Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate

Fish & Shellfish Immunology 30 (2011) 51e57

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Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate Luiz Carlos Kreutz*, Leonardo José Gil Barcellos, Stella de Faria Valle, Tális de Oliveira Silva, Deniz Anziliero, Ezequiel Davi dos Santos, Mateus Pivato, Rafael Zanatta Universidade de Passo Fundo, Faculdade de Agronomia e Medicina Veterinária, Campus I, Bairro São José, BR 282, km 171. 99052-900 Passo Fundo, RS, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 August 2010 Received in revised form 17 September 2010 Accepted 18 September 2010 Available online 29 September 2010

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC50e96h) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24 h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24 h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24 h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Silver catfish Glyphosate Blood cells Phagocytosis Natural immunity

1. Introduction Intensive crop production requires the use of agrichemicals with selective effect on unwanted invasive weeds. Glyphosate, a worldwide used herbicide, consist of an isopropylamine salt (IPA) mixed with a non-ionic polyethoxylated amine surfactant (POEA) and water, that has been used mostly in rice and glyphosate-resistant transgenic soybean culture [1]. Glyphosate-based herbicides used as expected and under field conditions is considered of low risk to humans, other mammals and birds [2]. Nonetheless, even though the active ingredient of glyphosate is readily dissipated in water, and soil micro flora contributes to its biodegradation, there is a potential for toxic effects mainly for aquatic organisms present in water ponds and springs found in agricultural areas, that are much more sensitive to glyphosate that other species [2,3]. Glyphosate

* Corresponding author. Tel. þ55 54 33168444; Fax. þ55 54 3316 8163. E-mail address: [email protected] (L.C. Kreutz). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.09.012

and several other agrichemicals have been detected in soil and water in south America [4,5] and the adverse effect of commercial formulation of glyphosate on fish species has been a matter of increasing interest [6e10]. The toxicity of glyphosate to silver catfish (Rhamdia quelen), a South American teleostean fish from the Heptapteridae family, has been recently demonstrated [6]. Low concentrations of glyphosate, such as those used in rice and soybean fields, might cause changes in metabolic and enzymatic parameters of silver catfish [8] and other fish species [9,10] like inhibition of brain acetylcholinesterase (AChE), lipid peroxidation and protein catabolism. Sublethal concentration of glyphosate increases the level of silver catfish seric cortisol [11], but does not interfere with the ability to cope with an additional stress [12]. In addition, silver catfish females kept in earthen pound and exposed to sublethal concentrations of glyphosate had higher liver-somatic index (LSI), lower concentration of 17b-estradiol and decreased egg viability [7]. The effects of glyphosate in other fish species have also been investigated. In piava (Leporinus obtusidens) exposed to glyphosate,

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a significant increase in hepatic glycogen and glucose was observed [9], concomitant with a reduction of hematological parameters and brain AChE. Following exposure of Prochilodus lineatus to glyphosate contaminated water, a significant increase in plasma glucose and catalase activity were reported [13]; in the same study, hepatocyte morphology and histological alterations were also detected. Hepatocyte histological changes were also reported in Oreochromis niloticus [14] after glyphosate exposure. In goldfish (Carassius auratus), glyphosate caused a significant reduction in superoxide dismutase, glutathione S-transferase, glutathione reductase and glucose-6-phosphatase dehydrogenase, and a substantial increase in liver catalase [10]. In the neotropical fish Prochilodus lineatus glyphosate induced oxidative stress and lipid peroxidation [15]. Thus, the data reported so far indicates that commercial formula of glyphosate causes a typical stress response on fish with a likely effect on immune response. Indeed, altered protein biosynthesis and immune function were observed in Tilapia (Tilapia nilotica) exposed to glyphosate [16]. Even though the adverse effect of commercial glyphosate on fish physiological and biochemical parameters have been reported, few studies aimed to investigate the natural immune response of aquatic organisms exposed to herbicide contaminated water [16, 17]. Recently, we demonstrated that silver catfish exposed to sublethal concentrations of glyphosate were more susceptible to intracoelomic challenge with Aeromonas hydrophila; in addition, in these fish, macrophage collected from the coelomic cavity had reduced phagocytic activity [18]. Thus, because the use of commercial glyphosate has dramatically increased in recent years and many of the adverse effect are related to oxidative stress and a likely effect on immune response, we aimed to investigate whether hematological and natural immune parameter were compromised on silver catfish exposed to water containing nonlethal concentrations of glyphosate.

