Springer 2005 Hydrobiologia (2005) 537: 25–33 Haematological parameters in a neotropical fish, Corydoras paleatus (Jenyns, 1842) (Pisces, Callichthyidae), captured from pristine and polluted water Jimena Cazenave1, Daniel Alberto Wunderlin2, Andrea Cecilia Hued1 & Marı́a de los Ángeles Bistoni1,* 1 Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Fı´sicas y Naturales, Diversidad Animal II. Av. Ve´lez Sársfield 299, 5000 Córdoba, Argentina 2 Universidad Nacional de Córdoba, Facultad de Ciencias Quı´micas, Dpto. Bioquı´mica Clı´nica, Medina Allende y Haya de La Torre, Ciudad Universitaria, 5000 Córdoba, Argentina (* Author for correspondence: Tel.: +54-351-4332090, Fax: +54-351-4334162, E-mail: mbistoni@gtwing.efn.uncor.edu) Received 12 April 2004; in revised form 19 July 2004; accepted 26 July 2004 Key words: aquatic pollution, biomarkers, Corydoras paleatus, haematology Abstract We report normal ranges of haematological indices in healthy Corydoras paleatus from an unpolluted area. Haematological parameters studied include: erythrocyte counts (Er), haematocrit (Ht), haemoglobin concentration (Hb), mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). Normal red blood parameters did not change according to maturation stages, sex or seasons. Then, we compared them with those coming from fish captured in a site polluted by sewage. Fish exposed to pollution presented significantly higher values of Er, Ht, Hb, MCH and MCHC than those captured in an unpolluted area. Discriminant analysis showed that Hb is a key parameter to point out differences between populations exposed to different environmental conditions. We suggest that haematological values of C. paleatus, registered during this study, could be used as biomarkers in future works evaluating the incidence of environmental stress on fish as well as pointing out changes in the water quality. Introduction Fish are useful bioindicators to evidence environmental degradation (Fausch et al., 1990). Their haematology provides an important tool in the evaluation of its physiological status, reflecting the relative health of the aquatic ecosystem. Therefore, it is necessary to know the normal range of the blood parameters previous to use them as biomarkers (Lusková, 1995). The haematological parameters in fish may be influenced by intrinsic factors such as sex, reproductive stage, age, size and health (Joshi, 1982; Ranzani Paiva & Godinho, 1985; Hlavová, 1993a; Lusková et al., 1995; Nespolo & Rosenmann, 2002). They are also affected by external factors like seasonal dynamics, water temperature, environmental quality, food, stress, etc. (Mahajan & Dheer, 1979; Tisa et al., 1983; Sandström, 1989; Van Vuren et al., 1994; Witeska, 1998; TavaresDias et al., 1999; Rios et al., 2002). Fish blood characteristics change in response to environmental conditions, thus the variation of haematological features could serve as a biomarker of sublethal environmental stress (Bridges et al., 1976). Numerous haematological studies have been published on species that are used in pisciculture (Ranzani Paiva & Godinho, 1986, 1991; TavaresDias & Sandrim, 1998; Tavares-Dias et al., 1999), however, less data are available on wild 26 populations. Corydoras paleatus, locally called liampiafondos, was chosen for this study because it is a widely distributed neotropical fish with ornamental value (Gómez et al., 1993, 94). This benthic species is a facultative air-breathing fish, which is able to tolerate low-oxygen concentrations (Gómez, 1993). In the central region of Argentina, the urban activities of Córdoba city produce serious and negative impacts on Suquı́a River water quality. The locations downstream from the city sewage discharge have been pointed out as polluted sites, recording low dissolved oxygen levels (Bistoni et al., 1999; Pesce & Wunderlin, 2000; Wunderlin et al., 2001; Hued & Bistoni, 