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Effects of cyanobacterial biomass on the Japanese quail
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Toxicon 49 (2007) 793–803 www.elsevier.com/locate/toxicon
Effects of cyanobacterial biomass on the Japanese quail B. Skocovskaa, K. Hilscherovab, P. Babicab, O. Adamovskyb, H. Bandouchovaa, J. Horakovaa, Z. Knotkovaa, B. Marsalekb, V. Paskovab, J. Pikulaa, a
Department of Veterinary Ecology and Environmental Protection, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1-3, 612 42 Brno, Czech Republic b Centre for Cyanobacteria and their Toxins, Institute of Botany, The Academy of Sciences of the Czech Republic and RECETOX, Masaryk University, Kamenice 126/3, CZ62500, Brno, Czech Republic Received 22 September 2006; received in revised form 29 November 2006; accepted 29 November 2006 Available online 8 December 2006
Abstract Mortality of wild aquatic birds has recently been attributed to cyanobacterial toxins. Despite this, no experimental studies on the effects of defined doses of microcystins administered orally to birds exist. In this experiment, four groups of male Japanese quails daily ingesting 10 ml of Microcystis biomass containing 0.045, 0.459, 4.605 or 46.044 mg of microcystins, respectively, for 10 and 30 days, showed no mortality. Histopathological hepatic changes in birds after the biomass exposure included cloudy swelling of hepatocytes, vacuolar dystrophy, steatosis and hyperplasia of lymphatic centres. On subcellular level, shrunken nuclei of hepatocytes containing ring-like nucleoli, cristolysis within mitochondria and vacuoles with pseudomyelin structures were present. Vacuolar degeneration of the testicular germinative epithelium was found in two exposed males. Statistically significant differences in biochemical parameters were on day 10 of exposure only. They comprised increased activities of lactate dehydrogenase and a drop in blood glucose in birds receiving the highest dose of the biomass. Principal component analysis revealed a pattern of responses in biochemical parameters on day 10 that clearly separated the two greatest exposure groups from the controls and lower exposures. The results indicate that diagnosis of microcystin intoxication solely based on clinical biochemical and haematological parameters is hardly possible in birds. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cyanobacterial water bloom; Avian toxicity tests; Peroral exposure; Biochemistry; Histopathology; Electron microscopy; Mitochondria; Microcystis; Microcystins; Coturnix coturnix japonica
1. Introduction Water blooms of cyanobacteria represent a problem throughout the world due to the eutrophication of the aquatic environment in particular (Chorus and Bartram, 1999; Marvan and Marsalek, Corresponding author. Tel.: +420 5 41562655; fax: +420 5 49243020. E-mail address: [email protected] (J. Pikula).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.11.032
2004). The most serious consequences of bloom formation are poisonings by secondary metabolites of cyanobacteria—cyanotoxins (Carmichael, 1992), which fall into five groups (Wiegand and Pflugmacher, 2005) based on their primary toxicological target, i.e. hepatotoxins (microcystins and nodularins), neurotoxins (anatoxin-a, anatoxin-a(S), saxitoxins), cytotoxins (cylindropspermopsin), dermatotoxins (aplysiatoxins and lyngbyatoxin) and irritant toxins (lipopolysaccharides).
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A wide range of aquatic organisms including invertebrates, fish, mammalian and avian species may be exposed to cyanotoxins via consumption of or dermal contact with water contaminated by toxic cyanobacteria. A number of poisonings in animals have been attributed to cyanobacterial toxins (Duy et al., 2000). Well known is an incident reported in Switzerland concerning fatalities in cattle kept on alpine pastures (Mez et al., 1997) as well as a dog poisoning at Loch Inch, Scotland (Edwards et al., 1992). Toxic effects of cyanobacterial products have also been recognised in avian species. Mortalities of wild birds attributed to toxigenic taxa of cyanobacteria have been reported (Alonso-Andicoberry et al., 2002; Chittick et al., 2002; Henriksen et al., 1997; Krienitz et al., 2002; Matsunaga et al., 1999; Onodera et al., 1997). For example, mass deaths of wild birds including about 20 ducks (Anas poecilorhyncha zonorhyncha) have been attributed to the sudden appearance of toxic cyanobacteria Microcystis aeruginosa in a pond in Nishinomiya (Japan) in 1995 (Matsunaga et al., 1999). The biomass of cyanobacteria contained high levels of microcystins and autopsy revealed icteritic livers and necroses of liver tissue giving thus indication of the presence of hepatotoxins. Several bird deaths in Canadian and Belgian lakes have also been linked with occurrence of cyanobacterial blooms producing microcystins (Wirsing et al., 1998; Park et al., 2001; Murphy et al., 2003) or anatoxin-a (Murphy et al., 2000). Another case of avian mortality associated with cyanotoxins was the mysterious deaths of lesser flamingos (Phoenicopterus minor) around a thermal Lake Bogoria (Kenya). Birds manifested neurological signs prior to death and hepatotoxic microcystins LR, -RR, -LF and -YR and a neurotoxin (anatoxin-a) were found in the stomach content as well as in faeces. Intoxication is supposed to have occurred through drinking water from the lake and feather grooming performed as a daily activity near hot springs of the lake (Krienitz et al., 2002). Catastrophic mortality due to cyanobacterial toxins has also been reported in wild flamingos from a Spanish national park (AlonsoAndicoberry et al., 2002) and in captive Chilean flamingos in USA (Chittick et al., 2002). Onodera et al. (1997) confirmed that the cause of bird mortality in Danish lakes in 1993–94 was consumption of the cyanobacterium Anabaena lemmermannii containing anatoxin-a(s). Uncharacterized cyanobacterial neurotoxin was probably responsible for lethal avian
vacuolar myelinopathy observed in USA (Wilde et al., 2005). Previous documentation of the toxicity of cyanobacteria in birds has rather been based on indirect evidence such as observation of mortality in several avian species under natural conditions associated with the occurrence of water blooms and long-term stay and foraging of birds at the affected locality, detection of cyanobacterial toxins in the crop and liver and exclusion of alternative causes of mortality. Our intention was to complement field observations published by other workers with results from laboratory experiments. Therefore, the aim of this study was to perform evidence-based avian toxicity tests using standard experimental avian species and evaluate the effects of different doses of cyanobacterial toxins in birds. 2. Materials and methods Effects of cyanobacterial toxins in birds were evaluated according to the OECD 205 Guideline for the testing of chemicals—Avian Dietary Toxicity Test (1984) with some minor modifications to fit our experimental conditions. 2.1. Experimental substances Experimental birds were exposed to homogenate of cyanobacterial biomass dominated by Microcystis sp., which was collected using plankton net (25 mm) from Brno reservoir in autumn 2004. Colonial cyanobacteria of the Microcystis genus (M. aeruginosa 80%, Microcystis viridis 15% and Microcystis flos-aquae 5%) prevailed in the species composition. Concentration of cyanobacteria in experimental biomass was determined by cell counting under a fluorescent microscope and concentration of biomass dry weight was estimated in an aliquote of biomass after drying (50 1C). Cyanobacterial biomass was homogenized by repeated freeze thawing and by ultrasonic probe. Microcystin contents in biomass and homogenate were analysed using HPLC-DAD (Agilent 1100 Series) on Supelcosil ABZ+ Plus column, 150 4.6 mm, 5 mm according to Babica et al. (2006). Concentrations of microcystins in experimental biomass were 141.8 mg/g DW of microcystin-RR, 33.7 mg/g DW of microcystin-YR, 141.7 mg/g DW of microcystin-LR, and 56.1 mg/g DW of unidentified compound possessing microcystin-like UV spectrum. The total concentration of microcystins (including
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unidentified structural variant) in biomass was 373.3 mg/g DW. Four experimental concentrations of biomass were prepared by dilution of biomass homogenate with drinking water, aliquoted into plastic cups and stored frozen. Drinking water and water for the control birds was handled in the same way. After thawing on day of application, homogenate of experimental biomass or drinking water were provided orally to birds. 2.2. Experimental animals The Japanese quail (Coturnix coturnix japonica) is a common experimental bird. Due to its small size, the selection was economic regarding the quantity of the cyanobacterial biomass necessary for the experiment. Experimental quails (4 months old, weighing 205.25 g on average) were randomly divided into groups of five individuals (males only to eliminate gender differences). One control (C) and four exposure groups (E1-4) receiving different daily doses of the cyanobacterial biomass were formed. Commercially available feeds and drinking water were supplied ad libitum. 2.3. Experimental design Considering the daily dose of the biomass, experimental birds received cyanobacteria containing microcystins in amounts presented in Table 1. The highest dose was equivalent to daily consumption of 10 ml of biomass with concentration of 3 108 cell/ml (12334.8 mg DW/L, corresponded to microcystin concentration 4604.4 mg/l). Experimental birds were given the cyanobacterial biomass using a crop probe thrice a day to obtain a daily
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dose, which was the same both in the acute and subchronic exposure. We employed 10-day (acute) and 30-day (subchronic) experiments. Samples of blood for haematology and plasma chemistry profiles were collected one day prior to the start of the experiment and on day 10 (10-day and 30-day experiment), and 20 and 30 (30-day experiment). Blood (1 ml) was collected from the right jugular vein using the Omnican 0.30 12 mm insulin set (Braun, Germany). Whole blood was placed in heparinized tubes (Leciva inj., Prague), centrifuged immediately, and plasma removed and frozen (20 1C). Plasma was analysed using an automated analyzer (CobasMira, Roche) for total protein (TP; g/l), albumin (ALB; g/l), glucose (GLU; mmol/l), creatinine (CREA; mmol/l), uric acid (UAC; mmol/l), aspartate aminotransferase (AST; mkat/l), creatine kinase (CK; mkat/l), lactate dehydrogenase (LD; mkat/l), alkaline phosphatase (ALP; mkat/l), total cholesterol (T-Chol; mmol/l), triglycerides (TG; mmol/l), amylase (AMY; mkat/l), phosphorus (IP; mmol/l), potassium (K; mmol/l) and sodium (Na; mmol/l). Plasma calcium (Ca; mmol/l) concentrations were analysed with an Atomspec analyzer (Hilger 1550). Hematocrit (PCV) was measured using microhematocrit tubes. Haemoglobin (Hb) was determined by the cyanmethemoglobin method, and total red (RBC) and white (WBC) blood cell counts were determined manually using haemocytometer with Natt and Herrick’s solution. Blood smears on a glass slide, prepared immediately after blood collection, were air-dried and stained using May+ Grunwald and Giemsa-Romanowski stains. Two hundred leucocytes were counted for each smear and classified as heterophils, eosinophils, basophils, lymphocytes and monocytes.