2. Materials and methods

2.3. Culture of pathogens A. hydrophila [19] and Micrococcus luteus (ATCC 7468) were cultured in Brain Heart Infusion (BHI) for 18 h at 37  C. The broth cultures were centrifuged (600  g for 20 min), the supernatant discarded and the bacteria pellet was washed three times in PBS (pH 7.4). Final bacteria concentration was adjusted using a spectrophotometer: A. hydrophila was adjusted to 0.1 OD at 550 nm, and M. luteus to 0.5 OD at 450 nm. For both bacteria, the number of colony forming units (CFU)/ml1 was determined using standard dilution methodology and plating on Tryptic Soy Agar (TSA) for 24 h at 37  C. A local isolate of Candida albicans was grown on yeast extract (1%), peptone (2%) and glucose (2%) at 37  C for 18 h, then washed three times with PBS (pH 7.4) and coupled to Fluorescein Isothyocyanate (FITC), according to manufactures instructions (Sigma, St Louis, USA). A. hydrophila was used for the bactericidal and hemagglutination assays and M. luteus was used to determine lysozyme activity. FITC-labeled C. albicans was used to determine the phagocytic index (PI).

2.4. Collection of blood and phagocytic cells Following exposure to glyphosate all fish were captured and anesthetized with Eugenol (50 mg/L). For the hematological studies, blood samples were drawn from the caudal vein using sterile heparinized syringes. To determine immune parameter, blood was allowed to clot on ice-chilled water for 2 h, centrifuged (600  g, 4  C, 10 min) and serum aliquots were stored (18  C) until use. To collect phagocytic cells, anesthetized fish were placed on ice-chilled water, transferred to the laboratory and the coelomic cavity was injected with 3 ml of ice-cold sterile phosphate buffered saline (PBS, pH 7.4). After 1 min, the PBS-containing phagocytic cells was collected from the coelomic cavity and the cells pelleted by centrifugation (600  g), counted and suspended in RPMI media containing 1% fetal bovine serum (FBS; Cultilab, Brazil) to a final concentration of 106/ml1.

2.1. Fish Silver catfish fingerlings of both sexes, with an average weight of 18.0  8 g were used to determine immunological parameters; juveniles catfish with an average weight of 80e100 g were used for hematological studies. All fish were transferred from the experimental farm to laboratory tanks containing aerated water and acclimated for 7 days (7e10 fish/tank; specific density < 1 g of fish/ L). Water temperature was kept at 22.0  2.0  C, pH 7.40.6 units, dissolved oxygen 7.80.4 mg/L; total ammonia was lower than 0.01 mg l1 and total hardness and alkalinity were 66 and 22 mg l1 CaCO3, respectively.

2.2. Experimental design All experiments were carried out in triplicates with at least one control group. Following the adaptation period, commercial available glyphosate (N-phosphonomethyl glycine, 360 mg/L) was added to the water to a final concentration of 0.730 mg/L1 which corresponded to 10% of the previously reported CL50e96h [6] for silver catfish. The effects of glyphosate on hematological parameters were determined in fish kept in contaminated water for 96 h; immunological parameters were evaluated after 24 h or 10 days exposure to glyphosate contaminated water. Fish were fed twice daily with commercial fish pellets (42% crude protein, Supra, Brazil). Tanks were cleaned every two days and the water quality did not change during the experimental period.

