Erythrocytes and Hemoglobin of Fish: Potential Indicators of Ecological Biomonitoring

Abstract

Anthropogenic pollution of the freshwater basins is a serious environmental problem. This has necessitated the search for different approaches to the detection of different pollutants in water bodies. Many authors point out that the hematological parameters of freshwater fish are sensitive to the action of various pollutants in freshwater basins. This chapter summarizes the results of studies on the effects of current water pollutants (heavy metals, organic matter, etc.) on erythrocytes and hemoglobin in fish. An analysis of the possibility of the use of erythrocyte damage and the change in the hemoglobin content of the tested animals for the purposes of ecological biomonitoring of freshwater pollution will be made.

1. Introduction

The state of the environment is signaled by a group of different biological species known as bioindicators. They are responsible for demonstrating the impact of different types of pollutants. The selection of a specific biological species as an indicator should be done considering its sensitivity to various changes in the environment [1]. Fishes are valuable species as bioindicators for the pollution of water bodies. A very large number and essentially different methods can be applied to them, which allow assessment of the severity of toxic effects by determining the accumulation of toxic substances in the tissues, by using histological and hematological approaches. Thus, bioindication using fish represents a good tool for biomonitoring, regarding both pollution and river engineering aspects, e.g., river restoration and management [2].

In this chapter, our and other authors’ summarized data on the possibilities of using the erythrocyte indicators of teleost fishes for the purposes of biomonitoring will be presented.

2. Erythrocytes and hemoglobin in teleost in the norm and under toxic effects

2.1 Erythrocytes and hemoglobin in teleost in the norm

The erythrocytes of teleost fishes have a similar morphology to the erythrocytes of other non-mammalian vertebrates. They are nucleated cells with an elliptical to oval shape. Their cytoplasm is eosinophilic, and the nucleus is centrally located, deeply basophilic, with an oval to elliptical shape. Erythrocyte sizes range from 102 to 800 fl [3, 4]. Moderate anisocytosis and polychromasia are normal in teleost species.

Their life span is 13–500 days. The number of erythrocytes in the peripheral blood depends on many factors—fish activity, water temperature, and dissolved oxygen concentration, as well as other environmental factors and shows significant seasonal variability. Furthermore, it depends on age, sex, nutritional and reproductive status, and may differ between populations of the same species. It typically ranges from 0.5–1.5 × 10⁶/mm³ in less active species to 3.0–4.2 × 10⁶/mm³ in more active species. Antarctic icefishes, adapted to cold and well-oxygenated water, do not have erythrocytes.

The main erythropoietic organ in teleost is the head kidney. Erythropoiesis is similar to that of other vertebrates and involves the same precursors. Characteristically, in teleost fishes, the barrier between the hematopoietic tissue and the circulatory system is weak, and therefore in the circulating blood, many immature red blood cells are found, often constituting more than 10% of all erythrocytes. Immature erythrocytes are round rather than oval in shape, their cytoplasm is blue-stained, and their nucleus is larger and heterochromatic.

As in other vertebrates, fish erythrocytes contain tetrameric hemoglobins, but with different oxygen affinities. It is lower in species living in well-oxygenated water than in those exposed to hypoxia. Several hemoglobin isoforms with different oxygen affinities are often present in the blood of fish, which is considered an adaptation to variable oxygen concentration in the water. Fish erythrocytes are sensitive to environmental pollution, and their morphological assessment can be used as a bioindicator of toxicity [3].

2.2 Hematological disorders in teleost as a biomarker for water pollution

Hematologic studies in fish have been performed for more than 70 years now. Recently, they have gained great importance because they are an effective and sensitive indicator for evaluating physiological and pathological changes caused by natural or anthropogenic pollution of water sources. Hematological indicators are therefore considered an important tool to identify the functional state of the body in response to various stressors [5]. However, for the correct interpretation of the obtained results, it is necessary to consider a set of variables, such as reproductive cycle, age, sex, feeding behavior, stress, nutritional status, and water quality, as well as the habitat of the species, since poikilothermic animals are under the impact of environmental changes [6].

Hematological disorders in boteleost include anemia, polycythemia, abnormal morphology, and cytoplasmic and nuclear inclusions [4], and according to Witeska [3], they also include changes in the size and color of erythrocytes.

