The effects of acute boric acid treatment on gill, kidney and muscle tissues in juvenile rainbow trout

Abstract

Boric acid (BA) is an essential nutrient for plants and many organisms, but it has become an environmental contaminant because of widespread use. Pesticide and its compounds are a serious threat to aquatic organisms. This study was carried out to determine the histopathological effects of acute exposure to BA concentrations in rainbow trout. The fish were exposed to 10² and 10³ mg/L concentrations of BA. Tissues were sampled at 6, 12, 24, 48 and 96 h. Histopathological alterations occurring in tissues were common in both doses of BA. Gill tissues showed lamellar oedema, cellulary infiltration, lamellar disorganization, degenerative changes and lamellar thickening. Kidneys had glomerular oedema and glomerulonephritis, degeneration of the tubulary epithelium, interstitial fibrosis and a hyaline cast within the tubular lumens. Muscle tissues displayed interstitial oedema and degenerative and atrophic changes to varying degrees in the myofibrils. Our study shows that BA can be toxic for rainbow trout and cause histopathological damage in fish tissue.

Keywords:

• boric acid
• gill
• histopathology
• kidney
• muscle
• rainbow trout

1. Introduction

Boron is a naturally occurring compound found in low concentrations in natural surface waters and is an essential nutrient for plants and animals (Hu & Brown 1997; Nielsen 1997). Boron enters aquatic environments in varied ways such as anthropogenic and natural sources. The main sources of boron and its compounds are the weathering of clay sedimentary soils and volcanic activity (Nable et al. 1997; Polat et al. 2004). They are used for many different purposes in various products such as cleaners, detergents, personal care products, the manufacturing of ceramic, processes of metallurgy, glass and glass fibres, adhesives and industrial fluids (Schoderboeck et al. 2011). Boron is soluble in water and may cause toxic effects in aquatic animals. Fish are more sensitive than aquatic invertebrates to boron exposure during chronic exposures (Birge & Black 1977; Schoderboeck et al. 2011). The fish LC50 values (3.63 to >1000 mg B/L) show high variation due to test cases, life stages, fish size, fish species and test duration (Schoderboeck et al. 2011). The 96-h LC50 value of boron for rainbow trout is up to 1100 mg B/L (USEPA, Office of Prevention, Pesticides and Toxic Substances 1993b), but it has been reported that the LC50 value of boric acid (BA) is determined to be 138 mg/L and the lowest observed effect concentrations (LOECs) for rainbow trout are 0.10 mg/L (Loewengart 2001). In another study, LOECs for rainbow trout range between 0.001 and 0.008 mg B/L (Loewengart 2001). These values show that aquatic organisms like rainbow trout are very sensitive to boron treatment, and its compounds should be further investigated.

BA and its compounds are pesticides (USEPA, Office of Prevention, Pesticides and Toxic Substances 1993a), and additives for numerous industrial and pharmaceutical products and techniques (Turkez & Geyikoglu 2010). BA is found naturally in water and seas at 1–5 ppm concentrations, and it may change the biological functions of different animal species. Therefore, it is frequently used as a pesticide, but high doses of BA and boron compounds are toxic to fish such as rainbow trout and zebrafish (Rowe et al. 1998; Büyükgüzel et al. 2013).

Aquatic organisms may be exposed to herbicides in streams and ponds surrounded by agricultural areas. Rainbow trout are among these organisms, and it is one of the most studied fish species because of its importance as food (Ozaki et al. 2001). There is no information about the histopathological effects of BA in rainbow trout. Histological techniques are used to assess the unknown toxic effect of pollutants such as pesticides and heavy metals in the aquatic environment (Capkın et al. 2009). Therefore, it is important to investigate histopathological effects occurring after acute exposure to BA in juvenile rainbow trout. Thus, the objective of this study was to investigate the toxic effects of BA on the histopathological changes in the kidney, muscle and gill tissues of juvenile rainbow trout.

2. Materials and methods

2.1. Fish maintenance

Juvenile rainbow trout (Oncorhynchus mykiss) with an average weight of 3.3 ± 0.5 g were obtained from Atatürk University, Faculty of Fisheries and Inland Water Fish Breeding and Research Center. Fish were acclimatized for 30 days under laboratory conditions in dechlorinated tap water (12 h light–12h dark). During acclimation, fish were fed 2.5% of their body weight in commercial trout pellets. Water quality parameters were as follows: temperature 9.0 ± 2°C, pH 7.0 ± 0.2, dissolved oxygen 9.1 ± 0.3 mg/L, SO₄ ^–2 = 0.33 mg/L, PO₄ ^–3 = trace, NO₃ ^– = 1.45 mg/L and NO₂ ^– = trace. Two-thirds of the water in the aquarium was renewed every day.