2.5. Hematological parameters Blood smear were prepared immediately after sampling, airdried and submitted to Wright-Giemsa staining. Hematocrit, hemoglobin and erythrocyte counts were determined on whole blood within 2 h after sampling, as previously described [20].

2.6. Phagocytosis assay The phagocytic activity of coelomic cells was determined, for each fish, using C. albicans coupled to fluorescein isothiocyanate (FITC). For the phagocytic assay, 10 ml of FITC-labeled C. albicans (109/ml) were thoroughly mixed with 200 ml of coelomic cells (106/ml1) in RPMI medium (1% FBS) and two aliquots of 100 ml each were layered over a circular (13 mm diameter) coverslip glass lamina placed inside the wells of a 24-wells tissue culture plates, and incubated at 22  C for 15 min. Following that, the wells were washed three times with PBS to remove both nonadherent cells and non-phagocyted FITC-labeled C. albicans, the coverslip was removed, washed once more in PBS, quenched with Evans blue for 30 sec., fixed with standard histological solution and mounted over a microscopic lamina. To determine the phagocytosis index (PI), 100 cells were counted using an epifluorescent microscope, and the number of cells containing engulfed FITC-labeled C. albicans was registered, in duplicates, for each fish.

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2.7. Bactericidal activity The bactericidal assay was carried out in sterile micro-vials by mixing 18 ml of fish serum and 2 ml of A. hydrophila (2  105 CFU/ ml1). The serum-bacteria solution was incubated at 22  C for 90 min with occasionally mixing. Controls were prepared by replacing fish serum with sterile PBS. After incubation, 80 ml of sterile PBS (pH 7.4) was added to the mixture, and the total volume was pour-plated on TSA and incubated for 24 h at 37  C. The number of viable bacteria was determined by counting the colonies that grew onto the TSA plates. 2.8. Serum agglutination activity The natural agglutination activity of fish serum was investigated using “U” shaped 96-wells plates. Two-fold dilution of serum was made in PBS (pH 7.4) containing Caþþ and Mgþþ and an equal volume of formalin-killed A. hydrophila suspension (0.1 OD 550 nm) was added to each well. Then, the plates were incubated for 2 h at 25  C and then overnight at 4  C; the serum titer was determined as the logarithm of the highest dilution of the serum that caused complete agglutination of the bacteria cell. 2.9. Natural complement hemolytic activity The natural hemolytic activity of fish serum was determined using rabbit red blood cells (RaRBC) as described previously [21] with minor modifications. Briefly, freshly collected RaRBC were washed three times in phenol red free Hank’s solution and the final concentration adjusted to 2  108 ml1. For the assay, serum samples were two-fold serially diluted from 10% to 0.312%, in Hank’s solution containing 1 mM Mgþþ, 10 mM EGTA and 6.7 mM HEPES (pH 7,2) in a 100 ml final volume. Then, 100 ul of RaRBC were added and the mixture incubated at 22  C for 90 min with gently mixing every 15 min. The reaction was stopped by adding 1 ml of Hank’s solution containing 10 mM EDTA. The non-lysed RaRBC were pelleted by centrifugation (500  g for 5 min at 8  C) and 200 ml of the supernatant was transferred to flat bottom 96 well plates and the absorbance was determined using an ELISA reader (405 nm). The positive control (100% hemolysis) consisted of 100 ml of RaRBC lysed in 100 ml of distilled water; negative control (spontaneous hemolysis) consisted of 100 ml of RaRBC incubated with Hank’s. Both positive and negative controls were also mixed with 1 ml of Hank’s containing 10 mM EDTA. The degree of hemolysis was calculated as described [21]. 2.10. Lysozyme activity The assay for lysozyme was carried out using the turbidimetric assays. Fish serum (20 ml) was mixed with 180 ml of M. luteus (OD 0.5 at 450 nm) in 0.05 M sodium phosphate buffer (pH 6.2) at 25  C. The OD was taken with a spectrophotometer (450 nm) at 30 s and at 5 and 10 min after adding M. luteus. Lysozyme from white hen egg (Sigma Aldrich, St Louis, USA) was used as control. Lysozyme activity was expressed as units ml1; one unit was defined as a reduction in the OD of 0.001 min1.

