Using of Daphnia magna, Artemia salina and Tubifex tubifex for ...

Kyselková I., Maršálek B. (2000): Using of Daphnia magna, Artemia salina and Tubifex tubifex for cyanobacterial microcystins detection. Biologia 55(6): 637-643.

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Use of Daphnia pulex, Artemia salina and Tubifex tubifex for cyanobacterial microcystins toxicity detection Ivana KYSELKOVÁ1,2 & Blahoslav MARŠÁLEK1* 1

Institute of Botany, Academy of Science of the Czech Republic, Květná 8, CZ-60200 Brno, Czech Republic; tel.: ++420 5 43241911, e-mail: [email protected]; [email protected] 2 Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Technical University Brno, Czech Republic KYSELKOVÁ, I. & MARŠÁLEK, B., Use of Daphnia pulex, Artemia salina and Tubifex tubifex for cyanobacterial microcystins detection. Biologia, Bratislava, 55: 637-643, 2000; ISSSN 0006-3088. Four water samples from the Czech Republic and one sample from Slovakia containing cyanobacteria Microcystis wesenbergii, M. aeruginosa, Anabaena flos-aquae and Aphanizomenon flos-aquae were tested for microcystins. The content of total microcystins and microcystin-LR were analysed before testing by high performance liquid chromatography (HPLC). The need for more ethical, economic assays is increasing in these days. We used the acute toxicity tests (microbiotests) with two typical zooplankton crustacean species: Artemia salina and Daphnia pulex, and one typical zoobenthic species: Tubifex tubifex. Immobilization of D. pulex and mortality of A. salina and T. tubifex were determined with microcystin-containing and microcystinno-containing cyanobacterial extracts. Extraction and fractionation by C18 cartridges of test samples was undertaken to select and isolate microcystin-containing fractions and reduce interference from other fractions (pigments, carbohydrates, lipids etc.). The high correlation was found between toxic response of all species and the crude extract, and the fraction with toxins. There was no significant correlation between species and the fraction without toxins. The sensitivity of species decrease in the following order: Daphnia pulex, Artemia salina, Tubifex tubifex. The study also showed that fractionation of the crude extract of cyanobacterial biomass can distinguish toxic effects of microcystins proper from those of other toxic compounds present in the crude extract. Key words: cyanobacteria, microcystins, fractionations, acute toxicity tests, Artemia salina, Daphnia pulex, Tubifex tubifex, Czech Republic, Slovakia.

