THE EFFECTS OF PURE MICROCYSTIN LR AND BIOMASS OF BLUE-GREEN ALGAE ON BLOOD INDICES OF CARP ( Cyprinlls carpio L . )

Navratil S., M. Palikova. V. Vajcova: The Effect of PlIreMicrocystin LR and Biomass of Bille-green Algae Oil Blood Indices of Carp (Cyprinus carpio L.). Acta vet. Bmo 1998, 67: 273-279. The aim of this work was to tind out how the administration of the Microcystin LR and the biomass of blue-green algae with known amount of the Microcystin LR to juvenile carp influences indices of the red blood cells and activities of plasma enzymes. The experimental fish were given either pure Microcystin LR or the biomass of blue-green algae with known amount of Microcystin LR. The administration of pure Microcystin LR at the dose of 400!lg per I kg of body mass was conducted intraperitoneally, the biomass was applied per os. Microcystin LR volume in single doses was 3. 300. 600 and 1200 Ilg per I kg of body mass. After 24 and 48 h blood was taken from fish by cardiac punction. The following indices of the blood were evaluated: the red blood cell count (RBC). haematocrit (PCV), haemoglobin (Hb). mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH). Activities of alanine aminotransferase (AL T). aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) and total plasma proteins (TPP) were measured. Significant changes (p ::; 0.05 or p ::; 0.0 I) of RBC, PCV, Hb, MCV and MCH were found only after the application of the biomass of blue-green algae. These changes were usually within the physiological range of values variation or slightly under this range. The activities of ALT. AST and LDH were mostly increased after all fonns of administrations. The rate of increase depended on the route of administration, character of the material and amount of the toxin. A highly signit'icant increase (p::; 0.01) of the activities of ALT (60.07 ± 36.89 !lkat '1-1, controI2.98± 1.58!lkat '1-1). AST(38.91 ± 15.5lllkat . I-I, control 4.83 ±3.47 !lkat '1-1) and LDH (303.2 ± 110.7 Ilkat . I-I. control 10.4 ± 5.1 Ilkat . 1,1) was detected 24 h after i.p. administration of pure Microcystin LR. Significant (p ::; 0.05) increase of the activities of ALT was detected after oral administration of 3, 300 and 600 Ilg of Microcystin LR per I kg of body mass. highly significant (p::; 0.01) increase was detected after oral administration of 1 200llg. Significant (p ::; 0.05) or highly significant (p::; 0.0 I) increase of the activities of AST and LDH was detected only after oral administration of 1 200 Ilg of Microcystin LR. These changes were above the range of the normal values variation. The volume ofTPPwassignit'icantlydecreased (p::; 0.01) only 48 h after the administration of pure Microcystin LR (19.38 ± 4.90 g . I-I. control 30.68 ± 4.10 g . I-I). This change was at the limit of the normal values variation. Our investigation found out the intluence of the biomass of blue-green algae to indices of red blood cells and verified the intlllence of Microcystin LR and the bio"mass o{bllle-green algae to acti\'ities of plasma enzymes. Fish. evanotoxills. erythrocytes, plasma ell~ymes, total plasma proteill The blue-green algae can produce and incidentally release into their environment substances having a biological activity such as enzymes, vitamines, toxins, Address for correspondence: Doc. :-..tVDr. S :\a\Titil. esc. Department ad Game. Fish and Bee Discase<; Univer<;ity ofVt'terinary and Pharmaceutical Sdences. Palark.cho 1-3 612 -l.~ Sma. Czech Republic Phone: +420 5 -l-l56 2586

extracelullar polysaccharides.attractants, amino acids and other organic acids, antibiotics and hormones.Toxins of the blue-green algae (cyanotoxins) are secondary metabolic products (Marsalek and Turanek 1996).They may be divided according to various points of view.Carmichael (1992) divides the cyanotoxins according to methods of detection into cytotoxins and biotoxins.The cytotoxins are toxic to cell lines.The biotoxins are toxic to animal individuals.The biotoxins may be divided according to their biological activities into neurotoxins, hepatotoxins, cytotoxins, genotoxins, immunotoxins and embryotoxins (Marsalek and Turanek 1996).Biological activities of these toxins are often combined and a population of a single species of blue-green algae may be producing several toxins (M a r s a Ie k and Turanek 1996).
There are a number of different hepatotoxins produced by species and strains within the genera Anabaena, Cylindrospermopsis, Microcystis, Nodl/faria, Oscillatoria.Nostoc, Aphani;:.omenon,Gfoeotrichia and Coefosphaeriul1l.Hepatotoxins are the most common of the cyanobacterial toxins and the ones which are most commonly involved in acute toxicoses.They are slower-acting than the neurotoxins.Death can occur anywhere between 10 min to a few d after intoxication depending on a number of factors including size of animal, species, toxin dose, etc. (Re s so met al. 1994).Clinical signs in homoiotherm vertebrates are characterized by weakness, inappetence and shivering.There are necrobiotic processes in enlarged liver (Falconer and Yeung 1992).Kidneys are enlarged, too.Activities of alanine aminotrasferase (AL T), glutamine trans peptidase (GTP) and alkaline phosphatase (ALP) in blood serum are increased (Marsalek and Turanek 1996).The activity of gamma glutamyl transferase (GGT) is increased in man after drinking water from sources with massive development of water flowers of blue-green algae (Falconer et al. 1983).A suitable indicator of liver damage in sheep is glutamate dehydrogenase (GLDH).
The activities of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) are increased, too (C ar b is et al. 1994).R a be r g h et al. (1991) described an increase of activities of AST, ALD and LDH after i. p. administration of microcystin LR to the carp.Since there is no information available on possible changes of the red blood indices in fish exposed to or treated with microcystins, we decided to elucidate these questions.