Table 1 Daily doses of cyanobacterial biomass administered to birds of four exposure and one control groups and its characteristics Exposure and control groups E4 E3 E2 E1 C
Cyanobacterial biomass
Microcystin structural variants (mg)
Cells
mg of dry weight
MC-RR
MC–unidentif.
MC–YR
MC–LR
Sum of MCs
3 109 3 108 3 107 3 106 —
123.348 12.336 1.233 0.123 —
17.496 1.749 0.174 0.018 —
6.915 0.690 0.069 0.006 —
4.158 0.417 0.042 0.003 —
17.475 1.749 0.174 0.018 —
46.044 4.605 0.459 0.045 —
Birds were given the cyanobacterial biomass or drinking water using a crop probe thrice a day to obtain a daily dose of 10 ml which was the same both in the acute (10-day) and subchronic (30-day) exposure.
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After the experiment, birds were euthanized by decapitation, examined regarding pathoanatomical changes and used to collect tissue specimens (liver, CNS, kidney, spleen, pancreas, heart and both pectoralis muscles) for standard histopathology as well as electron microscopy. Samples for histopathology were fixed in buffered 10% formalin, prepared in a standard way and stained by hematoxylin eosin. Samples for electron microscopy were stored in glutaraldehyde and prepared in a standard way for transmission electron microscopy. Immediately after euthanasia, a second set of tissue specimens were collected for analyses of microcystin concentrations. For these purposes, tissue specimens were placed in plastic bags and deep frozen (80 1C). 2.4. Analyses of microcystin in tissues Microcystin concentrations in tissues of exposed animals were determined after repeated (3 ) extraction of fresh tissue by methanol under sonication. Methanolic extracts were partitioned three times with hexane:water. Methanolic layers were dried (50 1C), residues re-dissolved in distilled water and concentrations of microcystins analysed using direct competitive ELISA. The ELISA assay employed monoclonal antibody MC10E7 (Zeck et al., 2001a, b) and was performed as previously described in Babica et al. (2006). 2.5. Statistical evaluation Statistical analyses were performed with Statistica for Windowss 7.0 (StatSoft, Tulsa, OK, USA). Data normality and homogeneity of variances were evaluated by Kolmogorov–Smirnov test and the Levene’s test, respectively. One-way analysis of variance (ANOVA) and the nonparametric Kruskal–Wallis test were used for statistical comparisons. Values po0.05 were considered statistically significant for all tests. Multivariate variation of the analysed biochemical parameters as well as the haematology was further summarised in the principal component analysis (PCA) as an effective technique simplifying the correlation structure through linear transformation of the original variables. PCA based on correlation matrix was performed to provide: (1) component loading vectors sufficient for the explanation of relationships among the parameters and their role in the evaluation of the samples and
(2) component score vectors as pairwise uncorrelated variables that were used for the final exploratory survey of the data from the examined animals. The length and direction of the lines represent the significance of the associated variables for the plotted components and for the discrimination of the samples based on component scores.
3. Results There was no mortality of control and experimental birds both during the 10- and 30-day experiments and the birds showed no clinical signs of disease. Autopsy revealed no gross pathological changes of inner organs. No histopathological changes could be noted in experimental birds receiving lower doses of the cyanobacterial biomass (groups E1, E2, E3) as well as in the control group (C) after the 10-day exposure. There were lesions classified as cloudy swelling of hepatocytes in three birds of the E4 group receiving the highest dose of cyanobacteria for 10 days. Histopathology of birds euthanized after 30 days of oral intake of the cyanobacterial biomass revealed hyperplasia of lymphatic centres in the liver tissue (one bird in group E2 and another in E3). Vacuolar dystrophy of the liver tissue (cf. Fig. 1A) accompanied by steatosis was found in one bird in the E2 group. Steatosis was also noted in two birds from the E4 group receiving the highest dose of cyanobacteria. Normal histologic appearance of liver tissue in the Japanese Quail (C—untreated controls) is shown in Fig. 1B for comparative reasons. There was vacuolar degeneration of the germinative epithelium in the testicular tissue in two males (one bird from group E1 and another from E3). The above-mentioned histopathological lesions were noted only in experimental birds and lacking in control ones. Using electron microscopy, subcellular changes of liver cells could be noted only in the experimental group of birds receiving the highest dose of the cyanobacterial biomass (E4) during the 10- and 30day exposure. Nuclei of hepatocytes were shrunken and contained ring-like nucleoli (cf. Fig. 2). Cristae mitochondriales were completely absent in some mitochondria or showing severe damage (cf. Fig. 3A). Pseudomyelin structures of phospholipids were found within vacuoles. For normal subcellular appearance of nucleus, nucleolus and mitochondria
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Fig. 1. (A) Histologic appearance of liver showing vacuolar dystrophy. Vacuoles within hepatocytes (arrow). E2 group, subchronic exposure. Original magnification 400 . (B) Histologic appearance of normal liver tissue in the Japanese Quail (C—untreated controls). Thirty-day subchronic test. Original magnification 400 .
within a hepatocyte of untreated Japanese Quail controls confer to Fig. 3B. Results of microcystin concentrations in the liver presented in Table 2 show accumulation in the highest exposure groups. The low levels of micro-
cystins observed in control can be attributed to nonspecific interactions of the complicated tissue matrix with the ELISA reagents and thus represent background values. It can also be seen that the microcystin concentrations in the liver tissue are not
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Fig. 2. Electron micrograph of a hepatocyte, its nucleus and a ring-like nucleolus (arrow). E4 group, subchronic exposure.
to scale with the 10-fold increment in the dose of the cyanobacterial biomass ingested by birds of each consecutive experimental group (cf. Tables 1 and 2). Despite the same daily doses of the cyanobacterial biomass administered to experimental birds during the 10- and 30-day exposures, microcystin concentrations found in the liver of birds after 30 days were much lower than after only 10 days (cf. Table 2). Evaluation of biochemistry resulted in finding increased activities of LD and a drop in the blood GLU of statistical significance only in group E4 on day 10 (10- and 30-day experiments; (po0.05), cf. Figs. 4 and 5, respectively). The PCA also revealed the greatest changes in biochemical parameters on day 10 of exposure. The changes in haematology were much less pronounced. There was an apparent pattern of responses in biochemical parameters on day 10 that clearly separated the two greatest exposure groups (E3, E4) from the controls (C) and lower exposures (E1, E2), as shown in Fig. 6A. Fig. 6B shows the component weights of individual biochemical parameters used for the PCA of data obtained from experimental birds on day 10. The first principal component that clearly separated the higher exposure groups (E3, E4) from control and lower exposures is driven mainly by LD, Ca and P.