2002). C. paleatus is present in the Suquı́a River basin (Córdoba, Argentina) (Haro et al., 1986) and inhabits both polluted and non-polluted areas in a relative high number of individuals (Hued, 2003). The main goals of the present study were to establish normal ranges of haematological parameters in healthy Corydoras paleatus as well as to test the use of such parameters as potential biomarkers of aquatic pollution. The collected data provide the first information on the natural composition of blood in this species in relation to sex, gonadal maturation stages and seasonal dynamics as well as the first indications on the use of these parameters as biomarkers. Materials and methods Study area Fish were collected from the Suquı́a River 20 km upstream (Station 1) and 30 km downstream (Station 2) from Córdoba city. The Station 1 is situated in the locality of La Calera (3121¢ S; 6421¢ W) and was identified as pristine or quasipristine conditions by Hued & Bistoni (2002). The Station 2, named Villa Corazón de Marı́a (3127¢ S; 6359¢ W), is located downstream from the city sewage discharge. This site has been classified as a highly polluted site by Pesce & Wunderlin (2000), whose pointed out an important water quality drop. Among other effects, sewage pollution results in a drop of dissolved oxygen as well as increase in levels of ammonia and nitrite nitrogen (Wunderlin et al., 2001). Fish collection and water quality evaluation Adult specimens of Corydoras paleatus were captured by a backpack electrofisher (Coffelt, model Mark 10) and transported to the laboratory within 2 h after capture into tanks containing 20 l of water of the capture area. Together with fish collection, we verify the water quality of each sampling area by measuring the following parameters: pH, temperature (C), alkalinity (mg l)1), dissolved oxygen (DO, mg l)1), total solids (mg l)1), ammonia (mg l)1), nitrites (mg l)1), nitrates (mg l)1), 5-days biological oxygen demand (BOD, mg l)1), total phosphorus (mg l)1), hardness (mg l)1), calcium (mg l)1), magnesium (mg l)1), sulphates (mg l)1), chlorides (mg l)1), total coliforms (MPN 100 ml)1: most probable number per 100ml), faecal coliforms (MPN 100 ml)1). Analytical methods were standard (APHA, 1995). Water was sampled and transported to the laboratory according to standard procedures previously described (Pesce & Wunderlin, 2000). Normal haematological characterization Haematological parameters were investigated in healthy specimens collected seasonally during a year (2001–2002) from the Station 1. Blood was sampled in the laboratory within 24 h after the capture and it was extracted individually by dissection of the caudal peduncle (Reichenbach-Klinke, 1980; Roberts, 1981), using heparinized tools. Each fish was anesthetized with benzocaine (Summerfelt & Smith, 1990) previous to blood collection, which was completed within 3 min. Afterwards, each fish was killed by a direct blow to the head and dissected. Erythrocyte counts (Er) per mm3, were performed with a Neubauer chamber, using physiological solution (NaCl 0.15 M) diluted 1:200. Haematocrit (Ht) values were determined by the micromethod using capillary tubes and centrifuged at 3000 rpm for 10 min. Ht is expressed in percentage. Haemoglobin concentration (Hb) (g 100 ml)1) was measured by the cyanomethaemoglobin procedure (Houston, 1990). These basic blood parameters were then used to derive the following haematimetric indices: mean cell volume (MCV, fl), mean cell haemoglobin (MCH, pg) and mean cell haemoglobin concentration (MCHC, %). 