Anemia is well described in teleost. It is defined as a decrease in hemoglobin concentration below the reference level of an established threshold for a population of healthy organisms [7]. In teleost it is extremely difficult to establish reference values for hematological parameters, due to their strong variation caused by their poikilothermism and high sensitivity to the action of various factors (age, sex, water quality, photoperiod, season, diet, etc.). For example, hematocrit values strongly depend on the level of biological activity of the fish. Thus, actively swimming species such as tuna and other pelagic species generally have much higher hematocrit values than bottom-dwelling fish such as flatfish. Therefore, hematological parameters are very relative, and there is no clear definition of normal and abnormal values [8]. Therefore, anemia in fish is usually recognized by a significant reduction in red blood parameters compared with the values obtained for a reference group of animals not exposed to the specific damaging factor. According to Clauss et al. [4], hematocrit values (PCV) below 20% are indicative of anemia. The hematological parameters that are affected when anemia occurs in fish are mainly: red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and mean corpuscular hemoglobin concentration (MCHC). Examination of these indicators (along with WBC values) is recommended for routine monitoring of fish health status in fish farms [9].

Anemia in teleost fishes occurs under both natural and aquaculture conditions and can be caused by various effects, including the toxic effect of a number of pollutants in water bodies (nitrates, pesticides, heavy metal ions, cyanobacterial toxins, etc.) [3].

Depending on the causes of anemia, it can be hemorrhagic, hemolytic, or hypoplastic. Depending on the manifestations, the different types of anemia in fish are divided into microcytic, normocytic, and macrocytic (depending on the size of the erythrocytes), as well as hypochromic or normochromic (depending on the hemoglobin concentration) [4].

2.3 Hematological disorders of fish under toxic effects

2.3.1 Hematological disorders caused by the action of nitrites

Toxic concentrations of nitrites in fish breeding ponds can occur in intensive aquaculture systems as a consequence of high stocking density and feeding intensity. In natural water bodies, elevated nitrite levels usually result from sewage pollution. Prolonged exposure to nitrite induces oxidative stress in fish [10]. According to Witeska, nitrite intoxication can result in anemia, followed by oxidation of hemoglobin to methemoglobin [3]. Da Costa et al. [11] reported methemoglobinemia in Colossoma macropomum, caused by nitrite intoxication, accompanied by hemolytic anemia due to reduced erythrocyte life span. Erythrocyte hemolysis with prolonged exposure to nitrites results from a high expenditure of cellular energy to reduce methemoglobin, which shortens the average life span of red blood cells [10]. There is much evidence to show that the symptoms of anemia caused by nitrites can be different, which depends both on the severity of intoxication and on the biological species of the fish. Witeska [3] reported that in different types of fish, there can be a decrease in different parameters. Thus, in Dicentricus labrax, only the hemoglobin content tends to decrease, but in other species decrease is observed also in RBC and/or hematocrit, and in Colossoma macropotum and Sander vitreus, increase is even observed of MCHC (probably as a compensatory reaction to the impaired oxygen transport from the blood). According to Zhelev et al. [12], elevated nitrate levels in a natural water basin (Sazliyka River, Bulgaria) induce erythrocytosis and hyperchromia in adult Carassius gibelio females.

Data on the effect of nitrate pollution of water bodies on the morphology of erythrocytes of fish are scarce.

2.3.2 Hematological disorders caused by the action of metals and microelements

The impact of toxic concentrations of metal ions in water bodies causes morphofunctional changes in erythrocytes, which can result in various negative effects: direct damage to erythrocyte cells, reduction of their life span, acceleration of hemolysis or inhibition of erythropoiesis.

There is a large number of publications on this subject in the literature, and in most cases the authors describe and explain the mechanisms of action of each individual heavy metal toxicant. According to Fedeli et al. [13], copper induces oxidative damage to erythrocytes and increases their susceptibility to hemolysis. Our studies confirm this, showing that the hematocrit of C. gibelio is reliably reduced even by the action of copper at concentrations lower than the threshold limit values (TLVs) for freshwater bodies (0.1 mg/l), but by the action of high concentrations (2.0 mg/l), its values are comparable to those of the controls. In our opinion, this is due to a compensatory increase in erythropoiesis, as a result of which young erythrocytes with insufficient hemoglobin content are found in the peripheral blood of the fish. Enhanced erythropoiesis under the action of high concentrations of copper was indirectly proven by histological examination of the spleen of C. gibelio [14]. Unlike the control group, in fish exposed to 2.0 mg/l copper sulfate, no presence of hemosiderin was detected in the spleen. This is an indication of mobilization of iron reserve in fish organisms. The type of anemia was different under the influence of different concentrations of copper sulfate: under the influence of concentrations of 0.1 and 2.0 mg/l, it was microcytic and hypochromic, under the influence of a concentration of 0.5 mg/l, it was microcytic and normochromic, and under the influence of a concentration of 1.0 mg/l, normocytic and normochromic.