___________________ *Author to whom correspondence should be addressed.


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Introduction The development of massive blooms of cyanobacteria in eutrophicated waters is a current problem in many countries. Freshwater species of cyanobacteria can produce a wide range of toxins including hepatotoxic peptides (microcystins, nodularin), neurotoxic alkaloids, lipopolysaccharides. Other compounds with potentially useful bioactivities, including cytotoxic, antifungal and antiviral effects, have led to the discovery and identification of numerous novel bioactive metabolites including peptides, macrolipids and glycosides (SIVONEN & JONES, 1999; JAMES, 1996; PATTERSON et al., 1994; FAWEL et al., 1993). The occurrence of toxic cyanobacterial blooms in water bodies used for water recreation and potable water supplies can also constitute a hazard to human health through contact and ingestion of cells or released toxins (FALCONER, 1998). Specific, straightforward and rapid procedures are required for the detection, identification, and quantification of the potent low molecular weight toxins that are produced by cyanobacterial blooms in water bodies. The cyanobacterial toxins, as the product of secondary metabolism, that was studied most frequently are mamalian neuro- and hepatotoxins (CARMICHAEL, 1997). At least some of these toxins are also toxic to many invertebrate species (HARADA et al., 1999). Testing, monitoring and quantification of cyanobacterial hepato- and neurotoxins have traditionally been carried out upon the intraperitoneal mouse bioassay (PREMAZZI & VOLTERA, 1993). The chromatographic techniques, especially high-performance liquid chromatography (HPLC) was developed for purification and quantification of cyanobacterial hepatotoxins (JAMES, 1996; HARADA et al., 1999). However, the use of instrumental analysis of hepatotoxins currently requires specialized equipment and skills, and can be very time-consuming. We have examined a short-term (24-hour) toxicity tests with invertebrate species. The tests with Artemia salina (ARTTOXKIT, 1995) and Daphnia pulex (DAPHTOXKIT F PULEX, 1995) are commercially available and standardized as microbiotests. The test using zoobenthic species Tubifex tubifex is very useful because of the different life strategy of this species versus zooplankton crustacean species. The problem is that the short-term test using Tubifex tubifex is not commercially available, so we adopted and modified the short-term test with this species. These tests are typical non-specific tests. The toxicity signal results from the whole spectrum of consequences. Most toxicity studies on aquatic biota are indeed performed under test conditions which are close to optimal ones. In nature, on the contrary, aquatic organisms must cope with environmental conditions which may fluctuate considerably during the year (PERSOONE et al., 1989). The crude extracts of cyanobacterial biomass also give interpretations problems (MARŠÁLEK & BLÁHA, 2000). The aim of this study was to investigate if there was any significant relation (expressed as a correlation coefficient), between content of microcystins (for each sample and fraction) and the toxicological response of each species (expressed as 24 h LC50). Material and methods Collection of cyanobacterial samples In this study five samples of cyanobacterial water blooms from the Czech and Slovak Republic were tested. Each sample was taken from a water reservoir (Tab. 1), located in four regions of the Czech Republic: Brno, Jedovnice, Skalka, Nová Říše, and one from the Slovak Republic: a fishpond in Bratislava-Železná Studienka. The samples were stored thicken and frozen. The samples were analyzed for the content of cyanobacterial microcystins by high performance liquid chromatography (HPLC) according to method described by LAWTON et al. (1994). The quantity of microcystins (Tab. 1) was calculated in µg microcystins to g of dry weight of the sample (µg.g d.w1 ). Table 1 also shows the dominant species in each sample.


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Table 1. Characterizations of samples. Samples Localities No.

Dominate species in sample

Dry part of sample (g. mL-1)

1

Microcystis wesenbergii

0.039

Quantity total-MCYST (µg.g d.w-1) 0

Anabaena flos-aquae

0.0152

0

Aphanizomenon flos-aquae

0.035

8

Microcystis aeruginosa

0.0436

319

Microcystis aeruginosa

0.0172

1440.5

2 3 4 5

Brno recreational Jedovnice recreational Skalka recreatioanal Nová Říše drinking water Železná Studienka, fishpond

Table 2. Colours of the samples. Samples Localities Brno Jedovnice Skalka Nová Říše Železná Studienka

Colour of sample Yellow Light blue-green Dark blue Dark green Green

Fractionation of cyanobacterial biomass For better extrapolations of laboratory bioassays three fractions from each sample were prepared. The first fraction was a crude extract of cyanobacterial biomass, which contained disintegrated cell walls and cell content including microcystins. Preparation of a Crude extract: the samples were defrosted at the time of extracts preparation 20 mL of defrosted sample was taken for homogenization and after homogenization divided into 4 test tubes, 5 mL each; the test-tubes were shaken for 2 min and after 5 min of extraction shaken for 2 min again the samples were centrifuged for 20 min at 5000 cycles per min; after centrifugation the sediment is distinctly stratified from supernatant (crude extract) the supernatant was collected and 5 mL of distilled water was added to the sediment; shaking of test tubes for 2 min and after 5 min of extraction shaking for two minutes followed again; the samples were sonicated for 5 minutes; the last step was centrifugation again and collecting the supernatant (crude extract). The crude extracts of the selected samples were split into 2 subsamples: the first part was stored frozen for subsequent toxicity testing, the second part was fractionated with SPE C18 cartridges. Three sub-parts of the original cyanobacterial biomass were thus prepared and used for toxicity testing: 1. Crude extract 2. Pigment fraction: a water-soluble fraction without microcystins, but containing various compounds such as e.g. pigments, proteins, carbohydrates, lipids, acids, salts, etc; this fraction was prepared from crude extract, which was passed through C18 cartridges.