4. Discussion Both our studies using experimental birds that received four different daily doses of the cyanobacterial biomass for 10 and 30 days resulted in no mortality. This may be due to the selection of experimental birds, i.e., the Japanese quail. Reported avian wildlife toxicities concerned aquatic species, which namely, may be more susceptible or the susceptibility is due to their feeding modes or habits of filtering the surface water layer containing higher concentrations of cyanobacteria and toxins by several orders of magnitude. On the other hand, they may be exposed for a much longer period due to their bond to the specific aquatic habitat. Nevertheless, experimental birds in the E4 group ingested considerably high doses of the cyanobacterial biomass corresponding with the maximal environmental concentration of the dense cyanobacterial scums (Falconer et al., 1999), (cf. Table 1) and, for example, levels similar to those determined in the event of mass mortality of flamingos (AlonsoAndicoberry et al., 2002). Comparing, however, the concentration of microcystins found in the liver tissue of our E4 experimental birds (i.e., 43.7 ng MC/g of liver tissue; cf. Table 2) and greater flamingos chicks (i.e., 81 mg MC equivalent/ml of
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Fig. 3. (A) Electron micrograph of a hepatocyte with numerous mitochondria showing severe damage (arrows). E4 group, subchronic exposure. (B) Electron micrograph showing the normal subcellular appearance of both the nucleus and nucleolus (arrow a) as well as mitochondria (arrow b) within a hepatocyte of untreated Japanese Quail controls. Thirty-day subchronic test.
liver tissue; Alonso-Andicoberry et al., 2002), there is a difference in the magnitude of three orders which may help in explaining why the former survived and the latter died. Considering the fact that the birds in the E1–E4 groups ingested 10-fold
higher doses in each consecutive experimental group (cf. Table 1), results concerning the concentration of microcystins in the liver tissue are not to scale with this (cf. Table 2). It seems that during the 10-day experiment birds had to have been able to detoxify
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Table 2 Mean concentrations of microcystins (ng/g f.w. tissue) in the liver of experimental Japanese quails from acute and subchronic test Test
Exposure duration
Acute Subchronic
10 days 30 days
Treatment group C
E1
E2
E3
E4
2.2 0.47
3.1 0.96
2.2 0.54
4.2 2.74
43.7 7.5
Lactate dehydrogenase (µkat.L-1)
C, control; E1-4, exposure groups 1–4.
10 9
*
8 7 6 5 4 C
E1
E2
E3
E4
Treatment group
Fig. 4. Lactate dehydrogenase levels in four exposure and one control groups of experimental birds on day 10 of exposure in the acute test. C—control, E1-4—exposure groups 1–4. Values represent the mean7SD (* ¼ po0.05). 15
Glucose (mmol.L-1)
14 13 12 * 11 10 9 8 C
E1
E2
E3
E4
Treatment group
Fig. 5. Blood glucose levels in four exposure and one control groups of experimental birds on day 10 of exposure in the acute test. C—control, E1-4—exposure groups 1–4. Values represent the mean7SD (* ¼ po0.05).
and/or excrete microcystins with the threshold level being somewhere between the doses given to E3 and E4 groups. Interestingly, despite the same daily
doses of the cyanobacterial biomass administered to experimental birds during the 10-day and 30-day exposures, microcystin concentrations found in the liver of birds after 30 days were much lower than after only 10 days (cf. Table 2). Speculation on this paradoxical finding may include the initiation of detoxification mechanisms and enhancement of faecal excretion in experimental birds exposed to the toxins for a longer period. Takahashi and Kaya (1993) report an LD50 of 256 mg MC–RR per kg administered by intraperitoneal injection in the Japanese quail. Their experimental birds died mostly between 14 and 18 h after the injection. Despite repeated high doses of microcystins (225 mg/kg day at the highest experimental dose, group E4) administered orally to our experimental birds for 10 and 30 days, there was null mortality, possibly due to the different route of exposure. This observation well corresponds to the results of toxicological experiments with mammals, where the acute toxicity after i.p. application of microcystin was substantially higher than after p.o. exposure (Fawell et al., 1994, 1999). Subchronic or chronic oral exposure of mammals to purified microcystins or to cyanobacterial extracts containing microcystins also did not cause mortalities at levels equivalent to our highest experimental dose (Falconer et al., 1994; Fawell et al., 1994, 1999; Schaeffer et al., 1999). The lower toxicity observed after oral administration of microcystins might result from the slow and limited carrier-mediated gastrointestinal uptake of microcystins. Microcystins probably cannot penetrate the cell membrane through simple diffusion, and they are uptaken actively through the bile acid transport system (Runnegar et al., 1991). Death of birds following the intraperitoneal injection (Takahashi and Kaya, 1993) was, in our opinion, rather due to its antigenic action (demonstrated by accumulation of lymphocytes in the spleen) than toxicity. Unfortunately, Takahashi and Kaya (1993) did not perform histopathological examination that could be compared with our results. Reported pathological changes in birds include haemorrhagic and necrotic foci in the gizzard, liver, intestine and spleen, spleen enlargement by accumulation of lymphocytes (Alonso-Andicoberry et al., 2002; Takahashi and Kaya, 1993). Contrary to the above findings, birds in this study, euthanized after the experiment, showed no gross pathological lesions. Histopathologically there was a range of hepatic changes comprising cloudy swelling of
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A
801
4 3
Factor 2: 16.2 %
2 1 0 -1 -2 -3
-4
-3
-2
-1
0
1
2
3
4
5
Factor 1 : 31.2 %
B
1.0
Factor 2 : 16.2 %
0.5
0.0
-0.5
-1.0
-1.0
-0.5
0.0
0.5
1.0
Factor 1 : 31.2 % Fig. 6. Component score (A) and component weight (B) plots from principal component analysis: distribution of different types of samples (A) based on the pattern of biochemical parameters in blood (B) on day 10 of exposure (subchronic test). Abbreviations: C (control group), E1-4 (exposure groups 1–4), TP (total protein), GLU (glucose), CREA (creatinine), UAC (uric acid), AST (aspartate aminotransferase), CK (creatine kinase), LD (lactate dehydrogenase), ALP (alkaline phosphatase), T-Chol (total cholesterol), TG (triglycerides), AMY (amylase), IP (phosphorus), Ca (plasma calcium).
