Hepatotoxic effects of the herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) after short exposure on adult zebrafish Danio rerio

doi:10.21203/rs.3.rs-4682259/v1

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Hepatotoxic effects of the herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) after short exposure on adult zebrafish Danio rerio

https://doi.org/10.21203/rs.3.rs-4682259/v1

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2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide widely used around the world. It has been detected in water samples, with a half-life ranging from 15 to 300 days depending on environmental conditions. This study aimed to assess the effects of short-term exposure to the herbicide 2,4-D on the liver of Danio rerio (zebrafish) through histopathological and histochemical analyses, as well as markers related to oxidative stress. The results revealed structural and vascular lesions in the livers of zebrafish across all groups exposed to 2,4-D (at concentrations of 0.03, 0.3 and 3.0 mg/L). Analysis of the Histopathological Alteration Index suggests severe (3.0 mg/L) or moderate (0.03 and 0.3 mg/L) liver impairment in zebrafish exposed to 2,4-D. Exposure to the herbicide also led to a reduction in acid polysaccharides (0.03 and 3.0 mg/L) and glutathione (GSH) levels (at concentrations of 0.03 and 3.0 mg/L), and increased levels of the oxidized glutathione (GSSG) (at concentrations of 0.03 and 0.3 mg/L). No significant changes in lipid peroxidation levels were observed. These findings suggest that just 7 days of exposure to permissible concentrations of 2,4-D (0.03 mg/L) or higher (0.3 and 3.0 mg/L) can have a detrimental impact on biochemical, histochemical, and histopathological parameters in the liver of adult zebrafish (Danio rerio).

aquatic environment

pollution

pesticide

liver

histopathological

ecotoxicology

The large-scale agricultural production model has historically relied on the use of pesticides to address issues stemming from soil degradation and to manage pests that pose threats to crops (Moraes 2019). Consequently, there has been a notable increase in pesticides sales in recent years. Only in Brazil, over 800 thousand tons of pesticides were sold in 2022 (IBAMA 2023), while global pesticide consumption currently stands at 2.6 million tons per year (Alan et al. 2023). Among these pesticides, herbicides are intensively used to control plant diseases and eliminate weeds (Gupta 2019).

2,4-dichlorophenoxyacetic acid (2,4-D) is an herbicide widely used throughout the world, and the second most sold pesticide in Brazil (IBAMA 2023). It serves as the primary active ingredient in more than 1,500 chemicals formulations (Islam et al. 2018). Highly selective, 2,4-D is utilized as pre-/post-emergent systemic herbicide across various crops. Functioning as a growth regulator, it mimics the plant hormone auxin, thereby influencing plant growth regulation and ultimately leading to plant death (Martins et al. 2024). Despite being one of the earliest herbicides developed, its high efficacy and low cost continue to render it a commercially appealing option to this day.

In the aquatic environment, the half-life of 2,4-D can range from 15 to 300 days, depending on environmental factors (Islam et al. 2018). Typically, the concentration of 2,4-D in water falls within the range of 4 to 24 µg/L (Atamaniuk et al. 2013; Islam et al. 2018). Due to its intense and widespread use, several countries, including Spain, the United States, Mexico, Greece and Iran, have detected 2,4-D concentrations in both surface and drinking water (Hernández et al. 2011; Rodil et al. 2012; Ensminger et al. 2013; Yamini and Saleh 2013; Tsaboula et al. 2016; Martins et al. 2024).

While the European Union adopts a more conservative stance and establishes a maximum concentration of 0.1 µg/L (Zuanazzi et al. 2020; Pereira et al. 2022), in Brazil the maximum permitted limit of 2,4-D for consumption is 30 µg/L, or 0.03 mg/L (CONAMA 2008). An assessment of water quality conducted in the interior of São Paulo state revealed the presence of 2,4-D in regions adjacent to sugarcane, corn, coffee, and other crops, with concentrations ranging from 143.1 to 366.6 µg/L (CETESB 2018). In another study, this time involving samples from Paraná state, the presence of 2,4-D (ranging from 0.193 to 0.387 µg/L) was detected in water samples from surface and underground sources, as well as in treated water, raising concerns about its presence and toxic effects on non-target organisms (PEVASPEA 2023).

