The effect of acute exposure to herbicide Gardoprim Plus Gold 500 SC on haematological and biochemical indicators and histopathological changes in common carp (Cyprinus carpio L.)

The study was focused on the assessment of effects of herbicide preparation Gardoprim Plus Gold 500 SC (terbuthylazine and S-metolachlor as active substances) on haematological and biochemical indices as well as tissue histopathological changes in common carp (Cyprinus carpio L.). Forty eight oneto two-year-old fish were divided into two groups, i.e. 24 fish were treated with 13.0 mg·l-1 of Gardoprim Plus Gold 500 SC and 24 fish were used in control. The experiment was conducted according to OECD method No. 203 Fish, Acute Toxicity Test. Experimental carp showed a significant decrease in leukocyte and lymphocyte counts (P < 0.01) and haematocrit value (P < 0.05) in haematological profile. In biochemical indices, a significant increase in glucose, aspartate aminotransferase, ammonia (P < 0.01), and lactate dehydrogenase (P < 0.05) and a significant decrease in inorganic phosphorus, triglycerides (P < 0.01), and chlorides (P < 0.05) were found in exposed carp compared to control. Histopathological examination revealed lesions in gills and liver. The decline in both leukocyte and lymphocyte counts indicates decreased nonspecific immunity of treated common carp. Increase in ammonia and glucose concentrations, and in catalytic activities of lactate dehydrogenase and aspartate aminotransferase can be related to stress burden, and alteration of liver cell function, respectively, in experimental carp compared to control fish. The study uniquely contributes to the evaluation of the effect of two-component herbicide preparation on common carp. Terbuthylazine, S-metolachlor, fish, acute toxicity Triazine herbicides belong to the most well known and widely used pesticide group. Triazine substance terbuthylazine has been replacing the better known and longer used and studied atrazine in several regions of the world. It is used as a broad spectrum preor post-emergence. It is stable in neutral, weakly acid and weakly alkaline media. Water solubility of terbuthylazine is 8.5 mg·l-1 at 20 °C (Gangolli 1999b). There are studies on terbuthylazine toxicity to terrestrial animals (Lang et al. 1997), but less is known about its toxicity to aquatic organisms, including fish (Dezfuli et al. 2006). Chloracetanilide herbicides are used to control annual grasses and broad leaf weeds. Chloracetanilide metolachlor has been used to control annual grass and broad-leaved weeds following pre-emergence application for more than 20 years. Metolachlor is relatively soluble in water with 488 mg ·l-1 at 25 °C (Gangolli 1999a). There are several studies on the toxicity of metolachlor to terrestrial and aquatic organisms (Wan et al. 2006). The aim of this study is to assess the effect of the herbicide Gardoprim Plus Gold 500 SC (187.5 g·l-1 of triazine terbuthylazine and 312.5 g·l-1 of chloracetanilide S-metolachlor) on both haematological and biochemical indicators, and tissue histopathological changes of exposed common carp. The hypothesis of synergistic effect of triazineand acetanilidebased active substances contained in composite commercial preparation on fish immune system status, stress burden and tissue impairment was evaluated. ACTA VET. BRNO 2011, 80: 359–363; doi:10.2754/avb201180040359 Address for correspondence: MVDr. Radka Dobšíková, Ph.D. Department of Veterinary Public Health and Toxicology Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno Palackého 1/3, 612 42 Brno, Czech Republic Phone: + 420 541 562 784 Fax: + 420 541 562 790 E-mail: dobsikovar@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm Materials and Methods Oneto two-year-old common carp (Cyprinus carpio L.) of 93.66 ± 31.77 g and 18.28 ± 2.15 cm body weight and body length, respectively, were used for the assessment. Twenty four carp (experimental group) were exposed to 13.0 mg·l-1 of Gardoprim Plus Gold 500 SC (corresponding to 2.25 mg·l-1 and 3.75 mg·l-1 of terbuthylazine and S-metolachlor, respectively). Other twenty four carp (control group) were kept in water. Experimental and control groups of carp were divided into 2 duplicates, i.e. twelve fish individuals were kept in each 200 l aquarium (two aquaria with 13.0 mg·l-1 of the herbicide, two control aquaria). A 96-hour toxicity test was performed semistatically with water renewal every 24 h. According to the OECD No. 203 Fish, Acute Toxicity Test, the concentration of active substances in test water was maintained above 80%. Terbuthylazine and S-metolachlor concentrations were determined using GC/MS/MS method. Blood samples were taken by cardiocentesis. Heparinized blood samples were used for the evaluation of haematological indicators including haematocrit (PCV), erythrocyte count (RBC), haemoglobin concentration (Hb), mean erythrocyte volume (MCV), mean erythrocyte haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leukocyte count (WBC), and differential leukocyte count (Svobodova et al. 1991). A part of heparinized blood was centrifuged at 855 g for 10 min and used for biochemical analysis. Biochemical indicators including glucose (GLU), lactate (LACT), total proteins (TP), albumins (ALB), ammonia (NH3), triacylglycerols (TRIG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), calcium (Ca2+), inorganic phosphate (PHOS) were measured by a COBAS MIRA automatic analyser (Hoffman, La Roche, Co., Switzerland) using BioVendor Tests. Natrium (Na+) and chloride (Cl-) concentrations were analysed using Easy Lyte Analyzer (Medica Corp., Bedford, MA, USA). Tissue samples of gills, liver, and kidneys were fixed in buffered 10% neutral formalin, dehydrated, embedded in paraffin wax, sectioned on a microtome at a thickness of 4 μm, and stained with haematoxylin and eosin (HE). The sections were examined by light microscopy and photographed by a digital camera. Statistical analysis of the data was conducted using Statistica 8.0 for Windows (StatSoft, Inc., USA). Values were tested for normal distribution using Kolmogorov–Smirnov test and log-transformed to improve the homogeneity of variance. A one-way analysis of variance (ANOVA) and Tukey-HSD t-test were applied.