27 Each fish was measured (total length, LT, mm) and weighed (somatic mass, M, g). The Fulton condition factor was calculated as K ¼ M/ LT3 · 100 000, according to Anderson & Neumann (1996). The sex of each fish was determined by dissection. The state of female gonadal maturation was determined by direct observation and the gonadosomatic index was calculated according to GSI ¼ MG/M, where MG is the weight of gonads (Strange, 1996). Haematological parameters of specimens captured at a polluted area In order to test the potential use of blood characteristics as biomarkers, all the blood parameters mentioned above, were measured in C. paleatus collected from a polluted site on Suquı́a River (Station 2). Blood parameters measured in fish from station 2 were compared with those obtained from station 1. Statistical analyses Descriptive statistics of blood parameters (mean, standard deviation, range) were evaluated by grouping individuals according to gonadal maturation stage, sex, season and capture station. Further statistical analyses were performed on data normalized to zero mean and unit variance (standardized data) (Johnson & Wichern, 1992). One way analysis of variance was performed to evaluate changes in haematological variables between groups. Differences were considered statistically significant when p < 0.05. Discriminant Analysis (DA), stepwise mode, was performed to evaluate the haematological parameters point out differences between both sampling stations. Table 1. Water quality data from Station 1 (quasi-pristine area) and Station 2 (polluted area) Variable Station 1 Station 2 Temperature (C) 18 ± 6 17 ± 7 pH Alkalinity (mg l)1) 8.3 ± 0.6 92 ± 8 7.5 ± 0.2 168 ± 11 Dissolved Oxygen 11 ± 2 4±1 2±1 4±2 (mg l)1) BOD (mg l)1) Ammonia (mg l)1) 0.3 ± 0.1 2±3 Nitrites (mg l)1) 0.1 ± 0.1 0.4 ± 0.2 Nitrates (mg l)1) 1.2 ± 1.5 2.5 ± 1.1 Total phosphorus (mg l)1) 0.03 ± 0.01 1.0 ± 0.4 Hardness (mg l)1) 82 ± 20 228 ± 49 Calcium (mg l)1) 22 ± 4 64 ± 13 7±2 17 ± 4 Magnesium (mg l)1) Chorides (mg l)1) 7±1 63 ± 22 Sulphates (mg l)1) 19 ± 4 142 ± 41 Total solids (mg l)1) 122 ± 32 589 ± 253 Total coliforms (MPN 100 ml)1) 1476 ± 2139 666 750 ± 845 512 Faecal coliforms 434 ± 581 170 937 ± 132 363 (MPN 100 ml)1) Values are mean ± SD. total phosphorous, chlorides, total and faecal coliforms bacteria. Most evaluated parameters presented the worst values at station 2, showing the deterioration of water quality at this point. Both urban non-point pollution and the city sewage discharge contribute to this drop in water quality. Moreover, the low levels of dissolved oxygen observed at station 2 are the result of the increase in BOD, bacterial activity as well as to the rise in the ammonia nitrogen, leading to oxygen consumption downstream from the city sewage discharge. Results Normal haematological characterization Water quality Major changes in the water quality of Suquı́a River were observed between the studied sites. The station located downstream from Córdoba City (Station 2 – Villa Corazón de Marı́a) was characterized by degraded water quality conditions. Table 1 shows changes in parameters like dissolved oxygen, BOD, ammonia, nitrites, nitrates, Blood analyses were carried out on 97 individuals (48 females and 49 males). Their somatic data are reported in Table 2. The different gonadal maturation stages were: resting (n ¼ 5), maturation (n ¼ 6), mature (n ¼ 23) and spent (n ¼ 14) (Table 3). Statistical comparison of the studied haematological parameters did not show significant differences 28 Table 2. Somatic data for Corydoras paleatus from a quasipristine area (La Calera, station 1) and a polluted area (Corazón de Marı́a, station 2) Total length (mm) Sex Station 1 Male 53 ± 6 Female Somatic mass (g) Condition factor 61± 10 Station 2 63 ± 3 70 ± 7 Total 57 ± 9 68 ± 7 Male 2.4 ± 0.9 3.9 ± 0.9 Female 4.3 ± 2.3 5.9 ± 1.8 Total 3.3 ± 2.0 5.2 ± 1.8 Male 1.5 ± 0.2 1.6 ± 0.2 Female 1.7 ± 0.2 1.7 ± 0.2 Total 1.6 ± 0.2 1.6 ± 0.2 Values are mean ± SD. among maturation stages. Descriptive statistics of blood red components, grouped according to seasons and sex, are given in Table 4. The means of haematological parameters were usually higher for females than for males, but