the increased activity of CAT as a consequence of pesticide exposure. Fish gill CAT was activated by the exposure of Catla catla, Astyanax altiparanae and Tor putitora to a carbaryl/methyl parathion mixture [⁴⁸]; atrazine [⁴⁹] and cypermethrin [⁵⁰]. This activation can be explained by the fact that when reactive oxygen species (ROS) production exceeds the cell's endogenous protective mechanisms, oxidative stress occurs, causing damage to cellular components. This protection is conferred by specific degradative enzymes, such as CAT, and also by antioxidant vitamins and other radical scavengers [⁵¹].

This reduced capacity to scavenge hydrogen peroxide produced in the gills may be an indication of the decreased activity of CAT, leading to an increase in ROS and consequently, the oxidative stress, with deleterious effects. Therefore, CAT is sensitive to free radicals, particularly superoxide anions, which can inactivate the enzyme [⁴²]. This observation may explain the decrease in CAT activity in the presence of sediment. It is likely that an excessive increase in ROS was triggered by the ingestion of sediment particles containing adsorbed compounds by the fish, which may explain the reduction in CAT activity in the presence of sediment.

The literature reports on the diminishing activity of gill CAT due to exposure to pesticides and emerging pollutants. According to Al-Ghanim and coauthors [⁵²], gill CAT activity decreased significantly in fish exposed to the insecticide fenvalerate when compared to this in the control fish, in both the gills and liver. Uçar and coauthors [⁵³] reported that short-term exposition concentrations of fipronil (50-200 μg L^-1) inhibited the catalase (CAT) activity in the gills of rainbow trout. Gill CAT activities decreased significantly in Clarias gariepinus exposed to sub-chronic levels of Ibuprofen [⁵⁴].

The alteration of CAT activity by one pesticide can be affected in the presence of another. Thus, the CAT activities in the gills of Prochilodus lineatus exposed to the herbicide 2,4-D increased by 95%. However, in fish exposed to the mixture of 2,4-D and fipronil, their enzyme activity did not alter when compared with that of the control group [⁵⁵]. These findings are consistent with the data in Figure 2.

Hypoxia can lead to acidification and increased oxidative stress, both of which have profound consequences on cell physiology [⁵⁶]. This seems to be associated with the adverse effects on fish exposed sub-chronically to DFB, which demonstrated histopathological and biochemical alterations in the gills and erythrocytes [⁵⁷]. Therefore, we assume that any change in the normal status of oxygen uptake and its assimilation can indirectly influence CAT activity.

The hydrolysis of various phosphate-containing compounds are catalyzed by AcP and ALP enzymes. Both act in the transphosphorylation processes in acid and alkaline mediums, respectively [⁵⁸]. The enzymes are involved in a variety of metabolic processes such as cell differentiation, permeability of molecules, signal transduction, autophagic digestion, and decomposing organic phosphates [³⁶,⁵⁸,⁵⁹]. While AcP has a role in certain detoxification functions, AlP is linked to the transportation of intermediate compounds in glycogenesis or glycogenolysis [⁶⁰]. In these contexts, gill phosphatases have been used as indicators of contamination of polluted areas. For example, all tested concentrations of tannery wastewater significantly elevated the activities of gill ALP and other enzymes in Poecilia reticulata [⁶¹]. According to Seitkalieva and coauthors [⁶²], the AcP specific activity increased by a factor of 1.5-3 in the gills of mussels exposed to seawater samples from coastal areas characterized by different degrees of anthropogenic impact.

In this study, AcP activity decreased for PCA and their mixtures in the presence of sediment. However, these changes were not of great magnitude compared to the substantial increase in activity observed in the 75% PCA mixture, in the absence of sediment.

Variations in ALP activity, both increases and decreases, were also observed for the pure compounds and mixtures. However the most significant alteration (≥150%) was recorded as an increase in activity in the 50% PCA mixture in the absence of sediment.

Increased phosphatase activity in the gills may indicate the rupture of cell membranes and lysosomes [⁶³,⁶⁴]. According to Kong and coauthors [¹⁶] enhanced peroxidation of lysosomal membranes can lead to membrane lysis and release of ACP, as well as an increase in ACP activity. These authors also observed that both AcP and AlP activities increased in Carassius auratus embryos after exposure (120 h) to mercury concentrations ranging from 0.2 to 10 µg L^-1.

The gills are in direct contact with DFB and PCA in the water, and these compounds can inactivate phosphatases reducing their activity through this exposure route [⁶⁵,⁶⁶]. In previous work, our observations indicated that Hg²⁺ inhibited AcP activity in the fish Mettynis argenteus [⁶⁷]. AcP and AlP activities decreased in the gills of Channa punctatus when exposed to lindane (0.05 - 0.1 mg L^-1) [⁶⁸] and in Tilapia gills when exposed to the pollutant fumaronitrite [⁶⁹]. Some mechanisms might explain the variations in phospahatese activities, including both increases and decreases.

According to Jing and coauthors 2006 [⁷⁰], higher gill AcP activity in mussels was associated with damage to lysosomal membranes. Therefore, in the gills of Pinctada fucata, the increased AcP activity upon exposure was likely due to the stimulation of cell defense mechanisms. Conversely, the decrease in enzyme activity when exposed to the same pollutant was probably because the pollutant interfered with these defense mechanisms. In this way, the enzymes may have been inactivated by reactive oxygen species ROS [⁷¹]. Additionally, changes in enzyme activity may be related to the phosphorylation or dephosphorylation of proteins involved in signaling pathways that regulate an altered metabolism caused by the toxicant [¹⁶].

CONCLUSION

Our results indicate that DFB, PCA, and their mixtures can affect enzymes essential to fish health even at concentrations far below the LC50.

The mixture containing 75% of the toxic compound PCA exhibited synergistic effects on organisms. In this case, the use and disposal of these substances ought to be managed with caution. An aquatic environment containing these compounds may induce synergistic effects and impact other organisms, potentially compromising the entire trophic chain.

DFB and PCA can be adsorbed onto sediment particles; however, their toxic interactions did not change significantly.

The observations strongly support the use of branchial catalases and phosphatases in tilapia as biological biomarkers of water pollution. In some cases, enzymatic activity changes exceeded a 150% increase.

The present study provides critical data that may contribute to establishing maximum allowable concentrations of DFB and PCA in Brazil and other countries, as well as aid in the diagnosis of aquatic environments contaminated with these pollutants.

• Use of Generative Artificial Intelligence:
  The authors declare that large language models and other generative artificial intelligence (AI) or AI-assisted technologies cannot be credited as authors and have not been listed as authors of this paper.
  The author declare that did not use the artificial intelligence.
• Funding:
  This Project received financial support from CAPES, FAPESP and FAEPEX.
• Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee of the STATE UNIVERSITY OF CAMPINAS (protocols code Nº 2756-1 and Nº 3641-1, approved: 25-Jun-2012).
• Informed Consent Statement: Not Applicable.

Acknowledgments:

The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for awarding a postgraduate scholarship to DDD. The authors also thank Mr. Jonathan R. S. Bradnick for critically reviewing the manuscript.

Data Availability Statement:

Research data are only available upon request for corresponding author.

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• Editor-in-Chief:
  Bill Jorge Costa

• Associate Editor:
  Luiz Gustavo Lacerda

Publication Dates

• Publication in this collection
  10 July 2026
• Date of issue
  2026

History

• Received
  06 May 2025
• Accepted
  02 Mar 2026