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3. Toxin fraction with concentrated peptides including microcystins; the toxic peptides (microcystins) were selectively concentrated on the sorbent of cartridge and separated with methanol from water-soluble compounds; methanol must be evaporated up before testing. For better and clear orientation in this problem the following scheme shows a simple design of preparation of all fractions: SAMPLE ↓Precondiciation of cartridge CRUDE EXTRACT ↓ C18 CARTRIDGE ↓ PIGMENT FRACTION ↓ REEXTRACTION C18 CARTRIDGE WITH 50% METHANOL ↓ EVAPORE 50% METHANOL ↓ TOXIN FRACTION Toxicity tests Acute toxicity test with Artemia salina: Acute lethality test with the marine crustacean A. salina. This test is based on larvae hatched from cysts. The 24 h LC50s were calculated and expressed in mg dry biomass per millilitre (ARTOXKIT, 1995). Acute toxicity test with Daphnia pulex: Acute immobilisation test with the fresh water crustacean D. pulex. This test is also based on larvae hatched from cysts. The 24 h LC50s were calculated and expressed in mg dry biomass per millilitre (DAPHTOXKIT F PULEX, 1995). Acute toxicity test with Tubifex tubifex: This test is based on lethality of the aquatic oligochaete Tubifex tubifex. We used the equipment for this test from the DAPHTOXKIT F PULEX (1995) with multiwell test plate composed of 6x5 wells. The tap de-chlorinated water was used as a standard testing medium. The temperature of the water and air was 20 0C. The organisms for testing were acquired from a shop with Aquatic Necessity. The organisms were kept in a glass pot for 24 h. The reference test with potassium dichromate was performed and because the organisms responded very sensitively after 24 h yet, the 24 h test of acute toxicity was shown very sensitive too. We gave 8 organisms to each testing well. Consequently, 32 organisms were tested for each concentration of a sample. Data treatment Toxicity data are expressed in LC50 values. The LC50 value indicates the quantity of dry weight (mg.mL-1) for crude extract and percentage dilution of toxin fraction, which causes 50% mortality (LC) of testing organisms: LC50-24 h values for the ARTOXKIT (1995), DAPHTOXKIT F PULEX (1995) and for the acute toxicity test with Tubifex tubifex were calculated using the MovingAverage methods (EPA/600/4-85/013, 1985). For better orientation in results, the LC50 -24 h acute toxicity values were transformed to Toxicity Units – TU (DAMBORSKÝ et al., 1995): TU = (1/LC50)*100


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The relation between LC50 values obtained from the different tests and the content of total microcystins in each fraction were compared through a Correlation Analysis (Excel, 1995). This results are explained as Correlation coefficient. Results and discussion Three fractions were tested by short-term tests of acute toxicity in this study. Table 3 shows the LC50 values, for better orientation explained in milligrams of dry weight in one millilitre (mg.mL-1 d.w.) and charged into toxic units obtained from crude extract. Table 4 shows the values LC50 (explained in: mg.mL-1 d.w.) and transformed in toxic units, obtained from crude extract. Table 4 shows the LC50 values obtained from the toxin fraction and the values are explained as a percentage proportion of dilution of the clear toxin fraction, these values are also transformed into toxic units. Table 3. LC50 for the crude fraction, 95% confidence intervals (C.I.), and toxic units (TU). Species Artemia salina

Daphnia pulex

Tubifex tubifex

Sample Brno Jedovnice Skalka Nová Říše Železná Studienka Brno Jedovnice Skalka Nová Říše Železná Studienka Brno Jedovnice Skalka Nová Říše Železná Studienka

LC 50 0 0 7.9 1.1 0.7 9.2 0 0.8 1.5 0.5 14.5 0 4.8 7.2 2.5

C.I. 6.16-9.8 0.8-2.91 0.58-0.93 7.4-10.87 0.54-1.03 0.95-2.26 0.41-0.81 12.3-17.5 3.2-6.1 6.47-9.51 0.98-3.04

TU 0 0 12.65 90.9 142.8 10.86 0 125 66.66 200 6.89 0 20.83 13.88 40

Table 4. LC50 for the pigment fraction, 95% confidence intervals (C.I.), toxic units (TU). Species Artemia salina