hepatocytes, vacuolar dystrophy, steatosis and hyperplasia of lymphatic centres. Cloudy swelling of hepatocytes represents an early stage of toxic degenerative changes, which may still be reversible
when the cause is removed. It is understandable that these early changes developed after the shorter duration of exposure. More pronounced hepatic changes such as the vacuolar dystrophy and
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steatosis were found only in birds receiving cyanotoxins for 30 days. Hepatic lipidosis may thus be the result of long-term action of lower doses of cyanotoxins and toxic damage to the liver tissue (Schmidt et al., 2003) because no such changes were noted in control birds. Two males (E1 and E3 groups, subchronic exposure) suffered from vacuolar degeneration of the germinative epithelium in the testicular tissue. Considering this finding it would be necessary to evaluate the effect on the reproduction of birds. Primary effects of microcystins leading to hepatocellular changes and even cell death are morphological changes in mitochondria initiated by mitochondrial depolarisation (Ding et al., 2000; Ding and Ong, 2003; Majsterek et al., 2004). Mitochondrial changes have mostly been described using in vitro tests on isolated and cultured mammalian mitochondria, our study documents these changes in vivo in birds. Despite the obvious difference of the overall pattern of biochemical parameters between controls and greatest exposure groups on day 10, both biochemistry and haematology showed not very pronounced changes, except of the E4 group receiving the highest dose of cyanotoxins on day 10 (both 10- and 30-day experiments). Although AST is considered to be the intracellular enzyme most useful for diagnosing hepatocellular damage in birds (Harris, 2000), the elevation of AST in experimental Japanese quails was statistically insignificant contrary to the increased activities of LD and a drop in the GLU. LD is found in skeletal and cardiac muscle, liver, kidney, bone and erythrocytes and elevations can be observed with disruption of any of these or in haemolysis (Harris, 2000). It is known that LD levels may be used in following the progress of liver disease because they change more quickly. Distinction of the source of LD elevation depends on measuring CK originating mainly in skeletal and cardiac muscle. Elevated LD levels without concurrent elevation in CK in our experimental birds are suggestive of hepatocellular damage. Leakage of LD from hepatocytes follows even functional alterations of mitochondria (Ding and Ong, 2003) so its elevations may reflect hepatocellular damage of low degree. The drop in blood GLU may also be due to hepatopathy (Lewandowski et al., 1986). Even though the Japanese quail proved rather less susceptible to the effect of cyanotoxins, evidence of hepatocellular damage was demonstrated. Clinical
diagnosis of intoxication by cyanobacterial toxins solely based on biochemical and haematological parameters is not possible and needs to be supported by other evidence, such as massive microcystin-containing blooms in the birds’ habitats and/or traces of microcystins found in their tissues. Acknowledgement Supported by the Ministry of Education, Youth and Sports of the Czech Republic (Project MSM No. 6215712402) and project No. 1M6798593901 of the programme ‘‘Research Centres PP2—DP01’’ (1M)), and by project AVOZ60050516 granted to Institute of Botany, the Academy of Sciences of the Czech Republic. References Alonso-Andicoberry, C., Garcia-Villada, L., Lopez-Rodas, V., Costas, E., 2002. Catastrophic mortality of flamingos in a Spanish national park cause by cyanobacteria. Vet. Rec. 151, 706–707. Babica, P., Kohoutek, J., Blaha, L., Adamovsky, O., Marsalek, B., 2006. Evaluation of extraction approaches linked to ELISA and HPLC for analyses of microcystin-LR, -RR and YR in freshwater sediments with different organic material contents. Anal. Bioanal Chem. 285, 1545–1551. Carmichael, W.W., 1992. Cyanobacterial secondary metabolites—the cyanotoxins. J. Appl. Bacteriol. 72, 445–449. Chittick, E., Puschner, B., Walsh, M., Gearhart, S., St Leger, J., Skocelas, E., Branch, S., 2002. Blue-green algae microcystin toxicosis in captive Chilean flamingos. Proceeding of the American Association of Zoo Veterinarians, Milwaukee, USA, pp. 115–116. Chorus, I., Bartram, J., 1999. Toxic Cyanobacteria in Water. WHO, E&FN Spon, London, 416pp. Ding, W.X., Ong, C.N., 2003. Role of oxidative stress and mitochondrial changes in cyanobacteria-induced apoptosis and hepatotoxicity. FEMS Microbiol. Lett. 220 (1), 1–7. Ding, W.X., Shen, H.M., Ong, C.N., 2000. Critical role of reactive oxygen species and mitochondrial permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes. Hepatology 32 (3), 547–555. Duy, T.N., Lam, P.K.S., Shaw, G.R., Connell, D.W., 2000. Toxicology and risk assessment of freshwater cyanobacterial (blue-green algal) toxins in water. Rev. Environ. Contam. Toxicol. 163, 113–186. Edwards, C., Beattie, K.A., Scrimgeour, C.M., Codd, G.A., 1992. Identification of anatoxin-a in a benthic Cyanobacteria (blue-green-algae) and in associated dog poisoning at Loch Inch, Scotland. Toxicon 30 (10), 1165–1175. Falconer, I., Bartram, J., Chorus, I., Kuiper-Goodman, T., Utkilen, H., Burch, M., Codd, G.A., 1999. Safe levels and safe practices. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management. WHO & E&FN Spon, London, pp. 155–178.
ARTICLE IN PRESS B. Skocovska et al. / Toxicon 49 (2007) 793–803 Falconer, I.R., Burch, M.D., Steffensen, D.A., Choice, M., Coverdale, O.R., 1994. Toxicity of the blue-green alga (cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ. Toxicol. Water Qual. 9, 131–139. Fawell, J.K., James, C.P., James, H.A., 1994. Toxins From BlueGreen Algae: Toxicological Assessment of Microcystin-LR and a Method for its Determination in Water. Water Research Centre, Medmenham, Buckinghamshire, UK, pp. 1–46. Fawell, J.K., Mitchell, R.E., Everest, D.J., Hill, R.E., 1999. The toxicity of cyanobacterial toxins in the mouse: I MicrocystinLR. Hum. Exp. Toxicol. 18, 162–167. Harris, J.D., 2000. Clinical tests. In: Tully, T.N., Lawton, M.P.C., Dorrestein, G.M. (Eds.), Avian Medicine. Butterworth Heinemann, Oxford, pp. 43–51. Henriksen, P., Carmichael, W.W., An, J., Moestrup, Ø., 1997. Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds. Toxicon 35, 901–913. Krienitz, L., Ballot, A., Kotut, K., Wiegand, C., Pu¨tz, S., Metcalf, J.S., Codd, G.C., Pflugmacher, S., 2002. Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya. FEMS Microbiol. Ecol. 43 (2), 141–148. Lewandowski, A.H., Campbell, T.W., Harrison, G.J., 1986. Clinical chemistries. In: Harrison, G.J., Harrison, L.R. (Eds.), Clinical Avian Medicine and Surgery. W.B. Saunders, Philadelphia, USA, pp. 192–200. Majsterek, I., Sicinska, P., Tarczynska, M., Zalewski, M., Walter, Z., 2004. Toxicity of microcystin from cyanobacteria growing in a source of drinking water. Comp. Biochem. Phys. C 139 (1–3), 175–179. Marvan, P., Marsalek, B., 2004. Nutrients and their realisation in water ecosystem. In: Marsalek, B., Halouskova, O. (Eds.), Cyanobacteria, January 21, 2004, Brno, Czech Republic, pp. 79–84. Matsunaga, H., Farada, K.I., Senma, M., Ito, Y., Yasuda, N., Ushida, S., Kimura, Y., 1999. Possible cause of unnatural mass death of wild birds in a pond Nishinomiya, Japan: sudden appearance of toxic cyanobacteria. Nat. Toxins 7 (2), 81–84. Mez, K., Beattie, K.A., Codd, G.A., Hanselmann, K., Hauser, B., Naegeli, H., Preisig, H.R., 1997. Identification of microcystin in benthic cyanobacteria linked to cattle deaths on alpine pastures in Switzerland. Eur. J. Phycol. 32, 111–117.
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Murphy, T., Lawson, A., Nalewajko, C., Murkin, H., Ross, L., Oguma, K., McIntyre, T., 2000. Algal toxins—initiators of avian botulism? Environ. Toxicol. 15, 558–567. Murphy, T.P., Irvine, K., Guo, J., Davies, J., Murkin, H., Charlton, M., Watson, S.B., 2003. New microcystin concerns in the lower great lakes. Water Qual. Res. J. Can. 38, 127–140. OECD Guideline for testing of chemicals 205, 1984. Avian dietary toxicity tests. OECD Guidelines for Testing of Chemicals. Organisation for Economics Cooperation and Development, vol. 1(2), pp. 1–10. Onodera, H., Yasutaku, O., Henriksen, P., Yasumoto, T., 1997. Confirmation of anatoxin-a(s), in the cyanobacterium Anabaena lemmermannii, as the cause of bird kills in Danish lakes. Toxicon 35 (11), 1645–1648. Park, H., Namikoshi, M., Brittain, S.M., Carmichael, W.W., Murphy, T., 2001. [D-Leu1] microcystin-LR, a new microcystin isolated from waterbloom in a Canadian prairie lake. Toxicon 39, 855–862. Runnegar, M.T., Gerdes, R.G., Falconer, I.R., 1991. The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes. Toxicon 29, 43–51. Schaeffer, D.J., Malpas, P., Barton, L., 1999. Risk assessment of microcystin in dietary of Aphanizomenon flos-aquae. Ecotox. Environ. Saf. 44, 73–80. Schmidt, R.E., Reavill, D.R., Phalen, D.N., 2003. Pathology of Pet and Aviary Birds. Iowa State Press, Iowa, USA, 234pp. Takahashi, S., Kaya, K., 1993. Quail spleen is enlarged by microcystin RR as a blue–green algal hepatotoxin. Nat. Toxins 1 (5), 283–285. Wiegand, C., Pflugmacher, S., 2005. Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review. Toxicol. Appl. Pharm. 203 (3), 201–218. Wilde, S.B., Murphy, T.M., Hope, C.P., Habrun, S.K., Kempton, J., Birrenkott, A., Wiley, F., Bowerman, W.W., Lewitus, A.J., 2005. Avian vacuolar myelinopathy linked to exotic aquatic plants and a novel cyanobacterial species. Environ. Toxicol. 20, 348–353. Wirsing, B., Hoffmann, L., Heinze, R., Klein, D., Daloze, D., Braekman, J.C., Weckesser, J., 1998. First report on the identification of microcystin in a water bloom collected in Belgium. Syst. Appl. Microbiol. 21, 23–27. Zeck, A., Eikenberg, A., Weller, M.G., Niessner, R., 2001a. Highly sensitive immunoassay based on a monoclonal antibody specific for [4-arginine] microcystins. Anal. Chim. Acta 441, 1–13. Zeck, A., Weller, M.G., Bursill, D., Niessner, R., 2001b. Generic microcystin immunoassay based on monoclonal antibodies against Adda. Analyst 126, 2000–2007.