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peroxidase reaction was stopped after 5 min by adding 35 ml of hydrochloric acid (HCl, 3 M). Plates were read with a spectrophotometer at 492 nm. Samples without serum were added as negative control.

2.12. Statistical analysis The results shown in the text, tables and figures are expressed as the mean  standard error (SEM). Differences among treatment groups were tested by one way analysis of variance (ANOVA). The statistical analysis was carried out using the software program BioEstat 5.0 and GraphPad Prism 5.

3. Results 3.1. Hematological parameters of R. quelen exposed to glyphosate Results from the hematological analysis are depicted in Table 1. Total leukocytes, lymphocytes, thrombocytes and erythrocytes counts were significantly lower (p < 0.01), and the number of circulating immature cells were significantly higher (p < 0.01) in the blood of fish from the glyphosate exposed group relative to the same type of cells counted in the blood of non-exposed fish. The values of hematocrit, monocytes and neutrophil were similar amongst exposed and control groups. Glucose and total plasma proteins were also similar amongst groups (not shown).

3.2. Effect of glyphosate on coelomic cell phagocytic index Coelomic cells from fingerlings exposed to glyphosate for 24 h had a significant reduction (p < 0.05) in phagocytic activity compared to the control group; in contrast, cells from fingerlings exposed to glyphosate for 10 days had a slight but not significant increase in the phagocytic index compared to the respective control group (Fig. 1).

3.3. Bactericidal activity Exposure of fingerlings to glyphosate for 24 h or 10 days did not alter the serum bactericidal activity compared to the respective control group (Fig. 2). Because the serum bactericidal activity of each group was carried out at different times, a recently grown new bacteria batch was used in each experiment, which might account for the different number of CFU/ml1 observed when different groups (e.g. 24 h versus 10 days) are compared.

Table 1 Hematological parameters of silver catfish non-exposed (control group) or exposed for 96 h to 10% of the LC50e96h of glyphosate (0.730 mg/L). The results represent the mean  SEM for each group (n ¼ 7). Blood parameter

Treatment group Non-exposed

2.11. Total peroxidase activity of fish serum The peroxidase content of fish serum was performed following the protocol of Quade and Roth [22] with some modifications. Exactly 10 ml of fish serum was diluted with 90 ml of Caþþ, Mgþþ, and phenol red free Hank’s solution in flat bottomed 96 well plates. Then, 35 ml of OPD (o-phenylenediamine dihidrochloride), in citrate (0, 2 M) and phosphate buffer (0, 1 M, pH 5, 3) were added. The

Hematocrit (%) Erythrocytes (106/ml) Immature cells (%) Trombocytes (103/ml) Total leukocytes (103/ml) Neutrophils (103/ml) Monocytes (103/ml) Linfocytes (103/ml)

38.1 1.67 0.15 6.26 4.48 0.33 0.49 3.64

(2.9)a (0.09)a (0.04)a (0.7)a (0.4)a (0.10)a (0.17)a (0.25)a

Glyphosate exposed 36.7 1.35 0.36 3.61 2.65 0.15 0.25 2.22

(1.7)a (0.05)b (0.07)b (0.4)b (0.26)b (0.03)a (0.07)a (0.20)b

Significant differences between groups are indicated by different letters (p < 0.01).