2.2. Experimental design

BA (molecule formula H₃BO₃) was purchased from Sigma Chemical Co. It was dissolved in water, and the exposure concentrations of the boric acid in the stock solutions were 10² and 10³ mg/L for each group. We chose lower and upper values of lethal concentrations for rainbow trout because both doses of BA may occur in an aquatic environment. The LC50 value for rainbow trout of BA was 138 mg/L (Loewengart 2001). After the acclimation, 30 fish in three groups were assigned to treatments into each glass aquarium containing 25 L of water. The first group was Control I (untreated fish). The other two groups (II, III) were exposed to 10² and 10³ mg/L BA concentrations, respectively, under laboratory conditions. At the end of the exposure period, the control and all exposed fish were chosen from each aquarium randomly and were sampled at 6, 12, 24, 48 and 96 h. At the end of the experiment, fish were euthanized by cervical section, and the kidney, gill and muscle tissues were immediately removed. Muscles were taken from the dorsal side of the fish.

2.3. Histopathological process

Tissue samples from the gill, kidney and muscles were fixed in a 10% buffered formalin solution. After the routine histopathology process, paraffin sections in 5 µ were prepared and stained with haematoxylin and eosin. Histopathological lesions were semi-quantitatively assessed under the light microscope (Olympus BX51 with DP72 camera attachment system). For the histopathological score, six high-power (at 20X–40X magnification) fields were randomly chosen in two different fish (6 + 6 = total 12 areas). The scores were derived semi-quantitatively, considering the number of microscopic are with lesions and were reported as follows: none: − (0 lesion), mild: + (1–4 lesions), moderate: ++ (5–8 lesions) and severe: +++ (≥9 lesions).

3. Results

3.1. Control group

No histopathological changes were observed in the gill and muscle tissues of the control group (Figures 1 and 2).

Figure 1. Normal architecture of gill sections from control (a) and histopathological changes in experimental groups. Clumping and thickening of lamellas because of inflammation (arrow in b). Lamellary edema and celluary infiltrations and clumping (arrows in e). HE.

Figure 2. Normal architecture of kidney section from the control group (a). Glomerular edema and hyperemia (arrow in b). Hyaline casts (black arrows in c and d) and glomerulitis (white arrow in c). Interstitial fibrosis around the tubule (white arrow in d) HE.

3.2. Experimental groups

Lamellary oedema, cellulary infiltrations, thickening because of cellulary infiltrations or hypertrophy and clumping were observed in gill sections to varying degrees. Kidney tissues showed glomerular oedema, glomerulonephritis, degeneration of tubular epithelium, hyaline cast within the tubulary lumens and interstitial fibrosis to varying degrees. In muscle tissue, interstitial oedema as well as degenerative and atrophic changes in myocytes to varying degrees were observed.

Time- and dose-dependent comparisons of gill, kidney and muscle tissues were displayed in Tables 1–3 and Figures 1–3.

Table 1. Time- and dose-dependant comparison of histopathology in gill tissue.

	Histopathological changes in gill tissue
Groups	Hours	Lamellar oedema	Degenerative changes	Lamellary disorganization and clumping	Cellulary infiltration	Lamellar thickening
Control	All hours	–
Group II 10^2 mg/L	6	+++	–	–	–	–
	12	++	+	–	–	–
	24	++	++	–	+	–
	48	+	+++	+	++	+
	96	–	+++	+	++	+
Group III 10^3 mg/L	6	+++	–	–	–	–
	12	++	+	–	+	+
	24	++	++	++	++	++
	48	+	+++	++	+++	++
	96	–	+++	++	+++	++

Table 2. Time- and dose-dependant comparison of histopathology in kidney tissue.

	Histopathological changes in kidney tissue
Groups	Hours	Glomerular oedema	Glomerulonephritis	Degenerative changes	Hyaline casts within the tubules	Fibrous proliferation
Control	All hours	–
Group II 10^2 mg/L	6	–	–	–	–	–
	12	+	–	–	–	–
	24	++	+	+	–	–
	48	++	++	++	+	–
	96	++	++	++	++	–
Group III 10^3 mg/L	6	–	–	–	–	–
	12	+	–	–	–	–
	24	++	+	++	+	–
	48	+++	+++	+++	++	–
	96	+++	+++	+++	++	+

Table 3. Time- and dose-dependant comparison of histopathology in muscle tissue.