Materials and Methods
A total of 83 juvenile carp with the average body mass of 168g (110 -380g) were used in this study.The fish had been kept at least for five days before the experiment at 18 °c in laminated 1m 3 tanks equipped with aeration.
The fish v. ere fed commercial pelleted food for carp.Feeding of experimental fish had been always finished two days before the experiment.They experimental and control groups consisted of 6 -8 individuals each.The experiments lasted 24 and 48 h.For the i.p. administration we used pure Microcystin LR (Veterinary Research Institute.Brno) in a solution of HEPES at a dose of 400)Jg per kg of b.m. (I ml containing 100)Jg of Microcystin LR).Three kinds of biomass with known amount of Microcystin LR were used for oral administration.Analysis of the biomass was conducted by means of HPLC.Individual doses of the biomass were applied according to the content of ~1icrocystin LR orally in doses of 3,300.600and I 200 )Jg per kg of body mass.The biomass was diluted in distilled water to obtain a volume of 3.0 -3.6 m1.It was divided into two doses applied in 24-h intervals as to obtain a final dose of I 200)Jg of Microcystin LR.The red blood cells count (RBC), haematocrit (PCV), haemoglobin (Hb), mean corpuscular haemoglobin concentration (MCHC).mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were determined by standard methods according to S v 0 bod 0 v a et a1.(1986).Total plasma proteins were determined using the commercial Bio-Lachema-Test (Lachema Diagnostika.Czech Republic).The activities of AST, ALT and LDH were determined by commercial tests of HUMAN (Germany).
The results were evaluated by Student's t•test.

Results
The results are given in the Tables 1,2,3,4 and 5  Statistically significant (p::; 0.05 orp::; 0.01) changes ofRBC, PCV, Hb, MCHC, MCV and MCH were found only after oral administration of the biomass containing Microcystin LR.These changes were influenced by the dose and the time after treatment.They have often shown various trends.RBC significantly (p ::; 0.05) decreased after low but also high doses (1 200 fJg of Microcystin LR), while they were significantly (p ::; 0.05) increased 48 h after the dose of 600 fJg per 1 kg of body mass.PCV was significantly (p ::; 0.05 or p ::; 0.01) decreased after low 24h 48h TPP were decreased only after i.p. of pure Microcystin LR (p:S; 0.01).This change was on the limit of the normal values variation.
The activities of ALT, AST and LDH were practically increased after all routes of administration.The rate of increase depended on the method of administration, the character of the material and the amount of the toxin.A highly significant increase (p:S; 0.01) of ALT, AST and LDH activities was detected 24 h after i.p. administration of the pure Microcystin LR.Significant (p :s; 0.05) increase of the activities of ALT was detected after oral administration of 3, 300 and 600 J..lg of Microcystin LR., highly significant (p :s; 0.01) increase was detected after oral administration of 1 200 J..lg of Microcystin LR.Significant (p:S; 0.05) or highly significant (p:S; 0.01) increase of AST and LDH activities was detected only after oral administration of 1 200 J..lg of Microcystin LR per 1 kg of body mass.These changes were above the range of the normal values variation.There is no information in the literature on changes ofRBC, PCV, Hb, MCHC, MCV and MCH in fish after the administration of microcystins.Significant changes of these variables only after oral administration of Microcystin LR in biomass of blue-green algae may be explained by a certain amount of other substances present in this biomass.Various trends of RBC and PCV may be explained by patho-morphological findings.These comprise extensive haemorrhage in the skin, in the eyes, in hepatopancreas and in swim bladder.These blood losses were compensated for by release of young stages of erythrocytes after the dose of 600 J..lg of the Microcystin LR per 1 kg of body mass.However, this compensational mechanism was probably exhausted after the dose of 1 200 J..lg per kg of body mass.The changes of MCV and MCH corresponded to the described mechanism.These changes of the red blood cell indices were mostly ranging within the variation of physiological values or lower.PCV, Hb, MCHC, MCV and MCV were ranging below this range also in control fish.Their evaluation is therefore very difficult.
The activities of ALT, AST and LDH were mostly increased.These findings correspond with literature data (Rabergh et a!. 1991).The changes were ranging above the normal values (Svobodova eta!' 1986).
It is very difficult to explain the decreased the total plasma protein.S vobodova et al. (1997) described similar changes after the administration of some toxic substances.
Our investigation elucidated the intluence of the biomass of blue-green algae on red blood cell indices and verified the influence of Microcyst in LR and the biomass of blue-green algae on activities of plasma enzymes.

Table 2
) increased 24 h after the dose of 600 J.lg per I kg of body mass.No changes of PCV were found after the dose of I 200 J.lg of Microcystin LR.Hb was significantly (p :s; 0.05 or p :s; 0.0 1) decreased only after the dose of 3 and 300 J.lg per kg of body mass.The MCHC was significantly (p:S; 0.0 1) increased only 48 h after the dose of 1 200 J.lg per kg of body mass.TheMCV was significantly (p:S; 0.01) decreased 48 h after the dose of 600 J.lg per kg of body mass.This variable was significantly (p:S; 0.05) increased also after the dose of 1 200 J.lg of Microcystin LR.MCH was significantly (p :s; 0.05) decreased 48 h after the dose of