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A

B

60 55

55

a

50

45 40 35

b

30 25 20 15

Phagoc y tic index (%)

Phagoc y tic index (%)

50

60

45 40 35 30 25 20 15

10

10

5

5 0

0 Control

Glyphosate exposed

Control

Glyphosate exposed

Fig. 1. Phagocytic index measured on cells collected from the coelomic cavity of silver catfish fingerlings non-exposed (control) or exposed to 10% of the LC50e96h of glyphosate for 24 h (A) or 10 days (B). Phagocytosis was measures in all fish from the experiment. The results are expressed as percentile of coelomic cells phagocyting FITC-labeled C. albicans and represent the mean  SEM (p < 0.05).

3.4. Serum agglutination activity The natural bacterial agglutination titer measured against formalin-inactivated pathogenic A. hydrophila was significantly lower (p < 0.05) in glyphosate exposed fingerlings, either at 24 h or 10 days, compared to the respective control group (Fig. 3). In this particular experiment the same batch of formalin-killed bacteria was used to measure agglutination activity; consequently, similar results were obtained in both control groups.

(Table 2); however, a significant reduction on lysozyme activity was observed in the serum of fingerlings following 10 days exposure to glyphosate. In contrast, serum myeloperoxidase was significantly lower (p < 0.05) in 24 h glyphosate-exposed fingerling compared to the respective control group. Non significant difference in myeloperoxidase activity was observed in the serum of 10 days glyphosate exposed fingerlings compared to its control group (Table 2). No statistically significant differences could be detected in the natural complement hemolytic activity of fingerlings after 24 h or 10 days exposure to glyphosate.

3.5. Serum lysozyme, myeloperoxidase and natural complement hemolytic activity

4. Discussion

Serum lysozyme activity from fingerlings exposed to glyphosate for 24 h was similar to that observed in the respective control group

Fish production is becoming a sustainable alternative income for small farms in southern Brazil. However, the predominant high

B

70

70

65

65

60

60

Number of c olony for ming units

Number of c olony for ming unit

A

55 50 45 40 35 30 25 20 15 10 5

55 50 45 40 35 30 25 20 15 10 5

0

0

Control

Glyphosate exposed

Control

Glyphosate exposed

Fig. 2. Bactericidal activity measured on the serum of silver catfish fingerlings non-exposed (control) or exposed to 10% of the LC50e96h of glyphosate for 24 h (A) or 10 days (B). Serum bactericidal activity, measured against pathogenic Aeromonas hydrophila, is indicated as the number of colony forming units obtained by plating on TSA media following incubation of bacteria with fish serum. The results represent the mean  SEM (p < 0.05) of the bactericidal activity determined in the serum of all fish from the experiment.

L.C. Kreutz et al. / Fish & Shellfish Immunology 30 (2011) 51e57

A

B

a

2.0

1.5 b 1.0

0.5

Agglutination ac tivity (Log)

2.0

Agglutination ac tivity (Log)

55

a

1.5

b

1.0

0.5

0.0

0.0 Control

Glyphosate exposed

Control

Glyphosate exposed

Fig. 3. Serum agglutination activity measured on the serum of silver catfish fingerlings non-exposed (control) or exposed to 10% of the LC50e96h of glyphosate for 24 h (A) or 10 days (B). The ability of fish serum to agglutinate bacteria was determined against pathogenic Aeromonas hydrophila, and is expressed as the logarithm of the last serum dilution that caused bacteria agglutination. The results represent the mean  SEM (<0.05) of the agglutination activity determined in the serum of all fish from the experiment.

production crop fields demand a large and continuous use of herbicides such as glyphosate that, in turn, can be found as a contaminant of soil and water creeks [4,5] that eventually feed ponds used in aquaculture. Hence, the presence of agrichemical in water and soil might present an impediment to fish production. Because aquatic organisms are much more sensitive to glyphosate that other species [2,3] and the harmful effects of glyphosate on fish species, including silver catfish, is under investigation, the use of glyphosate in the vicinity of water springs and ponds should be reevaluated. Herbicide application techniques currently in use allow for water contamination by direct overspray drift from aircraft, spray drift from crop fields adjacent to water bodies, runoff and leaching. Thus, the undesirable effect of herbicides on the highly susceptible aquatic environment commonly found near crop fields should be evaluated by risk assessment procedures using indigenous, non-target plant and fish species native to the water catchments [23] such as R. quelen. Such toxicity studies will provide data for the enforcement of regulatory rules aiming to protect autochthonous species and to periodically monitor traces of herbicides in soil and water. In addition, water bodies used for aquaculture