no significant differences were found between sexes for each season. The mean values of haematological variables did not show significant changes between seasons when considering all the studied individuals or grouping them according to the sex. Haematological parameters in specimens from a polluted area Somatic data of wild specimens collected from station 2 (n ¼ 15) are given in Table 2. The Er count ranged from 1.6 to 3.4 · 106 mm)3; Ht values were between 31 and 54%, while those for Hb were be- tween 9.2 and 14.2 g 100 ml)1. Minimum and maximum for MCV were 121 and 291 fl; 34.79 and 78 pg for MCH; 22.3 and 34.3% for MCHC respectively. Figure 1 shows the variation of each parameter, observed between fish captured in pristine site (station 1) and those coming from the polluted station (station 2). Except MCV, mean values of blood components in C. paleatus captured in the polluted area (station 2) were significantly higher than those corresponding to station 1 (p < 0.001). Discriminant Analysis (DA) based on haematological characteristics defined two groups: one corresponds to fish captured in station 1 (nonpolluted), while the other corresponds to station 2 (polluted). Stepwise DA affords a classification matrix showing 89% right assignation (Table 5). This result indicated that the two populations were clearly different. Discriminant functions showed that Hb is the main parameter to point out differences between both groups. Discussion The evaluation of haematological characteristics in fish has become an important issue to understand normal and pathological processes, supporting the characterization of fish species in a more complex manner within the framework of ichthyology (Hlavová, 1993a). An important cyclical process in adult fish is the spawning period. It is known that blood parameters can change depending on the maturation of the gonads (Joshi, 1982; Ranzani Paiva & Godinho, 1985). Results obtained during this work indicate that Corydoras paleatus spawn- Table 3. Haematological parameters in females of Corydoras paleatus, captured in an unpolluted area (Station 1), according to gonadal maturation stages (GSI: mean gonadosomatic index) Stage Er (· 106 mm)3) Ht (%) Hb (g 100 ml)1) MCV( fl) MCH (pg) MCHC (%) Resting 1.8 ± 0.5 32 ± 13 6.2 ± 1.5 169 ± 42 36 ± 11 22 ± 6 (GSI = 1.46) (1.2–2.5) (22–51) (4.8–8.7) (127–205) (22–53) (16–27) Maturation 2.1 ± 0.6 32 ± 4 7.5 ± 0.8 148 ± 46 36 ± 9 23 ± 4 (GSI = 2.31) (1.3–3.0) (27–35) (6.2–8.4) (96–204) (25–50) (18–27) Mature (GSI = 14.11) 1.9 ± 0.4 (1.2–2.6) 38 ± 8 (19–52) 7.0 ± 2.0 (4.1–11.2) 212 ± 80 (115–431) 37 ± 8 (26–61) 19 ± 5 (10–26) Spent 1.9 ± 0.4 38 ± 7 7.4 ± 1.8 219 ± 71 40 ± 9 20 ± 6 (GSI = 3.35) (1.0–2.6) (30–53) (3.8–10.2) (132–365) (27–65) (10–27) Values are means ± SD, maximum and minimum are given between parenthesis. 29 Table 4. Normal haematological characters in Corydoras paleatus according to seasons and sex Season Sex N Er Ht (%) (· 106 mm)3) Summer Autumn All seasons MCH (pg) MCHC (%) 5 2.0 ± 0.4 31 ± 7 6.9 ± 1.5 169 ± 62 36 ± 6 23 ± 5 Female 9 1.7 ± 0.3 39 ± 8 6.9 ± 1.7 236 ± 74 40 ± 4 19 ± 6 Total 14 1.8 ± 0.4 (1.0–2.5) 36 ± 8 (19–53) 6.9 ± 1.6 (3.8–9.0) 212 ± 75 (94–365) 38 ± 5 (26–48) 20 ± 6 (10–28) Male 14 1.7 ± 0.3 30 ± 5 6.1 ± 1.6 185 ± 32 38 ± 7 21 ± 4 5 1.9 ± 0.5 34 ± 12 6.5 ± 2.0 160 ± 35 31 ± 7 20 ± 6 19 1.8 ± 0.4 31 ± 8 6.2 ± 1.6 179 ± 33 37 ± 7 20 ± 5 (1.2–2.5) (20–51) (3.6–9.1) (127–242) (22–51) (15–29) 1.8 ± 0.4 1.9 ± 0.4 35 ± 8 41 ± 5 5.8 ± 1.1 7.4 ± 2.0 195 ± 55 201 ± 3 33 ± 7 39 ± 3 17 ± 3 20 ± 2 Total Spring MCV (fl) Male Female Winter Hb (g 100 ml)1) Male Female 10 4 Total 14 1.9 ± 0.3 36 ± 8 6.3 ± 1.5 197 ± 48 34 ± 7 18 ± 3 (1.3–2.6) (22–46) (3.4–9.4) (89–283) (21–43) (13–24) Male 20 1.9 ± 0.4 32 ± 7 6.5 ± 2.0 177 ± 57 35 ± 9 21 ± 6 Female 30 2.0 ± 0.5 36 ± 8 7.2 ± 1.8 199 ± 80 38 ± 10 21 ± 5 Total 50 1.9 ± 0.5 34 ± 8 6.9 ± 1.9 190 ± 71 37 ± 10 21 ± 5 (1.0–3.0) (19–52) (3.0–11.2) (96–431) (22–65) (8–29) 20 ± 5 Male 49 1.8 ± 0.4 32 ± 7 6.3 ± 1.7 183 ± 51 35 ± 8 Female 48 1.9 ± 0.4 37 ± 8 7.1 ± 1.8 204 ± 73 38 ± 9 20 ± 5 Total 97 1.9 ± 0.5 34 ± 8 6.9 ± 1.9 190 ± 71 37 ± 10 21 ± 5 (1.0–3.0) (19–52) (3.0–11.2) (96–431) (22–65) (8–29) Values are mean ± SD. Range (considering all specimens captured in the unpolluted area) is showed between parentheses. ing did not produce significant changes in the haemogram dynamic (Table 3); which is in agreement with findings for some other species (Ranzani Paiva & Godinho, 1991; Hlavová, 1993a; Parma de Croux, 1994). On the other hand, previous studies (Kavamoto et al., 1983; Ranzani Paiva & Godinho, 1991) have shown slight decreases in erythrocyte counts as fish advanced towards spent stage, which was not observed during this study. The influence of sex on certain red blood components has also been reported (Hlavová, 1993b; Lusková, 1995; Lusková et al., 1995). However, several neotropical species, such as Rhamdia hillari (Kavamoto et al., 1983), Prochilodus scrofa (Ranzani Paiva & Godinho, 1985), Brycon sp. (Ranzani Paiva et al., 1991), and Prochilodus lineatus (Parma de Croux, 1994) did not present differences between sexes. During the present study, there was no evidence on differences in blood parameters of Corydoras paleatus between males and females when evaluated with or without consideration of the season (Table 4). The seasonality of haematological characteristics in fish may be related to natural physiological cycles, environmental conditions, or both. Lusková et al. (1995) observed variations of some parameters in Chondrostoma nasus, coincident with both spawning and water temperature. Bridges et al. (1976) had demonstrated significant seasonal differences in red blood variables, occurring with relation to the reproductive activity of Pseudopleuronectes americanus. According to Joshi (1982), the lowest values of red blood parameters were usually recorded in spent fish, or during winters when the temperature was quite low and the food scarce. The red blood parameters of C. paleatus did not show significant changes associated with seasonality (Table 4). Considering that red blood parameters in C. paleatus are not sensitive to changes in the maturation stage, sex or seasons, we hypothesized that haematological differentiation observed between fish captured at different sites (Figure 1) could be attributed to different environmental 30 280 3. 6 260 3. 2 220 MCV (fL) Er (x 106·mm-3) 240 2. 8 2. 4 200 180 160 2. 0 140 1. 6 1. 2 1 2 120 Mean±SD Mean±SE Mean 100 1 STATION 2 Mean±SD Mean±SE Mean STATION 52 65 60 48 55 44 MCH (pg) Ht (%) 50 40 36 45 40 35 32 30 28 24 1 2 Mean±SD Mean±SE Mean 25 20 1 STATION 2 Mean±SD Mean±SE Mean STATION 14 34 12 30 -1 Hb (g·100mL ) 2 Mean±SD Mean±SE Mean MCHC (%) 10 8 6 26 22 18 4 1 2 Mean±SD Mean±SE Mean 14 1 STATION STATION Figure 1. Box-Plot of blood parameters of Corydoras paleatus captured from a pristine area (Station 1), and from a polluted area (Station 2). SD: standard deviation; SE: standard error. Table 5. DA based on haematological parameters. (a) classification matrix (stepwise mode); (b) classification functions for Station 1 (La Calera, non-polluted site) and Station 2 (Villa Corazón de Marı́a, polluted site) Real group Group assigned by DA Station 1 % Right assignation Station 2 Panal (a) Station 1 46 4 92 Station 2 3 12 80 89 Total 49 16 Parameter Station 1 Station 2 panel (b) Hb )0.76 2.59 Constant )0.46 )3.05 conditions (e.g. oxygen depletion, increased toxicity due to ammonia, etc.). Thus, changes in haematology could indicate that fish are exposed to environmental stress. Changes in haematological parameters associated with low levels of dissolved oxygen (DO) have been mentioned in a few field studies. Lochmiller et al. (1989) found that most of the haematological parameters measured in a population of Morone saxatilis were significantly increased as DO decrease. Likewise, blood constituents of Colossoma macropomum revealed an increase in both haemoglobin content and erythrocyte count, when fish were exposed to low oxygen concentrations (Saint-Paul, 1984). 