Therefore, there is significant concern about aquatic ecosystems, as agricultural chemicals undoubtedly constitute the largest group of hazardous substances causing widespread pollution (Atamaniuk et al. 2013). Fish, occupying higher trophic levels in aquatic food chains, are more vulnerable to the presence of contaminants due to the biomagnification effect, making them crucial indicators of environmental pollution (Prusty et al. 2011). Indeed, anthropogenic pollution of freshwater ecosystems by pesticides, particularly herbicides, has led to declines in fish populations and other aquatic organisms in recent decades (Burkhardt-Holm et al. 2002; Davidson and Knapp 2007; Amenyogbe et al. 2021; Brain and Prosser 2022; Islam et al. 2022). In this context, the liver serves as an important bioindicator due to its functions in nutrient metabolism and xenobiotic detoxification processes in vertebrates (Camargo and Martinez 2007; Martins et al. 2021).

Considering the high level of commercialization of 2,4-D in Brazil and globally, its persistence in the environment, and its presence in rivers and lakes, it is evident that concerns persist regarding contamination of non-target organisms in aquatic ecosystems (Martins et al. 2024). Therefore, the objective of this study was to evaluate the hepatotoxic effects of short-term exposure to the herbicide 2,4-D through histopathological and histochemical analysis, as well as markers related to oxidative stress in adult Danio rerio zebrafish.

2.1. ANIMALS

Adult fish of the species Danio rerio were obtained from aquarium stores in Santa Catarina. They were acclimated for two weeks in the vivarium of the Federal University of Santa Catarina (UFSC). The fish were housed at a stoking density of 1 gram (g) of fish per liter (L) under standardized laboratory conditions. These conditions included dechlorinated and filtered water, a controlled temperature (27 ± 1ºC), a light-dark cycle of 14-10h. They were fed twice a day with commercial fish food administered twice daily (composition: 46% protein, 6% fat, 1.8% fiber and 9.5% inorganic matter - Ca, F and Mg). Water parameters were monitored daily and maintained within the following ranges: pH 7.0 (± 0.5), toxic ammonia (NH3) (0 ~ 0.25ppm), and nitrite (NO2) (0.001 ~ 0.01ppm). The animal husbandry conditions described above were in compliance with the Ethics Committee on the Use of Animals – UFSC (protocol approved by CEUA / UFSC nº 6778251119).

2.2. HERBICIDE

The commercial formulation U46 BR (Nufarm Chemical and Pharmaceutical Industry S/A), containing 806.0 g/L of 2,4-D, was utilized in the experiments. The dilution was prepared using aquarium water, taking into account the concentration of 2,4-D in the formulation.

2.3. EXPERIMENTAL PROCEDURES

After the acclimatization period, the animals were randomly divided into 4 groups (n = 35/group), 0.0 (control) and exposed to 2,4-D concentrations: 0.03; 0.3; 3.0 mg/L. The designated exposure duration was 7 days, as there is a lack of literature regarding the effects of 2,4-D after this duration of exposure. On the 4th day of exposure, the water in all the aquariums was replaced. At the end of the 7th day of exposure, the specimens were euthanized to procure their livers. Euthanasia was conducted by immersing the specimens in eugenol at a concentration of 75 mg/L of water. All aforementioned procedures were executed in compliance with Normative Resolution nº 37 of the National Council for the Control of Animal Experimentation (CONCEA).

2.4. HISTOLOGICAL ANALYSIS

For using light microscopy analyzes, liver samples were dissected, fixed in alcoholic Bouin solution for 24 hours and subsequently preserved in 70% ethanol (n = 3/group). Following this, the samples were dehydrated in an ascending ethanolic series 70% -100%, clearing in xylene and embedding in paraffin. Tissue sections of 6µm thickness were obtained using a Leica RM2255 rotary microtome at the “Laboratório Multiusuário de Estudos em Biologia” (LAMEB). The resulting slides were then subjected for staining techniques with Hematoxylin and Eosin (HE), Toluidine Blue (TB).