Toxicon 49 (2007) 793–803 www.elsevier.com/locate/toxicon
Effects of cyanobacterial biomass on the Japanese quail B. Skocovskaa, K. Hilscherovab, P. Babicab, O. Adamovskyb, H. Bandouchovaa, J. Horakovaa, Z. Knotkovaa, B. Marsalekb, V. Paskovab, J. Pikulaa, a
Department of Veterinary Ecology and Environmental Protection, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1-3, 612 42 Brno, Czech Republic b Centre for Cyanobacteria and their Toxins, Institute of Botany, The Academy of Sciences of the Czech Republic and RECETOX, Masaryk University, Kamenice 126/3, CZ62500, Brno, Czech Republic Received 22 September 2006; received in revised form 29 November 2006; accepted 29 November 2006 Available online 8 December 2006
Abstract Mortality of wild aquatic birds has recently been attributed to cyanobacterial toxins. Despite this, no experimental studies on the effects of defined doses of microcystins administered orally to birds exist. In this experiment, four groups of male Japanese quails daily ingesting 10 ml of Microcystis biomass containing 0.045, 0.459, 4.605 or 46.044 mg of microcystins, respectively, for 10 and 30 days, showed no mortality. Histopathological hepatic changes in birds after the biomass exposure included cloudy swelling of hepatocytes, vacuolar dystrophy, steatosis and hyperplasia of lymphatic centres. On subcellular level, shrunken nuclei of hepatocytes containing ring-like nucleoli, cristolysis within mitochondria and vacuoles with pseudomyelin structures were present. Vacuolar degeneration of the testicular germinative epithelium was found in two exposed males. Statistically significant differences in biochemical parameters were on day 10 of exposure only. They comprised increased activities of lactate dehydrogenase and a drop in blood glucose in birds receiving the highest dose of the biomass. Principal component analysis revealed a pattern of responses in biochemical parameters on day 10 that clearly separated the two greatest exposure groups from the controls and lower exposures. The results indicate that diagnosis of microcystin intoxication solely based on clinical biochemical and haematological parameters is hardly possible in birds. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cyanobacterial water bloom; Avian toxicity tests; Peroral exposure; Biochemistry; Histopathology; Electron microscopy; Mitochondria; Microcystis; Microcystins; Coturnix coturnix japonica
1. Introduction Water blooms of cyanobacteria represent a problem throughout the world due to the eutrophication of the aquatic environment in particular (Chorus and Bartram, 1999; Marvan and Marsalek, Corresponding author. Tel.: +420 5 41562655; fax: +420 5 49243020. E-mail address: [email protected] (J. Pikula).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.11.032
2004). The most serious consequences of bloom formation are poisonings by secondary metabolites of cyanobacteria—cyanotoxins (Carmichael, 1992), which fall into five groups (Wiegand and Pflugmacher, 2005) based on their primary toxicological target, i.e. hepatotoxins (microcystins and nodularins), neurotoxins (anatoxin-a, anatoxin-a(S), saxitoxins), cytotoxins (cylindropspermopsin), dermatotoxins (aplysiatoxins and lyngbyatoxin) and irritant toxins (lipopolysaccharides).
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A wide range of aquatic organisms including invertebrates, fish, mammalian and avian species may be exposed to cyanotoxins via consumption of or dermal contact with water contaminated by toxic cyanobacteria. A number of poisonings in animals have been attributed to cyanobacterial toxins (Duy et al., 2000). Well known is an incident reported in Switzerland concerning fatalities in cattle kept on alpine pastures (Mez et al., 1997) as well as a dog poisoning at Loch Inch, Scotland (Edwards et al., 1992). Toxic effects of cyanobacterial products have also been recognised in avian species. Mortalities of wild birds attributed to toxigenic taxa of cyanobacteria have been reported (Alonso-Andicoberry et al., 2002; Chittick et al., 2002; Henriksen et al., 1997; Krienitz et al., 2002; Matsunaga et al., 1999; Onodera et al., 1997). For example, mass deaths of wild birds including about 20 ducks (Anas poecilorhyncha zonorhyncha) have been attributed to the sudden appearance of toxic cyanobacteria Microcystis aeruginosa in a pond in Nishinomiya (Japan) in 1995 (Matsunaga et al., 1999). The biomass of cyanobacteria contained high levels of microcystins and autopsy revealed icteritic livers and necroses of liver tissue giving thus indication of the presence of hepatotoxins. Several bird deaths in Canadian and Belgian lakes have also been linked with occurrence of cyanobacterial blooms producing microcystins (Wirsing et al., 1998; Park et al., 2001; Murphy et al., 2003) or anatoxin-a (Murphy et al., 2000). Another case of avian mortality associated with cyanotoxins was the mysterious deaths of lesser flamingos (Phoenicopterus minor) around a thermal Lake Bogoria (Kenya). Birds manifested neurological signs prior to death and hepatotoxic microcystins LR, -RR, -LF and -YR and a neurotoxin (anatoxin-a) were found in the stomach content as well as in faeces. Intoxication is supposed to have occurred through drinking water from the lake and feather grooming performed as a daily activity near hot springs of the lake (Krienitz et al., 2002). Catastrophic mortality due to cyanobacterial toxins has also been reported in wild flamingos from a Spanish national park (AlonsoAndicoberry et al., 2002) and in captive Chilean flamingos in USA (Chittick et al., 2002). Onodera et al. (1997) confirmed that the cause of bird mortality in Danish lakes in 1993–94 was consumption of the cyanobacterium Anabaena lemmermannii containing anatoxin-a(s). Uncharacterized cyanobacterial neurotoxin was probably responsible for lethal avian
vacuolar myelinopathy observed in USA (Wilde et al., 2005). Previous documentation of the toxicity of cyanobacteria in birds has rather been based on indirect evidence such as observation of mortality in several avian species under natural conditions associated with the occurrence of water blooms and long-term stay and foraging of birds at the affected locality, detection of cyanobacterial toxins in the crop and liver and exclusion of alternative causes of mortality. Our intention was to complement field observations published by other workers with results from laboratory experiments. Therefore, the aim of this study was to perform evidence-based avian toxicity tests using standard experimental avian species and evaluate the effects of different doses of cyanobacterial toxins in birds. 2. Materials and methods Effects of cyanobacterial toxins in birds were evaluated according to the OECD 205 Guideline for the testing of chemicals—Avian Dietary Toxicity Test (1984) with some minor modifications to fit our experimental conditions. 2.1. Experimental substances Experimental birds were exposed to homogenate of cyanobacterial biomass dominated by Microcystis sp., which was collected using plankton net (25 mm) from Brno reservoir in autumn 2004. Colonial cyanobacteria of the Microcystis genus (M. aeruginosa 80%, Microcystis viridis 15% and Microcystis flos-aquae 5%) prevailed in the species composition. Concentration of cyanobacteria in experimental biomass was determined by cell counting under a fluorescent microscope and concentration of biomass dry weight was estimated in an aliquote of biomass after drying (50 1C). Cyanobacterial biomass was homogenized by repeated freeze thawing and by ultrasonic probe. Microcystin contents in biomass and homogenate were analysed using HPLC-DAD (Agilent 1100 Series) on Supelcosil ABZ+ Plus column, 150 4.6 mm, 5 mm according to Babica et al. (2006). Concentrations of microcystins in experimental biomass were 141.8 mg/g DW of microcystin-RR, 33.7 mg/g DW of microcystin-YR, 141.7 mg/g DW of microcystin-LR, and 56.1 mg/g DW of unidentified compound possessing microcystin-like UV spectrum. The total concentration of microcystins (including
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unidentified structural variant) in biomass was 373.3 mg/g DW. Four experimental concentrations of biomass were prepared by dilution of biomass homogenate with drinking water, aliquoted into plastic cups and stored frozen. Drinking water and water for the control birds was handled in the same way. After thawing on day of application, homogenate of experimental biomass or drinking water were provided orally to birds. 2.2. Experimental animals The Japanese quail (Coturnix coturnix japonica) is a common experimental bird. Due to its small size, the selection was economic regarding the quantity of the cyanobacterial biomass necessary for the experiment. Experimental quails (4 months old, weighing 205.25 g on average) were randomly divided into groups of five individuals (males only to eliminate gender differences). One control (C) and four exposure groups (E1-4) receiving different daily doses of the cyanobacterial biomass were formed. Commercially available feeds and drinking water were supplied ad libitum. 2.3. Experimental design Considering the daily dose of the biomass, experimental birds received cyanobacteria containing microcystins in amounts presented in Table 1. The highest dose was equivalent to daily consumption of 10 ml of biomass with concentration of 3 108 cell/ml (12334.8 mg DW/L, corresponded to microcystin concentration 4604.4 mg/l). Experimental birds were given the cyanobacterial biomass using a crop probe thrice a day to obtain a daily
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dose, which was the same both in the acute and subchronic exposure. We employed 10-day (acute) and 30-day (subchronic) experiments. Samples of blood for haematology and plasma chemistry profiles were collected one day prior to the start of the experiment and on day 10 (10-day and 30-day experiment), and 20 and 30 (30-day experiment). Blood (1 ml) was collected from the right jugular vein using the Omnican 0.30 12 mm insulin set (Braun, Germany). Whole blood was placed in heparinized tubes (Leciva inj., Prague), centrifuged immediately, and plasma removed and frozen (20 1C). Plasma was analysed using an automated analyzer (CobasMira, Roche) for total protein (TP; g/l), albumin (ALB; g/l), glucose (GLU; mmol/l), creatinine (CREA; mmol/l), uric acid (UAC; mmol/l), aspartate aminotransferase (AST; mkat/l), creatine kinase (CK; mkat/l), lactate dehydrogenase (LD; mkat/l), alkaline phosphatase (ALP; mkat/l), total cholesterol (T-Chol; mmol/l), triglycerides (TG; mmol/l), amylase (AMY; mkat/l), phosphorus (IP; mmol/l), potassium (K; mmol/l) and sodium (Na; mmol/l). Plasma calcium (Ca; mmol/l) concentrations were analysed with an Atomspec analyzer (Hilger 1550). Hematocrit (PCV) was measured using microhematocrit tubes. Haemoglobin (Hb) was determined by the cyanmethemoglobin method, and total red (RBC) and white (WBC) blood cell counts were determined manually using haemocytometer with Natt and Herrick’s solution. Blood smears on a glass slide, prepared immediately after blood collection, were air-dried and stained using May+ Grunwald and Giemsa-Romanowski stains. Two hundred leucocytes were counted for each smear and classified as heterophils, eosinophils, basophils, lymphocytes and monocytes.