Som et al. [15] reported a decrease in the amount of erythropoietic precursors in the head kidney of Labeo rohita associated with the level of copper intoxication. Cadmium, like copper, damages hematopoietic precursors in the kidney of Ictalurus nebulosus [16]. According to Kondera and Witeska [17], this may be due to an increase in apoptosis in precursor cells in copper or cadmium intoxication.

Results of our study [18] allow indicating some features related to changes in the sizes of erythrocytes and their nuclei under the influence of different concentrations of copper in water. Under the influence of low concentrations of copper (even below TLV), a decrease in the size of the cells and of their nuclei is identified. However, under the influence of higher concentrations of copper, an increase in the size of the cells is caused, which can be explained by the activation of compensatory processes.

The bases of Prise-Jones curves gave the following results:

• At the big diameter of erythrocytes, anisocytosis in concentrations of 0.1 and 1.0 mg/l was found.

• At the small diameter of erythrocytes, there is a widening of the curves bases, especially well expressed in 1.0 mg/l concentration.

• At the big diameter of the nuclei, there was a tendency of decreasing anisocytosis.

• At the small diameter of the nuclei, the fluctuations are near to the values of the controls.

A conclusion can be made that copper, even in concentrations below TLV for waters, causes atrophic changes in the erythrocytes of C. gibelio. This fact can be used for the purposes of ecological biomonitoring of copper contamination of water bodies.

According to Akahori et al. [19] and Gabryelak et al. [20], zinc induced hemolytic anemia in fish, which resulted from its adverse effect on the lipid layer of the erythrocyte membrane. In our studies, a negative effect of the action of different concentrations of zinc on different hematological parameters of C. gibelio was found. Changes were found in RBC, erythrocyte morphology, and size, Hb, PCV, MCV, MCH, and MCHC. From the analysis of the obtained results, it can be concluded that zinc causes hypochromic anemia even in low concentrations (below TLV). A trend of transition from microcytic to macrocytic type of anemia was observed with increasing zinc concentration. Witeska and Kościuk [21] reported that hematocrit and frequency of abnormal erythrocytes in Common carp increased after a 3-hour exposure to 20 mg/dm⁻³ zinc (ZnO).

The changes in the shape (poikilocytosis) and in the sizes of erythrocytes were also different, under the action of the different concentrations of zinc. The beginning of the processes of karyopyknosis, hypertrophy and anisocytosis were detected [22, 23, 24]. Similar to the effect of copper, compensatory changes in erythrocyte size and erythrocyte indices were observed here as zinc concentration increased. In this case, similar to the effect of copper, an absence of hemosiderin was found in the spleen of fish subjected to the action of 2.0 mg/l zinc, which is an indication that a similar process of mobilization of the body’s iron reserves is taking place.

Lead reduces the activity of ALA-D (a key enzyme involved in heme synthesis) [25, 26]. In our studies, we found that carp lead caused erythrocyte deformations with a clear upward trend proportional to increase in concentration [27]. After short-term exposure (96 h LC50), lead caused increase in frequency of morphological anomalies in carp erythrocytes over the entire experimental period. Cromatin condensation at the nucleus border and nuclear malformation were the most common anomalies. No complete recovery took place until the end of the experiment [28].

The effect of other metal pollutants in water (nickel, mercury, chromium, selenium, etc.) has been less studied. In our studies, it was found that nickel causes erythrocyte changes in carp, including with concentrations far below TLV [29]. Its main effect is damage to the cell membrane (which causes the appearance of poikilocytosis) and changes in the shape of cell nuclei. Under the influence of concentrations above TLV, necrotic changes occur in the nuclei (karyopyknosis). The established erythrocyte changes may be explained by the finding of De Luca et al. [30] that nickel affects cellular metabolism, causing an enhancement of oxidative stress in erythrocytes. In Cirrhinus mrigala, nickel causes significant decrease of RBC, Hb, and PCV [31]. According to Moosavi et al. [32], hematological and biochemical parameters can be used as an indicator of nickel-related stress in fish on exposure to elevated nickel status.