Table 2 Natural immunological parameters of silver catfish exposed to 10% of the LC50e96h of glyphosate (0,730 mg/L). Fish were capture for blood sampling at 24 h or 10 days following exposure. For each sampling time a non-exposed, control group was sampled. Natural immune parameter

Glyphosate exposure 24 h Control

10 days Glyphosate exposed

Control

Glyphosate exposed

75.0 (7.0)a 51.6 (5.2)b Serum lysozyme 70.0 (11.4)a 67.1 (3.8)a (units/ml) 1074.5 (71.8)a 685.5 (38.0)b 847.4 (123.6)a 864.2 (98.5)a Peroxidase (OD450 nm) Complement 355.0 (77.5)a 270.8 (34.5)a 361.5 (57.6)a 332.2 (42.3)a hemolytic activity (ACH50) Data represent the mean  SEM. Significant differences between groups are indicated by different letters (p < 0.05).

purposes should be physically protected from overspray and spray drift, leaching and runoff water from agricultural areas. With this in mind, our group has concentrated efforts to evaluate the effects of commonly used agrichemical on hematology, biochemistry and hormonal balance, and oxidative stress [7,11,12,20,24] and to optimize raising conditions of silver catfish even in a polyculture system with exotic fish species [25]. Recently, it has also been demonstrated that glyphosate, at concentrations likely to be found in field conditions, increases the susceptibility of silver catfish to pathogenic A. hydrophila challenge [18]. The impact of residual agrichemicals on indigenous species has become a matter of concern to researchers and environmentalists. Thus, the experiments reported herein were designed to further evaluate the effect of sub-lethal concentrations of a glyphosate-based herbicide on hematology and natural immune parameters of silver catfish. Hematological parameters of fish might be affected by stressors and water pollutants [26]. Thus, evaluating fish blood parameters might be a useful tool to understand the impact of agrichemicals on fish health. The acute effects (up to 96 h exposure) of sublethal concentrations of several agrichemicals on hematological parameters of fish species have already been reported [27e32]. Therefore, for comparison purposes, in the present study, blood parameters were also measured after 96 h exposure rather than after 24 h or 10 days when immunological parameters were evaluated. Thus, by correlating the effects of glyphosate on blood cells related to immune functions and immunological parameters, a better picture could be drawn to understand the possible immunosuppressive effect of glyphosate. Herein, the values of blood parameters observed in the control group were within the range previously reported for R. quelen [20]. However, following short term exposure (96 h) of silver catfish to glyphosate-contaminated water, major changes on blood parameters related to immune functions could be observed. In the glyphosate exposed group, the number of neutrophils and monocytes was lower than that observed in the non-exposed fish, but significant differences were observed only in the number of circulating leukocytes and lymphocytes. In contrast, the number of immature cells was significantly higher in the glyphosate exposed group. The reduction of total circulating leukocytes in the glyphosate exposed fish might account for a decrease in the number of cells in the coelomic cavity as recently