Table 1 Daily doses of cyanobacterial biomass administered to birds of four exposure and one control groups and its characteristics Exposure and control groups E4 E3 E2 E1 C
Cyanobacterial biomass
Microcystin structural variants (mg)
Cells
mg of dry weight
MC-RR
MC–unidentif.
MC–YR
MC–LR
Sum of MCs
3 109 3 108 3 107 3 106 —
123.348 12.336 1.233 0.123 —
17.496 1.749 0.174 0.018 —
6.915 0.690 0.069 0.006 —
4.158 0.417 0.042 0.003 —
17.475 1.749 0.174 0.018 —
46.044 4.605 0.459 0.045 —
Birds were given the cyanobacterial biomass or drinking water using a crop probe thrice a day to obtain a daily dose of 10 ml which was the same both in the acute (10-day) and subchronic (30-day) exposure.
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After the experiment, birds were euthanized by decapitation, examined regarding pathoanatomical changes and used to collect tissue specimens (liver, CNS, kidney, spleen, pancreas, heart and both pectoralis muscles) for standard histopathology as well as electron microscopy. Samples for histopathology were fixed in buffered 10% formalin, prepared in a standard way and stained by hematoxylin eosin. Samples for electron microscopy were stored in glutaraldehyde and prepared in a standard way for transmission electron microscopy. Immediately after euthanasia, a second set of tissue specimens were collected for analyses of microcystin concentrations. For these purposes, tissue specimens were placed in plastic bags and deep frozen (80 1C). 2.4. Analyses of microcystin in tissues Microcystin concentrations in tissues of exposed animals were determined after repeated (3 ) extraction of fresh tissue by methanol under sonication. Methanolic extracts were partitioned three times with hexane:water. Methanolic layers were dried (50 1C), residues re-dissolved in distilled water and concentrations of microcystins analysed using direct competitive ELISA. The ELISA assay employed monoclonal antibody MC10E7 (Zeck et al., 2001a, b) and was performed as previously described in Babica et al. (2006). 2.5. Statistical evaluation Statistical analyses were performed with Statistica for Windowss 7.0 (StatSoft, Tulsa, OK, USA). Data normality and homogeneity of variances were evaluated by Kolmogorov–Smirnov test and the Levene’s test, respectively. One-way analysis of variance (ANOVA) and the nonparametric Kruskal–Wallis test were used for statistical comparisons. Values po0.05 were considered statistically significant for all tests. Multivariate variation of the analysed biochemical parameters as well as the haematology was further summarised in the principal component analysis (PCA) as an effective technique simplifying the correlation structure through linear transformation of the original variables. PCA based on correlation matrix was performed to provide: (1) component loading vectors sufficient for the explanation of relationships among the parameters and their role in the evaluation of the samples and
(2) component score vectors as pairwise uncorrelated variables that were used for the final exploratory survey of the data from the examined animals. The length and direction of the lines represent the significance of the associated variables for the plotted components and for the discrimination of the samples based on component scores.
3. Results There was no mortality of control and experimental birds both during the 10- and 30-day experiments and the birds showed no clinical signs of disease. Autopsy revealed no gross pathological changes of inner organs. No histopathological changes could be noted in experimental birds receiving lower doses of the cyanobacterial biomass (groups E1, E2, E3) as well as in the control group (C) after the 10-day exposure. There were lesions classified as cloudy swelling of hepatocytes in three birds of the E4 group receiving the highest dose of cyanobacteria for 10 days. Histopathology of birds euthanized after 30 days of oral intake of the cyanobacterial biomass revealed hyperplasia of lymphatic centres in the liver tissue (one bird in group E2 and another in E3). Vacuolar dystrophy of the liver tissue (cf. Fig. 1A) accompanied by steatosis was found in one bird in the E2 group. Steatosis was also noted in two birds from the E4 group receiving the highest dose of cyanobacteria. Normal histologic appearance of liver tissue in the Japanese Quail (C—untreated controls) is shown in Fig. 1B for comparative reasons. There was vacuolar degeneration of the germinative epithelium in the testicular tissue in two males (one bird from group E1 and another from E3). The above-mentioned histopathological lesions were noted only in experimental birds and lacking in control ones. Using electron microscopy, subcellular changes of liver cells could be noted only in the experimental group of birds receiving the highest dose of the cyanobacterial biomass (E4) during the 10- and 30day exposure. Nuclei of hepatocytes were shrunken and contained ring-like nucleoli (cf. Fig. 2). Cristae mitochondriales were completely absent in some mitochondria or showing severe damage (cf. Fig. 3A). Pseudomyelin structures of phospholipids were found within vacuoles. For normal subcellular appearance of nucleus, nucleolus and mitochondria
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Fig. 1. (A) Histologic appearance of liver showing vacuolar dystrophy. Vacuoles within hepatocytes (arrow). E2 group, subchronic exposure. Original magnification 400 . (B) Histologic appearance of normal liver tissue in the Japanese Quail (C—untreated controls). Thirty-day subchronic test. Original magnification 400 .
within a hepatocyte of untreated Japanese Quail controls confer to Fig. 3B. Results of microcystin concentrations in the liver presented in Table 2 show accumulation in the highest exposure groups. The low levels of micro-
cystins observed in control can be attributed to nonspecific interactions of the complicated tissue matrix with the ELISA reagents and thus represent background values. It can also be seen that the microcystin concentrations in the liver tissue are not
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Fig. 2. Electron micrograph of a hepatocyte, its nucleus and a ring-like nucleolus (arrow). E4 group, subchronic exposure.
to scale with the 10-fold increment in the dose of the cyanobacterial biomass ingested by birds of each consecutive experimental group (cf. Tables 1 and 2). Despite the same daily doses of the cyanobacterial biomass administered to experimental birds during the 10- and 30-day exposures, microcystin concentrations found in the liver of birds after 30 days were much lower than after only 10 days (cf. Table 2). Evaluation of biochemistry resulted in finding increased activities of LD and a drop in the blood GLU of statistical significance only in group E4 on day 10 (10- and 30-day experiments; (po0.05), cf. Figs. 4 and 5, respectively). The PCA also revealed the greatest changes in biochemical parameters on day 10 of exposure. The changes in haematology were much less pronounced. There was an apparent pattern of responses in biochemical parameters on day 10 that clearly separated the two greatest exposure groups (E3, E4) from the controls (C) and lower exposures (E1, E2), as shown in Fig. 6A. Fig. 6B shows the component weights of individual biochemical parameters used for the PCA of data obtained from experimental birds on day 10. The first principal component that clearly separated the higher exposure groups (E3, E4) from control and lower exposures is driven mainly by LD, Ca and P.