For morphological analyses of the liver samples, 10 photomicrographs were analyzed per animal, totaling 30 photos per group. Evaluations were performed by calculating the Histological Alteration Index (HAI), which considers the frequency and severity of each histological change (lesion). Lesions were categorized into progressive stages based on tissue function impairment, following the criteria outlined by Poleksic and Mitrovic-Tutundzic (1994), using the formula:

IAH = 10⁰. Σ^I + 10¹. Σ^II + 10². Σ^III

where Σ^I, Σ^II and Σ^III represent the total number of changes according to their respective stage, and 10⁰, 10¹ and 10² are the factors for calculating the HAI, based on the severity of the injury. HAI values ranging from 0 to 10 indicate normal organ function; 11 to 20 suggest slight damage; 21 to 50 indicate moderate damage; 50 to 100 indicate severe damage and values exceeding 100 indicate irreversible tissue damage, as adapted from Poleksic and Mitrovic-Tutundzic (1994). The stained sections were photographed using an Olympus BX41 upright microscope. and images were captured with the Q-imaging 3.3-megapixel color digital camera and the Q-imaging Q-capture Pro 5.1 image capture software.

Slides stained with TB were utilized to examine the histochemical profile of acidic polysaccharides, employing integrated density analysis with ImageJ software (Schneider et al. 2012; Hartig 2013). In this analysis, the photomicrograph is transformed into an 8-bit grayscale and squares of defined area were employed for all measurements. These squares were randomly positioned within the image and six areas of 4588.23 µm² each were measured in each section. Subsequently, data were extracted and plotted using GraphPad Prism statistical software for further analysis.

2.5. BIOCHEMICAL ANALYSIS

Liver samples (consisting of 5 livers/sample) were thawed (n = 6/group), homogenized in phosphate buffer (0.3 M, pH 7.4) and subsequently centrifuged (10 min, 10,000 rpm, 4°C). The resulting supernatant was used to determine protein concentration, reduced glutathione (GSH) level, oxidized glutathione (GSSG), lipid peroxidation (LPO) level, and NADPH oxidase activity. Protein concentration (mg mL-1) was assessed determined by the Bradford method (Kruger 1994) with bovine serum albumin as standard in an Infinite M200 TECAN microplate reader, with absorbance measured at 595 nm. GSH and GSSG measurement was done as previous described (Rahman et al. 2007). Briefly GSH reacts with dithionitrobenzoic acid (DTNB), forming a conjugate (GSH-TNB), which was measured by spectrophotometry (412 nm), It was used concentration curve of GSH as standard. The GSSG measurement as based on GSSG reduction to GSH by Glutathione reductase, reducing all GSSG to GSH, then free GSH levels was measured by reaction by DTNB. To calculate GSSG, the measurement of GSH was subtracted by GSH measurement after glutathione reductase, this difference was the GSSG concentration (Huber et al. 2008). The end product of lipid peroxidation is MDA (Malondialdehyde), which was measured in tissue homogenates based on a reaction with thiobarbituric acid (TBA) to form a pink-colored fluorescent complex. The MDA produced was determined by the fluorescence of the MDA-TBA complex with excitation at 515 nm and emission at 553 nm, using MDA as standard (Ohkawa et al. 1979). To evaluate the activity of the NADPH oxidase enzyme, 1.25 microliters of 10mM lucigenin were inserted per well together with NADPH buffer (50mM PBS, 0.01mM EDTA, pH 7.40). A first reading was taken to determine the background count. After that, 25 microliters of 10mM NADPH were inserted per well and read continuously for 15 min at 37°C and the luminescence was evaluated during reading (Janiszewski et al. 2002).

2.7. STATISTICAL ANALYSIS

Results are presented as mean ± standard error. The data were subjected to the Kolmogorov-Smirnov normality test and outliers were assessed using the ROUT method. Identified outliers resulted in the removal of the respective animal from the analysis. Statistical analyses were conducted utilizing one-way Analysis of Variance (ANOVA), followed by Tukey's post-test, using the GraphPad Prism version 8.0.2 software. Differences were considered significant at p < 0.05. Study images were captured using Adobe Photoshop CS6 software.

3.1 HISTOPATHOLOGICAL AND HISTOCHEMICAL ANALYSIS

The histopathological analysis of the liver of adult Danio rerio zebrafish is shown in Fig. 1. The normal liver structure primarily comprises hepatocytes, blood vessels, and blood cells traversing these vessels (Fig. 1a). In zebrafish exposed to concentrations of 0.03, 0.3 and 3.0 mg/L of 2,4-D, structural and vascular lesions were observed in the liver tissue. The predominant lesions included vacuolization, cellular and nuclear hypertrophy, nucleus deformation, tissue disarray, sinusoid dilation, hyperemia and in some instances, hemorrhages and necrosis (Fig. 1b-f). No deaths of zebrafish were found in any of the groups evaluated in the study.