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reported [18]. Glyphosate exposed and non-exposed silver catfish had similar hematocrit values. In contrast, piava (L. obtusidens) exposed to glyphosate (3e20 mg/L) had a discrete, non-significant reduction of total leukocytes [9] but significant decrease in hematocrit, erythrocytes, and plasma hemoglobin and protein values; however, it should be noted that silver catfish were exposed to 0.73 mg/l of glyphosate, a dose much lower than that used for piava, and that the LC50 of glyphosate varies according to target species [1,3,6,8,9,10]. R. quelen blood parameters were not altered after 2, 4 and 8 days exposure to sublethal concentration of cypermethrin except for hemoglobin and mean corpuscular hemoglobin concentration, which increased [31]. On the other hand, the hematocrit content was reduced in R. quelen exposed for 96 h to the herbicide clomazole [28]. Furthermore, acute exposure of European catfish (Silurus glanis L.) to diazinon, an organophosphorous pesticide, also reduced hematocrit and hemoglobin values, and reduced the number of circulating erythrocytes and leukocytes [30]. The presence of significantly higher number of circulating immature cells, as observed in the present work, and the observation that a smaller number of coelomic cells have been found on glyphosate exposed fish [18] might suggest that glyphosate might be directly toxic to leukocytes, or that the oxidative stress caused by the presence of glyphosate in water [8e10,24] contributes to the reduction of the number of circulating cells. Nonetheless, even though the effect of glyphosate on fish leukocyte biology and biochemical routes has not been investigated yet, there is enough data to suggest that glyphosate mitigates natural immune function by reducing leukocyte population and the ability of cells to remove infecting bacteria, causing and augmented susceptibility to opportunistic infections such that caused by A. hydrophila. The deleterious effect of glyphosate on immune cells cultivated in vitro should be a matter of further investigation. Resistance of fish to infection by microorganisms relies mostly on an immediate response carried out by cells and serum soluble molecules. To evaluate the effect of glyphosate on silver catfish cell mediated immunity, a phagocytic assay was carried out using FITClabeled C. albicans and phagocytic cells collected from the coelomic cavity. A significant reduction (p < 0.05) on phagocytic index was observed in cells from fish exposed to glyphosate for 24 h compared to cells from control fish. Interestingly, the phagocytic index of cells from fish exposed to glyphosate for 10 days was similar to that found on non-exposed fingerlings. Previously, we reported that both glyphosate and atrazine caused a significant reduction on phagocytic index after 24 h exposure, and that fingerlings mortality was recorded only up to the third day following agrichemical exposure and challenge with A. hydrophila [18]. Studies regarding the effect of glyphosate on immune system are scarce [16]. In the other hand, plenty of studies have been carried out on the effect of glyphosate on oxidative stress. In those studies, it has been reported that reactive oxygen species (ROS) increase after exposure to glyphosate for 24 h but return to basal levels at 96 h [8,10,13]. The reduction of ROS is driven mostly by catalase, an antioxidant hepatic enzyme that metabolizes hydrogen peroxide (H2O2). The hepatic activity of catalase has been found elevated in fish exposed to glyphosate-based herbicides during a 24 h period [13] in comparison to non-exposed fish, but returned to basal levels after 96 h indicating that ROS production has been reduced to basal level at this time. Oxidative stress caused by acute exposure to glyphosate causes damage to cell membrane and could interfere with the ability to phagocitize antigens. Thus, a similar conclusion might be drawn from the phagocytic studies, in that by 10 days following exposure to glyphosate and with ROS at basal levels, there would be no impediment for coelomic cells to engulf FITC-labeled C. albicans. In addition, because a very low amount of glyphosate was used, and considering glyphosate half-life on water