Daphnia pulex

Tubifex tubifex

Sample Brno Jedovnice Skalka Nová Říše Železná. Studienka Brno Jedovnice Skalka Nová Říše Železná. Studienka Brno Jedovnice Skalka Nová Říše Železná. Studienka

LC 50 0 0 7.5 8.4 1.6 0 0 1.5 1.9 0.4 0 0 14.7 12.6 3

C.I. 5.6-8.75 6.12-9.4 0.94-2.06 0.95-2.32 0.98-2.91 0.28-0.76 12.67-17.3 11.06-13.9 2.63-4.07

TU 0 0 13.33 11.9 62.5 0 0 66.66 52.63 250 0 0 6.8 7.9 33.33


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Table 5. LC50 for the toxin fraction, 95% confidence intervals (C.I.), toxic units (TU). Organism Artemia salina

Sample LC 50 Brno 0 Jedovnice 0 Skalka 12.9 Nová Říše 6.8 Železná Studienka 7.8 Brno 0 Jedovnice 0 Skalka 6.06 Nová Říše 4.13 Železná Studienka 2.01 Brno 0 Jedovnice 0 Skalka 21.85 Nová. Říše 17.1 Železná Studienka 8.3

Daphnia pulex

Tubifex tubifex

Toxic unites

250

C.I. 8.66-19.44 4.3-18.81 5.42-10.74 4.73-8.2 2.99-6.02 0.99-1.74 17.27-28.76 13.32-21.84 6.47-10.5

TU 0 0 7.75 14.71 12.82 0 0 16.5 24.24 49.8 0 0 4.58 5.8 12.05

Artemia

200

Daphnia

150

Tubifex

100 50

d.

Ž. St u

íš e .Ř N

lk a Sk a

do v Je

Př ís ta vi

št ě

ni ce

0

60 50 40 30 20 10 0

Artemia Daphnia Tubifex

Př ís

ta vi št ě Je do vn ic e Sk al ka N .Ř íš e Ž. St ud .

Toxic unites

Fig. 1. Crude extract: The effects of samples with different content of microcystins (µg.g-1 d.w.): Přístaviště 0; Jedovnice 0; Skalka 8; Nová Říše 319; Železná Studienka 1440.5; which were tested in three organisms. The toxic units were calculated from LC50 (mg.g-1 d.w.).

Fig. 2. Toxin fraction: The effects of samples with different content of microcystins (µg.g-1 d.w.): Přístaviště 0; Jedovnice 0; Skalka 8; Nová Říše 319; Železná Studienka 1440.5; which were tested in three organisms. The toxic units were calculated from LC50 (percentage proportion of dilution).


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The fractionation of cyanobacterial biomass was proved to be a useful method for discrimination between samples with different content of microcystins. For significant response evaluation of invertebrate species to dose of microcystins in the samples, the correlation analysis was calculated. Table 6 shows the results of correlation coefficient. Table 6. The Correlation coefficient between both crude fraction and toxin fraction and microcystins concentration for each species. n=9. Organism Artemia salina Daphnia pulex Tubifex tubifex