Triazine herbicides belong to the most well known and widely used pesticide group. Triazine substance terbuthylazine has been replacing the better known and longer used and studied atrazine in several regions of the world. It is used as a broad spectrum preor post-emergence. It is stable in neutral, weakly acid and weakly alkaline media. Water solubility of terbuthylazine is 8.5 mg·l -1 at 20 °C (Gangolli 1999b). There are studies on terbuthylazine toxicity to terrestrial animals (Lang et al. 1997), but less is known about its toxicity to aquatic organisms, including fish (Dezfuli et al. 2006).
Chloracetanilide herbicides are used to control annual grasses and broad leaf weeds. Chloracetanilide metolachlor has been used to control annual grass and broad-leaved weeds following pre-emergence application for more than 20 years. Metolachlor is relatively soluble in water with 488 mg ·l -1 at 25 °C (Gangolli 1999a). There are several studies on the toxicity of metolachlor to terrestrial and aquatic organisms (Wan et al. 2006).
The aim of this study is to assess the effect of the herbicide Gardoprim Plus Gold 500 SC (187.5 g·l -1 of triazine terbuthylazine and 312.5 g·l -1 of chloracetanilide S-metolachlor) on both haematological and biochemical indicators, and tissue histopathological changes of exposed common carp. The hypothesis of synergistic effect of triazine-and acetanilidebased active substances contained in composite commercial preparation on fish immune system status, stress burden and tissue impairment was evaluated.

Materials and Methods
One-to two-year-old common carp (Cyprinus carpio L.) of 93.66 ± 31.77 g and 18.28 ± 2.15 cm body weight and body length, respectively, were used for the assessment. Twenty four carp (experimental group) were exposed to 13.0 mg·l -1 of Gardoprim Plus Gold 500 SC (corresponding to 2.25 mg·l -1 and 3.75 mg·l -1 of terbuthylazine and S-metolachlor, respectively). Other twenty four carp (control group) were kept in water. Experimental and control groups of carp were divided into 2 duplicates, i.e. twelve fish individuals were kept in each 200 l aquarium (two aquaria with 13.0 mg·l -1 of the herbicide, two control aquaria).
A 96-hour toxicity test was performed semistatically with water renewal every 24 h. According to the OECD No. 203 Fish, Acute Toxicity Test, the concentration of active substances in test water was maintained above 80%. Terbuthylazine and S-metolachlor concentrations were determined using GC/MS/MS method.
Tissue samples of gills, liver, and kidneys were fixed in buffered 10% neutral formalin, dehydrated, embedded in paraffin wax, sectioned on a microtome at a thickness of 4 μm, and stained with haematoxylin and eosin (HE). The sections were examined by light microscopy and photographed by a digital camera.
Statistical analysis of the data was conducted using Statistica 8.0 for Windows (StatSoft, Inc., USA). Values were tested for normal distribution using Kolmogorov-Smirnov test and log-transformed to improve the homogeneity of variance. A one-way analysis of variance (ANOVA) and Tukey-HSD t-test were applied.