Histopathological analyses of zebrafish livers exposed to 2,4-D demonstrated an increase incidence of tissue lesions compared to the control group (Table 1). Calculation of the Histopathological Alteration Index (HAI) revealed a significant difference [F(3,8) = 7.60; p < 0.01] of the 3.0 mg/L 2,4-D group compared to the control group (p < 0.01) and also compared to the other exposed groups (p < 0.5). The HAI of the control group indicates healthy liver function (range of 0 to 10), the 0.03 and 0.3 mg/L groups exhibited higher values within the range of 20 to 50, indicating moderate liver tissue damage. Notably, the group exposed to 3.0 mg/L demonstrated an HAI value exceeding 100, indicating that the organ presents irreparable tissue damage and suggesting a serious impairment of the organ (Fig. 2).

Table 1

Frequency table of histopathological lesions according to their stage in the liver of zebrafish (*Danio rerio*) control and after exposure to the herbicide 2,4-D. 0 = Absent or rarely frequent; + = Infrequent; ++ = Moderately frequent; +++ = Very frequent
Histological changes	Stage	Groups (mg/L)
Histological changes	Stage	Control	0.03	0.3	3.0
Vacuolation	I	+	++	+++	+++
Tissue disarray	I	+	++	++	+++
Cytoplasmic hypertrophy	I	+	+	++	+++
Nuclear hypertrophy	I	-	+	+	+
Nuclear deformation	I	-	++	++	++
Cell membrane rupture	II	-	+	+	++
Sinusoidal expansion	I	+	++	+	+
Hyperemia (vascular congestion)	I	+	+	++	++
Bleeding	II	-	+	++	++
Necrosis	III	-	-	-	++

The analysis of acidic polysaccharides through (TB) labeling demonstrated that exposure to 2,4-D can affect the levels of these molecules in the liver of zebrafish [F(3,716) = 47.27, p < 0.001]. It was observed that the groups exposed to 0.03 and 3.0 mg/L of 2,4-D showed a decrease in relation to the control group (p < 0.001). Additionally, the 3.0 mg/L 2,4-D group exhibited lower levels of acidic polysaccharides compared to the other groups exposed to 2,4-D (p < 0.001, for both groups) (Fig. 3).

3.2 BIOCHEMICAL ANALYSIS

Exposure to the herbicide 2,4-D induced changes in the levels of the GSH in the liver of zebrafish [F(3,19) = 8.247, p < 0.001], resulting in decreased GSH levels in the 0.03 and 3.0 mg/L groups of 2. 4-D (p < 0.01). The levels of the GSSG were also affected by 2,4-D [F(3,18) = 4.404, p < 0.05], showing an increased in groups exposed to 0.03 and 0.3 mg/L of 2,4-D (p < 0.05). While evaluating the activity of the NADPH oxidase enzyme in the liver, no significant differences were found compared to the control group, but rather between the groups exposed to 2,4-D [F(3,20) = 3.124, p < 0.05], where the 0.3 mg/L group exhibited higher activity compared to the 0.03 mg/L group of 2,4-D (p < 0.05). Regarding lipid peroxidation levels (expressed as MDA), no changes were observed in the liver of zebrafish after exposure to the 2,4-D herbicide (Fig. 4).

In our study, exposure to the commercial formulation of the herbicide 2,4-D to permissible concentrations (0.03 mg/L) or higher (0.3 and 3.0 mg/L), for only a week, induced several histopathological changes in the liver, including increased vacuolation, tissue disarray, cellular and nuclear hypertrophy, nuclear deformation, cell membrane rupture and necrosis, with the latter being present at the concentration of 3.0 mg/L of 2,4-D. Additionally, vascular changes such as sinusoidal dilation, hyperemia characterized by vascular congestion, and hemorrhages were noted.

Similar histopathological changes have been reported in previous studies following exposure to 2,4-D. Cattaneo et al. (2008) observed that Rhamdia quelen fish presented abnormal arrangement of hepatic cords, cell membrane rupture, and vacuolation of hepatocytes after exposure to 700 mg/L of 2,4-D for 96 h. In guppies (Viviparous Poecilia), acute exposure (96 h) to 20 µl/L of 2,4-D resulted in an increase in vacuolation and cytoplasmic damage, while a concentration of 40 µl/L also caused vascular damage such as sinusoid vasodilation and vascular congestion (Vigário and Sabóia-Morais 2014).