In general, it can be summarized that metal pollutants in water bodies cause a decrease in the values of hematological parameters of fish—most often (Hb, RBC, and PCV, and in some cases also MCV, MCH, and MCHC), but for some metals, there may be an increase in MCV (under the influence of copper and chromium) or in MCH under the influence of cadmium [3].

In natural conditions, hematological disorders can be caused by the action of mixtures of metal pollutants, and their effect on fish is the result of the combination of mechanisms of action of the contained metal pollutants in water bodies [33].

In 2007–2008, we conducted a study of hematological parameters of three species of freshwater fish—bleak (Alburnus alburnus L.), common rudd (Scardinus erythrophtalmus L.), and European perch (redfin perch) (Perca fluviatilis L.), inhabiting Studen Kladenets Dam (Arda River area, Bulgaria) [34]. Elevated levels of manganese and lead have been found in the waters of the dam. Anemic changes were found in the blood of the three species in both the summer and winter seasons. Each species, however, developed a different type of anemia—macrocytic and hyperchromic in bleak, hypochromic type in common rudd, and normochromic anemia, which developed into microcytic normochromic anemia in winter in redfin perch. Morphological examination of the erythrocytes of all three species of freshwater fish demonstrated a wide range of erythrocyte changes, as well as a large number of “amitotic” erythrocytes in the peripheral blood of common rudd and redfin perch. These changes showed inter-species differences. Later Omar et al. [35] found that high concentrations of heavy metal mixtures (Cu²⁺, Zn²⁺, Pb²⁺, Fe²⁺ and Mn²⁺) have a potential genotoxic effect on erythrocytes, in studies of cultured and wild Nile tilapia, Oreochromis niloticus and mullet Mugil cephalus, inhabiting a water body, contaminated with agricultural wastewater and domestic untreated water discharges (Lake Qaroun, Egypt). It has been shown that the genotoxic effect (measured by a micronucleus test) depends proportionally on the type and concentration of pollutants in the water body. In addition to micronuclei, other nuclear and cellular abnormalities have been reported in fish erythrocytes—lobular, vesicular, binucleate, dentate, budded, vacuolated and other deformed nuclei, karyolysis, nuclear retraction, as well as microcytes and vacuolated cytoplasm. Degradation of the studied aquatic habitats reveals species-specific effects.

In 2014/2015, a study of the cytometric characteristics of the erythrocytes of C. gibelio Bloch, 1782, and Rutilus rutilus (Linnaeus, 1758), inhabiting the Zaporizhya Reservoir (Ukraine), was conducted [36]. The species characteristic of accumulation of heavy metals in the body of carp fish was also investigated. It was established that young specimens of C. gibelio and R. rutilus accumulate essential elements, especially zinc, to a greater extent than adults. The level of intensity of occurrence of erythropoiesis was higher in young fish of both species. Specific features of the cytometric characteristics of fish erythrocytes were identified: the relative amount of mature red blood cells predominated in R. rutilus, and the area of mature red blood cells was significantly higher in C. gibelio. In addition, a significantly higher percentage of polychromatophilic normoblasts was found among the immature forms of red blood cells in juvenile R. rutilus.

According to Zhelev et al. [12], elevated levels of heavy metals in a natural water basin (Topolnitsa River, Bulgaria) induce erythrocytosis and microcytic hypochromic anemia in adult female C. gibelio.

2.3.3 Hematological disorders caused by the action of pesticides

Various pesticides used in agriculture and draining into water bodies have also been reported as agents causing various hematological disorders [3, 37, 38]. According to Mikula et al. [39], the pesticide alachlor in subchronic doses induced pathological changes in hematopoietic organs of carps (Cyprinus carpio L.) As a result, all hematological parameters were lower compared with the control group fish, except for PCV. Dogan and Can [40] suggested that dimethoate probably also has a damaging effect on fish erythropoietic tissue. In Oncorhynchus mykiss, in sublethal concentrations this pesticide caused a significant decrease in RBC, Hb, PCV, MCV, and MCH, indicating the occurrence of microcytic hypochromic anemia.

As reported by Ghaffar et al. [41], Fipronil causes a decrease of RBC, Hb, and PCV. It is assumed that the decrease of Hb may be due to its oxidation to methemoglobin, poorer gas exchange, and damage caused by free radicals. The authors assume that anemic changes are a marker of the weak role of hematopoietic tissues, inappropriate osmoregulatory mechanisms, and increased damage of red blood cells in hematopoietic organs. In another case, in L. rohita, exposure to Fipronil for 9 days at a dose of 0.03–0.15 mg/L showed various nuclear changes in addition to RBC reduction in erythrocytes.