Results
During the course of fish exposure to 13.0 mg·l -1 of the herbicide preparation Gardoprim Plus Gold 500 SC (corresponding to 2.25 mg·l -1 and 3.75 mg·l -1 of terbuthylazine and S-metolachlor, respectively) neither mortality nor obvious clinical signs of behavioral, respiratory, and/or neurologic distress were observed in exposed carp.

Indicators
Control Experimental mean ± SD (n = 24) mean ± SD (n = 24) RBC (T·l -1 ) The results of haematological examination of control and experimental carp are given in Table 1. The exposure caused a significant decrease in PCV (P < 0.05) as well as in leukocyte count (WBC) and lymphocyte count (P < 0.01) in experimental carp. The rest of the indices showed no significant differences between experimental and control groups of fish.
The results of plasma biochemical indicators analysis are given in Table 2. A significant increase in glucose, ammonia, AST (P < 0.01), and LDH (P < 0.05) levels and a significant decrease in triglycerides, inorganic phosphorus (P < 0.01), and chlorides (P < 0.05) were found in common carp exposed to 13.0 mg·l -1 of the herbicide. The other indicators monitored were found comparable in both fish groups.
Histopathological examination of treated carp revealed pathological lesions in gills and liver (Plate III, Figs 1, 2).

Discussion
The decrease in PCV in experimental group of common carp could be interpreted as a compensatory mechanism reducing oxygen carrying capacity to maintain gas transfer (Jee et al. 2005). The changes in PCV could be caused by the release of catecholamines during the primary stages of stress, which can mobilize red blood cells swelling as a result of fluid shift into the intracellular compartment (Lebelo et al. 2001). In our study, PCV decrease presumably has a connection with a substantial decline in the RBC count.
Both the decreased concentration of TRIG and the increased activity of LDH can be explained as a consequence of pathological changes in hepatic tissue (Doubek et al. 2010). Catalytic activities of plasma enzymes (i.e. LDH, CK, ALT, and AST) may be used as a relevant indicator of stress reaction as the increased values indicate stress-based tissue impairment. Philip et al. (1995) reported that the stress conditions in general amplify the elevation of transamination pathway, i.e. the increase of the transaminases activities. In our study, a significant increase in AST (P < 0.01) and LDH (P < 0.05) activities was found in exposed carp (Table 2).
Gill lesions were represented by hypertrophy and hyperplasia of epithelial cells with lamellar fusion, hyperaemia (Plate III, Fig. 1, upper arrow) and the presence of multiple foci of coagulation necrosis (Plate III, Fig. 1, lower arrow). Gills usually appear to be one of the most damaged organs due to their direct contact with the toxic substance as they provide a very large interface between the external and internal environment of fish (Oliveira Ribeiro et al. 2002). Fusion of the secondary lamellae and the hyperplasia of gill epithelial cells were also described after the exposure to terbuthylazine (Dezfuli et al. 2006).
Moderate dystrophic lesions, i.e. morphological signs of an initial cell injury represented by swelling and hydropic vacuolar degeneration of hepatocyte, were evident in the liver (Plate III, Fig. 2, arrows). Similar histopathological changes in liver parenchyma (i.e. focal steatosis, hyperaemia, necrosis, fatty dystrophy, glycogen inclusions, vacuolisation) were found in the studies of Dezfuli et al. (2006) and Mikula et al. (2008).
The shifts in both haematological and biochemical indicators, and pathological alteration of tissues reflect the preparation effect on fish immune system (and stress reaction), and the alteration of metabolic pathways especially in fish gills and liver.