The most frequent lesions identified in our study were vacuolization, cytoplasmic hypertrophy and tissue disarray. Vacuolization is characterized by the formation of vacuoles in the cell cytoplasm, may signify stored energy in the form of glycogen or lipids, or it may represent a change in which there is disruption of organelles such as the rough endoplasmic reticulum and Golgi apparatus, and/or accumulation of fluid in the cytoplasm (Braunbeck 1998). While the exact mechanisms behind vacuolization in our study are not definitively established, the alterations in organelles or cytoplasmic fluid accumulation observed in fish exposed to 2,4-D suggest changes in energy metabolism-related molecules (Oruç and Üner 1999; Cattaneo et al. 2008). Therefore, it is reasonable to consider that this increase in vacuolation is due to changes in organelles and/or accumulation of fluids in the cytoplasm, induced by exposure to 2,4-D. More studies are needed to clearly understand the cellular events caused by 2,4-D that are involved in this change.

Cytoplasmic or nuclear hypertrophy indicates increased cellular activity in response to the presence of a chemical compound or absence of a specific substance, resulting in increased cell volume due to water and electrolyte accumulation (Ferguson 2006; Hinton et al. 2018). Cytoplasmic hypertrophy was moderately frequent at the 0.3 mg/L concentration and very frequent at the 3.0 mg/L concentration of 2,4-D in our study, consistent with its progressive nature wherein higher 2,4-D concentrations led to greater hepatocyte hypertrophy (Bernet et al. 1999).

Tissue disarray was observed across all studied groups, with moderate (0.03 and 0.3 mg/L) or high (3.0 mg/L) frequency. Liver architecture alteration may stem from cytoskeleton changes induced by 2,4-D, leading to structural reorganization, redistribution of microtubules and microfilaments, which generate organelle distribution disruption, increase intracellular space, and impaired hepatocyte interactions (Zhao et al. 1987). Similar cytoarchitecture changes have been observed in previous studies (Cattaneo et al. 2008).

Vascular changes observed in zebrafish exposed to 2,4-D included sinusoidal dilation, hyperemia, and liver tissue hemorrhage. These vascular alterations may signify an adaptive process linked to increased blood flow, facilitating defense cell transport and tissue oxygenation (Santos et al. 2018). Given the liver's roles in xenobiotic metabolism and circulation, it is considered a primary target organ for chemical-induced tissue damage. Elevated hepatic blood flow in fish exposed to toxic agents triggers vessel dilation and hyperemia to support hepatocyte catabolism and detoxification, enhancing tissue oxygenation (Hinton et al. 2001). These events can elevate hepatic vascular pressure, potentially resulting in vascular endothelium rupture. Furthermore, 2,4-D has been shown to reduce tight junction proteins expression and quantity in endothelial cells membranes, facilitating endothelium disruption and consequently bleeding (Sharifi Pasandi et al. 2017).

We also evaluated the impact of 2,4-D on the liver of zebrafish considering the severity of each lesion and its frequency using the Histopathological Alteration Index (HAI) (Poleksic and Mitrovic-Tutundzic 1994). HAI analysis revealed that the control group showed healthy functioning, the 0.03 and 0.3 mg/L of 2,4-D groups displayed moderate tissue damage, and the 3.0 mg/L group showed irreparable tissue damage and possibly functional impairment. The elevated HAI value in the 3.0 mg/L group primarily stemmed from tissue necrosis, a severe damage type carrying greater weight due to its severity. While the 0.03 and 0.3 mg/L groups did not yield statistically significant HAI result, their values indicate moderate tissue damage. Biologically, this is a concerning outcome, considering that 0.03 mg/L of 2, 4-D is the maximum concentration permitted for human consumption and also for bodies of water (Brazil, Ordinance GM/MS nº 888, of May 4, 2021: Brazil, resolution nº 357, of March 17, 2005). Additionally, concentrations exceeding 0.3 mg/L have been identified in aquatic environments near sugarcane crops in São Paulo state, Brazil (CETESB 2018), highlighting the risks to aquatic organisms or even human beings.