According to Tahir et al., [37] a large number of pesticides (cypermethrin, triazophos, butachlor, DDT, BHC, aldrin, dieldrin, chlordane, permethrin, cypermethrin, karate, delmethrin sulfane, endosulfan, etc.) cause anemic changes in various teleost, with L. rohita being a suitable model for studying the damaging effects of pesticides.

In some cases, the hematopoietic system of fish has the ability to compensate for the action of pesticides. This was found by Hii et al. [42], who reported that endosulfan induced in Monopterus albus a short-term increase of RBC, Hb, and PCV, which is followed by a significant decrease in the values of these indicators, due to the damaging effect of the pesticide on the erythrocyte membrane.

Some pesticides have also been found to cause DNA damage in fish erythrocytes [37], in addition to affecting the main hematological parameters (RBC, Hb, and PCV, etc.). Thus, naphthalene-2-sulphonate caused genotoxic effects on Channa punctatus, which was detected by comet assay and micronucleus assay. A mixture of endosulfan and Chlorpyrifos can also induce DNA damage in the erythrocytes of O. niloticus, which was found by Ambreen and Javed [43] by comet assay.

The data cited above show that the study of erythrocyte indicators in natural and laboratory conditions is a good opportunity to establish the harmful effect of newly synthesized pesticides, which would help to determine their harmful effects, and based on these studies, more less toxic and environmentally friendly chemicals could be used [37].

2.3.4 Hematological disorders caused by the action of cyanobacterial toxins

Cyanobacteria inhabit both freshwater and saltwater bodies throughout the world. In case of excessive growth leading to eutrophication, they can produce specific toxins (cyanotoxins) in quantities causing toxicity, including in humans. Cyanotoxins are cyclic peptides and alkaloids. Cyclic peptides include microcystins and nodularins. Alkaloids include anatoxin-a, anatoxin-a(S), cylindrospermopsin, saxitoxins (STXs), aplisiatoxins, and lingbiatoxin [44]. The effects of microcystin have been best studied on teleost. According to Witeska [3], there are species differences in the sensitivity of fish to the action of this toxin. According to Zhang et al. [45], microcystin in high doses caused in Carassius auratus a significant decrease of RBC, Hb, and PCV in the high-dose group and Hb, while the erythrocyte sedimentation rate (ESR) was significantly increased, indicating the occurrence of normocytic anemia. No significant deviations were found in MCV, MCH, and MCHC. Under the action of low doses of the toxin, hematological disorders are reversible. According to the authors, such hematological disorders are due to impairment of erythropoiesis. In the same species, Zhou et al. [46] found that microcystin significantly increased lipid peroxidation as well as the activity of antioxidant enzymes. These changes cause erythrocyte malformation, cell membrane damage that increases hemolysis, i.e., there is another mechanism causing anemia (oxidative damage to erythrocytes).

Based on data, provided by Navratil et al. [47], microcystin induces in Cyprinus caprio hemorrhagic anemia, which is a consequence of extensive hemorrhages in the skin, hepatopancreas, and eyes. On the other hand, however, there are data that microcystin formed both by natural eutrophication and by cyanobacterial isolates does not cause any anemic changes in Cyprinus caprio (according to Witeska [3]).

3. Conclusion

Teleost fishes possess a high compensatory potential for improving oxygen transport under the influence of adverse environmental factors [48]. It includes synthesis of erythrocytes and hemoglobin, amitotic division of erythrocytes, release of erythrocytes from blood depots (spleen and main kidney), as well as activation of hematopoiesis.

A low-cost response to oxygen deficiency is realized by increasing the MCV. The increase in cell volume, together with the increase in erythrocyte pH, improves oxygen transport under hypoxic conditions [3]. They result from adrenergic activation of Na⁺/proton exchange across the erythrocyte membrane [49]. Knowledge, on the one hand, of anemic and genotoxic effects of aquatic pollutants, and on the other hand, of adaptive-compensatory responses to the action of aquatic toxicants, is a good basis for using a large set of freshwater bony fish for ecological biomonitoring purposes.

Acknowledgments

This chapter is published with support of NPD-Plovdiv University “Paisii Hilendarski” under Project № PP22-BF-005.

Conflict of interest

The authors declare no conflict of interest.