4. Discussion Both our studies using experimental birds that received four different daily doses of the cyanobacterial biomass for 10 and 30 days resulted in no mortality. This may be due to the selection of experimental birds, i.e., the Japanese quail. Reported avian wildlife toxicities concerned aquatic species, which namely, may be more susceptible or the susceptibility is due to their feeding modes or habits of filtering the surface water layer containing higher concentrations of cyanobacteria and toxins by several orders of magnitude. On the other hand, they may be exposed for a much longer period due to their bond to the specific aquatic habitat. Nevertheless, experimental birds in the E4 group ingested considerably high doses of the cyanobacterial biomass corresponding with the maximal environmental concentration of the dense cyanobacterial scums (Falconer et al., 1999), (cf. Table 1) and, for example, levels similar to those determined in the event of mass mortality of flamingos (AlonsoAndicoberry et al., 2002). Comparing, however, the concentration of microcystins found in the liver tissue of our E4 experimental birds (i.e., 43.7 ng MC/g of liver tissue; cf. Table 2) and greater flamingos chicks (i.e., 81 mg MC equivalent/ml of
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Fig. 3. (A) Electron micrograph of a hepatocyte with numerous mitochondria showing severe damage (arrows). E4 group, subchronic exposure. (B) Electron micrograph showing the normal subcellular appearance of both the nucleus and nucleolus (arrow a) as well as mitochondria (arrow b) within a hepatocyte of untreated Japanese Quail controls. Thirty-day subchronic test.
liver tissue; Alonso-Andicoberry et al., 2002), there is a difference in the magnitude of three orders which may help in explaining why the former survived and the latter died. Considering the fact that the birds in the E1–E4 groups ingested 10-fold
higher doses in each consecutive experimental group (cf. Table 1), results concerning the concentration of microcystins in the liver tissue are not to scale with this (cf. Table 2). It seems that during the 10-day experiment birds had to have been able to detoxify
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Table 2 Mean concentrations of microcystins (ng/g f.w. tissue) in the liver of experimental Japanese quails from acute and subchronic test Test
Exposure duration
Acute Subchronic
10 days 30 days
Treatment group C
E1
E2
E3
E4
2.2 0.47
3.1 0.96
2.2 0.54
4.2 2.74
43.7 7.5
Lactate dehydrogenase (µkat.L-1)
C, control; E1-4, exposure groups 1–4.
10 9
*
8 7 6 5 4 C
E1
E2
E3
E4
Treatment group
Fig. 4. Lactate dehydrogenase levels in four exposure and one control groups of experimental birds on day 10 of exposure in the acute test. C—control, E1-4—exposure groups 1–4. Values represent the mean7SD (* ¼ po0.05). 15
Glucose (mmol.L-1)
14 13 12 * 11 10 9 8 C
E1
E2
E3
E4
Treatment group
Fig. 5. Blood glucose levels in four exposure and one control groups of experimental birds on day 10 of exposure in the acute test. C—control, E1-4—exposure groups 1–4. Values represent the mean7SD (* ¼ po0.05).
and/or excrete microcystins with the threshold level being somewhere between the doses given to E3 and E4 groups. Interestingly, despite the same daily
doses of the cyanobacterial biomass administered to experimental birds during the 10-day and 30-day exposures, microcystin concentrations found in the liver of birds after 30 days were much lower than after only 10 days (cf. Table 2). Speculation on this paradoxical finding may include the initiation of detoxification mechanisms and enhancement of faecal excretion in experimental birds exposed to the toxins for a longer period. Takahashi and Kaya (1993) report an LD50 of 256 mg MC–RR per kg administered by intraperitoneal injection in the Japanese quail. Their experimental birds died mostly between 14 and 18 h after the injection. Despite repeated high doses of microcystins (225 mg/kg day at the highest experimental dose, group E4) administered orally to our experimental birds for 10 and 30 days, there was null mortality, possibly due to the different route of exposure. This observation well corresponds to the results of toxicological experiments with mammals, where the acute toxicity after i.p. application of microcystin was substantially higher than after p.o. exposure (Fawell et al., 1994, 1999). Subchronic or chronic oral exposure of mammals to purified microcystins or to cyanobacterial extracts containing microcystins also did not cause mortalities at levels equivalent to our highest experimental dose (Falconer et al., 1994; Fawell et al., 1994, 1999; Schaeffer et al., 1999). The lower toxicity observed after oral administration of microcystins might result from the slow and limited carrier-mediated gastrointestinal uptake of microcystins. Microcystins probably cannot penetrate the cell membrane through simple diffusion, and they are uptaken actively through the bile acid transport system (Runnegar et al., 1991). Death of birds following the intraperitoneal injection (Takahashi and Kaya, 1993) was, in our opinion, rather due to its antigenic action (demonstrated by accumulation of lymphocytes in the spleen) than toxicity. Unfortunately, Takahashi and Kaya (1993) did not perform histopathological examination that could be compared with our results. Reported pathological changes in birds include haemorrhagic and necrotic foci in the gizzard, liver, intestine and spleen, spleen enlargement by accumulation of lymphocytes (Alonso-Andicoberry et al., 2002; Takahashi and Kaya, 1993). Contrary to the above findings, birds in this study, euthanized after the experiment, showed no gross pathological lesions. Histopathologically there was a range of hepatic changes comprising cloudy swelling of
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A
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Factor 2: 16.2 %
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Factor 1 : 31.2 % Fig. 6. Component score (A) and component weight (B) plots from principal component analysis: distribution of different types of samples (A) based on the pattern of biochemical parameters in blood (B) on day 10 of exposure (subchronic test). Abbreviations: C (control group), E1-4 (exposure groups 1–4), TP (total protein), GLU (glucose), CREA (creatinine), UAC (uric acid), AST (aspartate aminotransferase), CK (creatine kinase), LD (lactate dehydrogenase), ALP (alkaline phosphatase), T-Chol (total cholesterol), TG (triglycerides), AMY (amylase), IP (phosphorus), Ca (plasma calcium).
hepatocytes, vacuolar dystrophy, steatosis and hyperplasia of lymphatic centres. Cloudy swelling of hepatocytes represents an early stage of toxic degenerative changes, which may still be reversible
when the cause is removed. It is understandable that these early changes developed after the shorter duration of exposure. More pronounced hepatic changes such as the vacuolar dystrophy and
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steatosis were found only in birds receiving cyanotoxins for 30 days. Hepatic lipidosis may thus be the result of long-term action of lower doses of cyanotoxins and toxic damage to the liver tissue (Schmidt et al., 2003) because no such changes were noted in control birds. Two males (E1 and E3 groups, subchronic exposure) suffered from vacuolar degeneration of the germinative epithelium in the testicular tissue. Considering this finding it would be necessary to evaluate the effect on the reproduction of birds. Primary effects of microcystins leading to hepatocellular changes and even cell death are morphological changes in mitochondria initiated by mitochondrial depolarisation (Ding et al., 2000; Ding and Ong, 2003; Majsterek et al., 2004). Mitochondrial changes have mostly been described using in vitro tests on isolated and cultured mammalian mitochondria, our study documents these changes in vivo in birds. Despite the obvious difference of the overall pattern of biochemical parameters between controls and greatest exposure groups on day 10, both biochemistry and haematology showed not very pronounced changes, except of the E4 group receiving the highest dose of cyanotoxins on day 10 (both 10- and 30-day experiments). Although AST is considered to be the intracellular enzyme most useful for diagnosing hepatocellular damage in birds (Harris, 2000), the elevation of AST in experimental Japanese quails was statistically insignificant contrary to the increased activities of LD and a drop in the GLU. LD is found in skeletal and cardiac muscle, liver, kidney, bone and erythrocytes and elevations can be observed with disruption of any of these or in haemolysis (Harris, 2000). It is known that LD levels may be used in following the progress of liver disease because they change more quickly. Distinction of the source of LD elevation depends on measuring CK originating mainly in skeletal and cardiac muscle. Elevated LD levels without concurrent elevation in CK in our experimental birds are suggestive of hepatocellular damage. Leakage of LD from hepatocytes follows even functional alterations of mitochondria (Ding and Ong, 2003) so its elevations may reflect hepatocellular damage of low degree. The drop in blood GLU may also be due to hepatopathy (Lewandowski et al., 1986). Even though the Japanese quail proved rather less susceptible to the effect of cyanotoxins, evidence of hepatocellular damage was demonstrated. Clinical