Histopathological biomarkers are widely used to evaluate the toxic effects of xenobiotics in fish, being a sensitive tool to diagnose direct or indirect toxic effects that affect animals. It is considered a biomarker of higher-level responses and may reflect previous changes in metabolism, macromolecule binding and/or biochemical changes, providing insights into toxicity (Yancheva et al. 2016; Huggett et al. 2018). In the present study, we used Toluidine Blue (TB) dye to label acidic polysaccharides in zebrafish liver post 2,4-D exposure. We observed a decrease level of these molecules in the liver, with a more pronounced decrease in the 3.0 mg/L group. TB selectively stains acidic tissue components, suchcarboxylic radicals, sulfates and phosphates, having an affinity for nucleic acids and therefore, also binds to tissues with high DNA and RNA content (Sridharan and Shankar 2012). Acidic polysaccharides containing uronic acid are widely distributed in animal tissues and include glycosaminoglycans (GAGs), which are macromolecules mostly present in the extracellular matrix and mucous secretions (Cao et al. 2015). Among the GAGs, chondroitin sulfate, hyaluronic acid and heparin are included, with heparin being an intracellular component found in greater quantities in the liver of fish (Song et al. 2017). Studies have demonstrated that GAGs such as heparin and hyaluronic acid can undergo degradation due to oxidative stress (Sies 1987; Duan and Kasper 2011; Chowdhury and Saikia 2020). Given 2,4-D's demonstrated ROS induction potential, the observed decrease in acidic polysaccharides may be linked to ROS-mediated degradation, particularly affecting heparin and other liver-present GAGs (Tayeb et al. 2012; Sharifi Pasandi et al. 2017).

Hepatotoxic compounds can disrupt cellular processes via cell and tissue damage, decrease of antioxidant compounds and lipid peroxidation, leading to the onset of liver disease (Elufioye and Habtemariam 2019). Toxic effects of 2,4-D have been associated with oxidation or oxidation product oxidation, resulting in increased reactive oxygen species (ROS) production and changes in the levels of antioxidant enzymes and antioxidant molecules (Oruç and Üner 1999, 2000; Oruç et al. 2004). Our study also investigated 2,4-D's effects on oxidative stress and xenobiotic metabolism markers. The glutathione (GSH) molecule, vital for xenobiotics metabolism and cellular defense against oxidative stress (Huber et al. 2008), executes its protective function by promoting the reduction of reactive oxygen species, such as hydrogen peroxide and superoxide anion. GSH undergoes oxidation and is converted into GSSG. Subsequently, GSSG is regenerated back into GSH through the catalytic cycle (Huber et al. 2008). In our results, we observed a decrease in the levels of the GSH molecule in the groups exposed to 0.03 and 3.0 mg/L of 2,4-D and an increase in GSSG in the groups exposed to 0.03 and 0.3 mg/L of 2,4-D. Taken together, data of GSH and GSSG suggest that exposure to 2,4-D generates oxidative molecules, and the liver utilizes GSH to neutralize and block oxidative damage in tissue. Simultaneously, an increase in GSSG occurs due to GSH oxidation in this process.

Another point is that in addition to acting against oxidative stress, GSH also acts in the metabolization of xenobiotics, and this occurs through the conjugation of GSH/xenobiotic through the enzyme glutathione-S-transferase (GST) to make this less toxic compound and more soluble in water, thus facilitating its elimination (Huber et al. 2008; Chowdhury and Saikia 2020). Exposure to 3.0 mg/L of 2,4-D caused a decrease in GSH but did not change the levels of GSSG and NADPH, suggesting that this decrease may be due to the formation of GSH/xenobiotic conjugates and their elimination. In the present study, GST levels were not evaluated, however, it has already been demonstrated that zebrafish larvae exposed to 2.5 mg/L of 2,4-D for 48 h present increased GST activity, which reinforces our hypothesis for the exposed group 3.0 mg/L of 2,4-D (Martins et al. 2021).

Liver tissue serves several vital metabolic functions, including carbohydrate metabolism, lipid storage, synthesis and oxidation of fatty acids, glycogen storage, plasma protein synthesis, and detoxification (Hinton et al. 2001; Ferguson 2006; Yao et al. 2012; Heath 2018). Those roles render liver cells particularly susceptible to oxidative stress induced by toxic agents (Elufioye and Habtemariam 2019). Decreased antioxidant response and induction of oxidative stress led to cellular and tissue damage, as evidenced by increased MDA levels (Martins et al. 2024). MDA, a product of lipid peroxidation, especially of polyunsaturated fatty acids, serves as a common marker of oxidative stress and damage to lipids and cell membranes. While the assessment of MDA levels in zebrafish liver did not show significant changes at any tested concentrations, modifications in antioxidant defenses imply that 2,4-D induced ROS, which were effectively regulated by the antioxidant mechanism, probably due to the ability of GSH to defend the liver in this short period of exposure. A similar result was observed in fish Oreochromis niloticus exposed to 27 ppm of 2,4-D for 96 hours (Oruç and Üner 2000).