Crude fraction 0.925 0.853 0.852

Toxin fraction 0.979 0.992 0.962

The separation of water-thicked samples into three fractions by extractions of solid phase (C18 cartridges) demonstrates the toxicity of cyanobacterian extracts has not only been given the contents of microcystins. Various methodological problems with cyanobacterial samples often occur, which influence the outcome of the toxicity tests (MARŠÁLEK & BLÁHA, 2000). For example, testing of crude extracts of cyanobacterial biomass can be problematic because of the presence of various (generally non-toxic) compounds such as e.g. organic acids, amino-acids, ions, pigments and proteins which in high concentrations influence the physico-chemical characteristics of the test medium (e.g. pH or oxygen content). Test organisms can react to these changes very sensitively and in such cases the effects observed are not necessarily related to the presence or the quantity of cyanobacterial toxins (MARŠÁLEK & BLÁHA, 2000). The pigment fraction, the water soluble one, had toxic effect to organisms in our study too, because this fraction contained other compounds (described above), which should be toxic directly or indirectly too. Solid phase extraction method was also used by CAMPBEL et al. (1994). It is not quite clear, if all peptide toxins are separated by SPE-C-18 cartridges and this could be also the source of toxic signal in pigment fraction. In our study, the fractionation of biomass helped to remove the influences of the compounds described above. In consequence, the animals were stressed during the experiment, and may therefore have reacted even to small toxin concentrations (REINIKAINEN et al., 1995). For example the sample Skalka has very low content of microcystins (8 µg.g-1d.w.), but very high toxicology response in crude fraction. The colour of this sample was dark blue. After separation by C18 cartridges the toxicity response was more relative to content of microcystins. This study demonstrated the usefulness of bioassays in detecting the acute toxicity of three fractions of cyanobacteria biomass from water reservoirs. Three tests used in this study were found to be simple, rapid and especially cost–effective. All species provided significant discrimination ability between the presence and absence of microcystins. But although the correlation coefficients are very high for each species, especially for toxin fraction, we must be careful with conclusions because only five samples were tested. The crude extracts from samples Brno and Jedovnice had very light colour and no microcystin content, at least no toxicological response of organisms. We suppose, that light color of extracts (with no content of microcystins) can cause modest toxic responses of the testing organisms. The test using Daphnia pulex – an important grazer in small pond ecosystems – seems to be the most sensitive of the compared bioassays (Tab. 6) for screening detection of microcystins present in the cyanobacterial samples. The test using Artemia salina is time- and cost-effective for cyanobacterial toxicity screening because it is easy to cultivate, and because only small volumes of water are needed in toxicity tests. However, since the testing samples were from fresh water reservoir, whereas A. salina is a marine one, and since daphnids and some other cladocerans are generally more typical feeders on planktonic cyanobacteria than A. salina, we felt it is ecologically relevant to test the effect of this compound on D. pulex as well. D. pulex also gave the highest


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"detection power" in this study (Tab. 6). REINIKAINEN et al. (1995) investigated the effects of Microcystis aeruginosa on the survival of juvenile and adult D. pulex in different food concentration of green alga Scenedesmus quadricauta. Microcystis aeruginosa reduced survival in D. pulex and the toxic effect decreased with increasing concentration of cyanobacteria (REINIKAINEN et al., 1995). MARŠÁLEK & BLÁHA (2000) recommend using crustacean Artemia salina and Thamnocephalus platyurus, which were definitely the most sensitive biota for analysis of cyanobacterial toxins in their study. Both A. salina and T. platyurus exhibited good discriminative power between the toxic and non-toxic samples with respect to their content in cyanobacterial hepatotoxin microcystin-LR. Since A. salina is a marine organism, the use of T. platyurus appears to be the most promising assay of future testing of the acute toxicity of toxic cyanobacterial waterblooms (MARŠÁLEK & BLÁHA, 2000). KIVIRANTA & ABDEL-HAMEED (1994) tested larvae of the laboratory strain Aedes aegyptii, which was found to be sensitive to hepato- and neurotoxin producing cyanobacteria. MARŠÁLEK & BLÁHA (2000) used the acute lethality test with the rotifer Brachionus calyciflorus, but this organism was not sensitive to the hepatotoxins in the samples. They also tested twenty-four-hour growth inhibition test with the ciliate protozoan Tetrahymena pyriformis. Because the protozoan can also feed on nutritional compounds present in the extracts from cyanobacterial biomass which may lead to additional interferences as well, this microbiotest was not considered as a good tool for screening cyanobacterial toxins. The using of oligochaetes Tubifex tubifex for cyanobacterial toxicity screening is useful, because T. tubifex is an organism from the sediment with different life strategy than the plankton crustaceans. Acknowledgements Grateful acknowledgement is extended to the CYANOTOX project (ENV4-CT98 0802, EU DGXII). References ARTTOXKIT 1995. Crustacean Toxicity Screening Test for saline water. SOP, STANDARD OPERATION PROCEDURE. Creasel, Belgium. CAMPBELL, D. L., LAWTON L. A., BEATIE K. A. & CODD G. A. 1994. Comparative assessment of the specifity of the brine shrimp and Microtox assay to hepatotoxic (microcystin LR containing) cyanobacteria. Environm. Tox. Wat. Qual. 9: 71-77. CARMICHAEL, W. W. 1997. The Cyanotoxins. Advances in Bot. Research 27: 211-256. DAMBORSKÝ, J., ROJÍČKOVÁ, R., MARŠÁLEK, B. & HOLOUBEK, I. 1995. Využití alternativních biotestů pro ekotoxikologický monitoring II: Statistická analýza toxikologických, chemických a mikrobiologických dat. – Toxicita a biodegrebilita odpadů a látek významných ve vodním prostředí. Aquachemie, Ostrava, pp. 47-52. DAPHTOXKIT F PULEX.1995. Crustacean Toxicity Screening Test for fresh water. SOP, STANDARD OPERATION PROCEDURE. Creasel, Belgium. EPA/600/4-85/013. 1985. Methods for measuring the acute toxicity of effluents to freshwater and marine organisms, pp. In: PELTIER, W. H. &WEBER, C.I . (eds), Toxicity Data Analysis, Creasel, Belgium. FALCONER, I. R. 1998. Algal toxin and human health. The handbook of Environmental Chemistry, 5(c): 54-77.