diagnosis of intoxication by cyanobacterial toxins solely based on biochemical and haematological parameters is not possible and needs to be supported by other evidence, such as massive microcystin-containing blooms in the birds’ habitats and/or traces of microcystins found in their tissues. Acknowledgement Supported by the Ministry of Education, Youth and Sports of the Czech Republic (Project MSM No. 6215712402) and project No. 1M6798593901 of the programme ‘‘Research Centres PP2—DP01’’ (1M)), and by project AVOZ60050516 granted to Institute of Botany, the Academy of Sciences of the Czech Republic. References Alonso-Andicoberry, C., Garcia-Villada, L., Lopez-Rodas, V., Costas, E., 2002. Catastrophic mortality of flamingos in a Spanish national park cause by cyanobacteria. Vet. Rec. 151, 706–707. Babica, P., Kohoutek, J., Blaha, L., Adamovsky, O., Marsalek, B., 2006. Evaluation of extraction approaches linked to ELISA and HPLC for analyses of microcystin-LR, -RR and YR in freshwater sediments with different organic material contents. Anal. Bioanal Chem. 285, 1545–1551. Carmichael, W.W., 1992. Cyanobacterial secondary metabolites—the cyanotoxins. J. Appl. Bacteriol. 72, 445–449. Chittick, E., Puschner, B., Walsh, M., Gearhart, S., St Leger, J., Skocelas, E., Branch, S., 2002. Blue-green algae microcystin toxicosis in captive Chilean flamingos. Proceeding of the American Association of Zoo Veterinarians, Milwaukee, USA, pp. 115–116. Chorus, I., Bartram, J., 1999. Toxic Cyanobacteria in Water. WHO, E&FN Spon, London, 416pp. Ding, W.X., Ong, C.N., 2003. Role of oxidative stress and mitochondrial changes in cyanobacteria-induced apoptosis and hepatotoxicity. FEMS Microbiol. Lett. 220 (1), 1–7. Ding, W.X., Shen, H.M., Ong, C.N., 2000. Critical role of reactive oxygen species and mitochondrial permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes. Hepatology 32 (3), 547–555. Duy, T.N., Lam, P.K.S., Shaw, G.R., Connell, D.W., 2000. Toxicology and risk assessment of freshwater cyanobacterial (blue-green algal) toxins in water. Rev. Environ. Contam. Toxicol. 163, 113–186. Edwards, C., Beattie, K.A., Scrimgeour, C.M., Codd, G.A., 1992. Identification of anatoxin-a in a benthic Cyanobacteria (blue-green-algae) and in associated dog poisoning at Loch Inch, Scotland. Toxicon 30 (10), 1165–1175. Falconer, I., Bartram, J., Chorus, I., Kuiper-Goodman, T., Utkilen, H., Burch, M., Codd, G.A., 1999. Safe levels and safe practices. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring and Management. WHO & E&FN Spon, London, pp. 155–178.
ARTICLE IN PRESS B. Skocovska et al. / Toxicon 49 (2007) 793–803 Falconer, I.R., Burch, M.D., Steffensen, D.A., Choice, M., Coverdale, O.R., 1994. Toxicity of the blue-green alga (cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ. Toxicol. Water Qual. 9, 131–139. Fawell, J.K., James, C.P., James, H.A., 1994. Toxins From BlueGreen Algae: Toxicological Assessment of Microcystin-LR and a Method for its Determination in Water. Water Research Centre, Medmenham, Buckinghamshire, UK, pp. 1–46. Fawell, J.K., Mitchell, R.E., Everest, D.J., Hill, R.E., 1999. The toxicity of cyanobacterial toxins in the mouse: I MicrocystinLR. Hum. Exp. Toxicol. 18, 162–167. Harris, J.D., 2000. Clinical tests. In: Tully, T.N., Lawton, M.P.C., Dorrestein, G.M. (Eds.), Avian Medicine. Butterworth Heinemann, Oxford, pp. 43–51. Henriksen, P., Carmichael, W.W., An, J., Moestrup, Ø., 1997. Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds. Toxicon 35, 901–913. Krienitz, L., Ballot, A., Kotut, K., Wiegand, C., Pu¨tz, S., Metcalf, J.S., Codd, G.C., Pflugmacher, S., 2002. Contribution of hot spring cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya. FEMS Microbiol. Ecol. 43 (2), 141–148. Lewandowski, A.H., Campbell, T.W., Harrison, G.J., 1986. Clinical chemistries. In: Harrison, G.J., Harrison, L.R. (Eds.), Clinical Avian Medicine and Surgery. W.B. Saunders, Philadelphia, USA, pp. 192–200. Majsterek, I., Sicinska, P., Tarczynska, M., Zalewski, M., Walter, Z., 2004. Toxicity of microcystin from cyanobacteria growing in a source of drinking water. Comp. Biochem. Phys. C 139 (1–3), 175–179. Marvan, P., Marsalek, B., 2004. Nutrients and their realisation in water ecosystem. In: Marsalek, B., Halouskova, O. (Eds.), Cyanobacteria, January 21, 2004, Brno, Czech Republic, pp. 79–84. Matsunaga, H., Farada, K.I., Senma, M., Ito, Y., Yasuda, N., Ushida, S., Kimura, Y., 1999. Possible cause of unnatural mass death of wild birds in a pond Nishinomiya, Japan: sudden appearance of toxic cyanobacteria. Nat. Toxins 7 (2), 81–84. Mez, K., Beattie, K.A., Codd, G.A., Hanselmann, K., Hauser, B., Naegeli, H., Preisig, H.R., 1997. Identification of microcystin in benthic cyanobacteria linked to cattle deaths on alpine pastures in Switzerland. Eur. J. Phycol. 32, 111–117.
803
Murphy, T., Lawson, A., Nalewajko, C., Murkin, H., Ross, L., Oguma, K., McIntyre, T., 2000. Algal toxins—initiators of avian botulism? Environ. Toxicol. 15, 558–567. Murphy, T.P., Irvine, K., Guo, J., Davies, J., Murkin, H., Charlton, M., Watson, S.B., 2003. New microcystin concerns in the lower great lakes. Water Qual. Res. J. Can. 38, 127–140. OECD Guideline for testing of chemicals 205, 1984. Avian dietary toxicity tests. OECD Guidelines for Testing of Chemicals. Organisation for Economics Cooperation and Development, vol. 1(2), pp. 1–10. Onodera, H., Yasutaku, O., Henriksen, P., Yasumoto, T., 1997. Confirmation of anatoxin-a(s), in the cyanobacterium Anabaena lemmermannii, as the cause of bird kills in Danish lakes. Toxicon 35 (11), 1645–1648. Park, H., Namikoshi, M., Brittain, S.M., Carmichael, W.W., Murphy, T., 2001. [D-Leu1] microcystin-LR, a new microcystin isolated from waterbloom in a Canadian prairie lake. Toxicon 39, 855–862. Runnegar, M.T., Gerdes, R.G., Falconer, I.R., 1991. The uptake of the cyanobacterial hepatotoxin microcystin by isolated rat hepatocytes. Toxicon 29, 43–51. Schaeffer, D.J., Malpas, P., Barton, L., 1999. Risk assessment of microcystin in dietary of Aphanizomenon flos-aquae. Ecotox. Environ. Saf. 44, 73–80. Schmidt, R.E., Reavill, D.R., Phalen, D.N., 2003. Pathology of Pet and Aviary Birds. Iowa State Press, Iowa, USA, 234pp. Takahashi, S., Kaya, K., 1993. Quail spleen is enlarged by microcystin RR as a blue–green algal hepatotoxin. Nat. Toxins 1 (5), 283–285. Wiegand, C., Pflugmacher, S., 2005. Ecotoxicological effects of selected cyanobacterial secondary metabolites a short review. Toxicol. Appl. Pharm. 203 (3), 201–218. Wilde, S.B., Murphy, T.M., Hope, C.P., Habrun, S.K., Kempton, J., Birrenkott, A., Wiley, F., Bowerman, W.W., Lewitus, A.J., 2005. Avian vacuolar myelinopathy linked to exotic aquatic plants and a novel cyanobacterial species. Environ. Toxicol. 20, 348–353. Wirsing, B., Hoffmann, L., Heinze, R., Klein, D., Daloze, D., Braekman, J.C., Weckesser, J., 1998. First report on the identification of microcystin in a water bloom collected in Belgium. Syst. Appl. Microbiol. 21, 23–27. Zeck, A., Eikenberg, A., Weller, M.G., Niessner, R., 2001a. Highly sensitive immunoassay based on a monoclonal antibody specific for [4-arginine] microcystins. Anal. Chim. Acta 441, 1–13. Zeck, A., Weller, M.G., Bursill, D., Niessner, R., 2001b. Generic microcystin immunoassay based on monoclonal antibodies against Adda. Analyst 126, 2000–2007.