Groups	Hours	Interstitial oedema	Degenerative and atrophic changes in myofibrils
Control	All hours	–
Group II 10^2 mg/L	6	–	–
	12	–	–
	24	–	+
	48	+	++
	96	++	++
Group III 10^3 mg/L	6	–	–
	12	–	+
	24	+	++
	48	++	++
	96	++	+++

Figure 3. Normal architecture of musle sections from control and experimental groups (a). Intercelulary edemea (* in b), atrophic miyocytes (arrows in c). Severe edema with necrotic myocytes (black arrow in d) and myocyte losses (white arrows in d). HE.

4. Discussion

BA is often used as a pesticide, and various non-target organisms are exposed to it (Büyükgüzel et al. 2013). Sub-lethal concentrations of pesticides cause biochemical, histological and physiological changes in many tissues of aquatic organisms (Boran et al. 2010). In this study, histological alterations were observed in the gill, kidney and muscle tissues of rainbow trout after acute exposure to BA. Similar results have been shown with other pesticides (Dalela et al. 1979; Ortiz et al. 2003; Altınok & Capkın 2007).

It is known that the gills are important organs in fish because they have important roles, including respiration, osmoregulatory functions and acid-base regulation (Ghate & Mulherkar 1979). Gills are considered to be one of the organs affected by water pollution, and it has been noticed that gills are target organs because of the effects of toxic pollutants (Ba-Omar et al. 2011). Toxic pollutants affect gill tissues, and they cause the disruption of the osmoregulatory function and reduce the oxygen consumption of aquatic organisms (Ghate & Mulherkar 1979; Peebua et al. 2008). It has been reported that pesticides lead to respiratory distress in the gill tissues of fish (Richmonds & Dutta 1989). In the present study, we observed that gill tissues were the most affected organ due to direct contact with toxic substances. In our study, lamellar oedema, cellulary infiltration, lamellar disorganization, degenerative changes in lamellar epithelium and lamellar thickening because of inflammation were observed in the gill tissues. These results showed that histopathological damages are direct responses to BA. Our results have shown to be similar to other pesticides in different fish species. Cengiz (2006) reported lesions such as epithelial hyperplasia, oedema and fusion of the secondary lamellae in the gill tissues of common carp exposed to deltamethrin. Ba-Omar et al. (2011) observed hypertrophy, epithelial lifting, desquamation and lamellar fusion in the gill tissues of Aphanius dispar exposed to the pesticide temephos.

The kidney is a critical organ that is affected by contaminants in the aquatic environment (Thophon et al. 2003; Iqbal et al. 2004). At the same time, kidneys are considered a good indicator of aquatic pollution (Cengiz 2006). Histopathological changes in the kidneys of fish exposed to chemical pollutants such as metals occurred. Hyaline droplets, reduction of Bowman’s space, tubule degeneration, dilation of capillaries and changes in the corpuscle were the main findings in fish exposed to chemical pollutants (Camargo & Martinez 2007). Thus it has been reported that renal lesions may be considered indicators of environmental pollution (Cengiz 2006). In this study, dose- and time-dependent lesions were observed in the histopathology of kidney tissues. These lesions were glomerular oedema, glomerulonephritis, degeneration of the tubulary epithelium, mild interstitial fibrosis 96 h after treatment with 10³ mg/L BA and a hyaline cast within the tubular lumens. It has been suggested that the hyaline droplets found in renal tubules may be an indicator of the renal toxicity of pollutants (Chaudhuri et al. 1999). Ortiz et al. (2003) reported the effects of lindane used as pesticide on fish, and their results showed lesions consisted of desquamation, tubular necrosis and the vacuolisation of tubular epithelial cells in the kidney. In another study, Altınok and Capkın (2007) recorded that endosulfan showed histopathological effects such as exudates in tubules and between tubules, tubular necrosis, glomerular necrosis and renal tubular separation in the kidneys of rainbow trout. Interstitial fibrosis was observed only in the kidney sections of a fish. Fibrotic change may be associated with other chronic conditions. However, kidney fibrosis was reported in O. mykiss with the other histological changes in kidney tissue in the treatment of sub-lethal concentrations of ethylenediaminetetraacetic acid during 96 h (Ghorashi et al. 2013). Further studies are needed in kidney fibrosis regarding boric acid toxicity.

The histopathological effects of BA on muscle tissues have not been previously reported. The present study showed that boric acid caused interstitial oedema and degenerative and atrophic changes to varying degrees in the myofibrils of the muscle tissues. These results showed that BA may affect the muscle.

In conclusion, the results of this study suggest that BA can be toxic for rainbow trout and cause histopathological damage in fish tissue. In addition, histopathology may be used as a useful indicator for environmental pollutants and toxicity studies in fish.

Disclosure statement

No potential conflict of interest was reported by the authors.