31 According to the results obtained from the present study, Hb seems to be the best blood indicator of environmental stress. This finding is in agreement with Saint-Paul (1984) who suggested that the increase in Hb concentration could be an especially reliable first indicator of an adaptational improvement in the oxygen transporting capacity of the blood. In addition to behavioral and morphological adjustments, fish could respond to low oxygen levels by adjusting several physiological and biochemical parameters (Val et al., 1998). Besides being an air-breathing fish (Gómez, 1993), Corydoras paleatus may increase its respiratory blood function, mainly through an increase in haemoglobin content. Results obtained during this field study suggest increase in the blood oxygencarrying capacity of C. paleatus probably in response to chronic hypoxic conditions registered in the Station 2. A drop in the dissolved oxygen was registered during the present study. Previous studies have also reported low DO levels at this station (Bistoni et al., 1999; Pesce & Wunderlin, 2000; Wunderlin et al., 2001; Hued & Bistoni, 2002). However, to the extent of the present study, it is not possible to discard effects from other pollution sources (like increases in ammonia content or presence of toxic chemicals). Typically, haematological parameters are non-specific in their responses towards chemical stressors. However, it is well known that toxic substances can significantly damage the haematological system of fish (van der Oost et al., 2003). Some changes may be the result of a disorder in erythrocyte cell membrane permeability and/or the result of the activation of protective mechanisms. These mechanisms may include the release of erythrocytes from blood deposits and/or from haemopoetic tissues into the blood stream (Svodová et al., 1994). On the other hand the haematocrit gives an indication of the hemopoetic activity of the animal. Abnormal haematocrit also can indicate nutritional deficiencies, the presence of disease-causing microorganisms, and other health aberrations (Blaxhall, 1972 in Anderson, 1990). Therefore, haematology may provide important information on the general physiology and health status of the organisms living under environmental stress. It is difficult to determine during field studies whether a biochemical response reflects an adverse condition to the fish or an adaptational change in response to environmental stress. Health impairment should be more accurately determined from observations at several levels of biological organization (genetic, biochemical, histological, etc) (Adams, 2002). This clearly indicate the need for future studies to determine incidence of different environmental stressors on fish health. Further work is underway to experimentally evaluate the effect of different environmental conditions on C. paleatus, which should provide answers to the remaining questions. Meanwhile, the evaluation of haematology can be used as a first indication of fish living under environmental stress arising from a change in water quality. Acknowledgements This work was supported by grants from the Agencia Nacional de Promoción Cientı́fica y Técnica (FONCyT/PICT-99/13-7143), the Agencia Córdoba Ciencia, and the Alexander von Humboldt Foundation. J. Cazenave has a fellowship from the Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (National Research Council – CONICET). D. Wunderlin is member of the research career of CONICET. The authors wish to thank G. Bianchi, F. Marraro and A. 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