While lipid peroxidation was not observed in the liver, histopathological analysis indicated liver toxicity induced by 2,4-D. Therefore, it is reasonable to postulate that other events contribute to toxicity, such as protein and enzyme oxidation or covalent bonding of 2,4-D and its metabolite (2,4-dichlorophenoxyacetyl-S-acyl-CoA) to liver proteins, potentially compromising them and inducing degradation (Di Paolo et al. 2001; Li et al. 2003; Matviishyn et al. 2014; Tichati et al. 2020). It's worth emphasizing that despite being a very old pesticide with a long history of commercialization, the full extent of its effects on non-target organisms like fish remains incompletely understood (Mahmood et al. 2016; Marcato et al. 2017). However, despite of significant changes in various parameters evaluated in this study accentuating concerns regarding both the maximum allowed concentration and the effects of excessive and/or inappropriate pesticide use (WHO, 2017; Zuanazzi; Ghisi; Oliveira, 2020), exposure to 2,4-D did not result in lethality among Danio rerio fish, as no deaths were recorded in any of the evaluated groups.

In summary, our findings indicate that (i) the herbicide 2,4-D adversely affected the biochemical, histochemical and histopathological parameters of the liver in adult Danio rerio fish; (ii) allowed concentration of 2,4-D (0.03 mg/L) was able to alter parameters associated with ROS formation and caused moderate liver damage in Danio rerio following only 7 days of exposure; and (iii) high concentration of 2,4-D (3.0 mg/L) resulted in the formation of necrotic foci. We also suggest that while antioxidant mechanisms appeared effective in preventing lipid peroxidation in zebrafish liver, additional factors may contribute to liver damage. Therefore, the observed tissue damage suggests that 2,4-D is hepatotoxic to adult zebrafish, particularly at elevated concentrations. These results raise concerns about the escalating use of 2,4-D and its increased presence in aquatic ecosystems, emphasizing the need for further research to comprehensively understand its toxicity in non-target environments.

Acknowledgements

The authors would like to thank the Federal University of Santa Catarina (Brazil) for the facilities and the “Laboratório Multiusuário de Estudos em Biologia” (LAMEB) for the facilities and analyses.

Funding

B. R. F. Oliveira and D. H. Moreira are supported by a scholarship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). C. E. Davico was supported by a scholarship from CAPES. G.S.I. is supported by a postdoctoral fellowship from CAPES/PRINT.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Breno Raul Freitas Oliveira: Conceptualization, methodology,

investigation, formal analysis, writing and review

José Ribamar Soares Neto: Methodology, investigation and formal analysis

Carla Eliana Davico: Methodology and review

Daniele Hummel Moreira: Methodology and research

Lucas Cezar Pinheiro: Methodology, research, resources and acquisition financing

Aline Guimarães Pereira: Conceptualization, methodology, investigation and review

Geison Souza Izídio: Conceptualization, research, resources and financing, review and supervision

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval

This study was approved by the Ethics Committee on the Use of Animals (CEUA) protocol nº 6778251119 of Federal University of Santa Catarina (UFSC), Florianópolis (SC) Brazil.

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

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Hepatotoxic effects of the herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) after short exposure on adult zebrafish Danio rerio

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Abstract

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1. INTRODUCTION

2. MATERIAL AND METHODS

2.1. ANIMALS

2.2. HERBICIDE

2.3. EXPERIMENTAL PROCEDURES

2.4. HISTOLOGICAL ANALYSIS

2.5. BIOCHEMICAL ANALYSIS

2.7. STATISTICAL ANALYSIS

3. RESULTS

3.1 HISTOPATHOLOGICAL AND HISTOCHEMICAL ANALYSIS

3.2 BIOCHEMICAL ANALYSIS

4. DISCUSSION

5. CONCLUSION

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Supplementary Files

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