[1,3] there could be a significant reduction in the amount of glyphosate in water that would have no effect on recently produced cells that would be present of the coelomic cavity, at 10 days following exposure, when the cells were collected for the phagocytic assay. Unfortunately, blood cells were not evaluated at this time to investigate whether blood parameters had returned to basal levels. Serum bactericidal and agglutination activity were measured against pathogenic A. hydrophila. No significant changes on bactericidal activity were observed on the serum of glyphosate-exposed catfish compared to the respective control group. The serum bactericidal activity of each group of fish was measured at different time with recently grown bacteria; thus, even after adjusting the bacteria concentration using a spectrophotometer, the exact number of CFU/ml1 was slightly different which accounts for by the difference in the results when both control groups are compared. Bactericidal activity, measured as the amount of CFU/ml obtained following incubation of serum with a standard amount of bacteria, is attributed to several small peptides that interact with cell wall causing bacterial inactivation [33,34]. Thus, in the present work, bactericidal activity should not be attributed to the serum lysozyme content or complement bacteriolytic activity, in that lysozyme is most effective upon gram positive bacteria, and complement activity was similar amongst groups, as discussed below. In contrast, a significant difference was observed on the bactericidal agglutination titer. In this assay, the same batch of formalin-inactivated A. hydrophila was used, thus similar agglutination titers were found in the serum of non-exposed fingerlings. On the other hand, serum obtained from fingerlings exposed to glyphosate for 24 h or 10 days had lower agglutination activity. Lysozyme bactericidal activity against gram positive bacteria has been studied as a major component of non-specific humoral immunity in fish [35,36]. Lysozyme is produced by several types of leukocytes, including neutrophils and macrophages [34]. The amount of lysozyme detected in the serum of silver catfish exposed for 24 h to glyphosate was similar to that found on fish from the control group; however, a significant reduction on serum lysozyme was observed on fish exposed to glyphosate for 10 days (Fig. 2). The reduced amount of serum lysozyme is likely related to a reduction of the circulating lysozyme-producing cells (i.e. leukocytes) observed at 96 h or due to disruption of protein synthesis, a phenomenon reported in Tilapia exposed to glyphosate [16]. The peroxidase content in silver catfish serum was significantly lower after 24 h exposure to glyphosate compared to control fish. Glyphosate had no effect on fish serum peroxidase content after 10 days exposure. Serum peroxidases might be released by phagocytic cells upon stimulation by different pathogens [33] and, together with oxygen radical, comprises an important mechanism to control infections. In this context, lower serum peroxidase content is likely the result of a lower phagocytic activity of cells, as observed also after 24 h exposure do glyphosate. Although blood cell parameters were measured only after 96 h exposure to glyphosate, data reported recently indicated that the amount of coelomic cells in glyphosate exposed fish was lower than that of non-exposed fish, which might suggest that the number of circulating cell with phagocytic activity could be already lower even after 24 h exposure, thus compromising resistance to opportunistic infection. Because glyphosate caused oxidative stress and lipid peroxidation [8,10,15], the increased amount of serum peroxidase could also result from damage to phagocytic cell membranes which in turn could account for cell death and the lower number of circulating cells observed after short term exposure to glyphosate. The natural bacteriolytic activity of complement is effective towards a range of microorganisms, except those containing large quantities of sialic acid, and constitutes an important part of natural

L.C. Kreutz et al. / Fish & Shellfish Immunology 30 (2011) 51e57

humoral immunity of fish [37]. In the present work, natural complement hemolytic activity was similar in fish exposed to glyphosate, for 24 h or 10 days, compared to the respective control group. Serum of glyphosate exposed fish had a tendency to present a lower hemolytic activity, however, due to a high variability observed within fish from the same group, no statistically difference could be found. Even though glyphosate decreases the number of leukocytes, and has major effect on liver [9], both important sites of complement components synthesis [37], complement hemolytic activity did not change. Because the biological half-life of fish complement components are unknown, it is possible that even following 10 days exposure and major changes in liver and leukocytes, the basal amount of complement component remained similar to that prior to glyphosate exposure. In conclusion, the results of the present study indicate that sublethal concentration of glyphosate has a potential immunossupressor effect on silver catfish. Thus, because glyphosate and other agrichemicals are ubiquitously used in modern crop production, all water bodies on the vicinity of agricultural areas should be protected and periodically monitored for the presence of contaminants. In addition, the hazardous and immunotoxic effects of glyphosate on the expression of genes related to immune functions and in the specific immune response of indigenous fish species should be a subject of future investigation.

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