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FAWEL, J. K., HART, J., JAMES, H. A. & PARR, W. 1993. Blue-green algae and their toxins-analysis, toxicity, treatment and environmental control. Water Supply 11: 109-121. HARADA, K., KONDO, F. & LAWTON, L. 1999. Laboratory analysis of cyanotoxins, pp. 369-399. In: CHORUS, I. & BARTRAM, J. (eds.), Toxic Cyanobacteria in water – a guide to their public health consequences, monitoring and management, World Health Organization, London. JAMES, H .A. 1996: Analysis of blue-green algal toxins in drinking waters. Drinknet workshop on drinking water analysis, Praha, 23. 5. 1996. KIVIRANTA, J. & ABDEL-HAMEED, A. 1994. Toxicity of blue-green alga Oscillatoria agardhii to the mosquito Aedes aegypti and the shrimp Artemia salina. World J. Microbiol. Biotechn. 10: 517520. LAWTON, L. A., EDWARDS, CH. & CODD, G. A. 1994. Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analys. 119: 1525-1530. MARŠÁLEK, B., & BLÁHA, L. 2000. Microbitests for cyanobacterial toxins screening, pp. 519-525. In: PERSOONE, G., JANSSEN, C. & DE COEN, W. (eds), New microbiotests for routine toxicity screening and monitoring, Kluwer Academic/Plenum Publishers, London. PATTERSON, G. M. L., LARSEN, L. K. & MOORE, R. E. 1994. Bioactive natural products from bluegreen algae. J. Applied Phycol. 6: 151-157. PERSOONE, G., VAN DE VEL, A., VAN STEERTEGEM, M. & DE NAYER, B. 1989. Predictive value of laboratory tests with aquatic invertebrates: influence of experimental conditions. Aquatic Toxicology 14: 149-166. PREMAZZI, G. & VOLTERA, L. 1993. Microphyte toxins. A manual for toxin detection, environmental monitoring and therapies to counteract intoxications. Commission EC, Luxembourg. REINIKAINEN, M., KETOLA, M. & WALLS, M. 1995. Effects of the concentrations of toxic Microcystis aeruginosa and an alternative food on the survival of Daphnia pulex. Limnol. Oceanogr. 39: 424-432. SIVONEN, K. & JONES, G. 1999. Cyanobacterial toxins, pp. 41-91. In: CHORUS, I. & BARTRAM, J. (ed.), Toxic Cyanobacteria in water – a guide to their public health consequences, monitoring and management, World Health Organization, London.