Marine Pollution Bulletin 50 (2005) 956–964 www.elsevier.com/locate/marpolbul
Cyanobacterial blooms in estuarine ecosystems: Characteristics and effects on Laeonereis acuta (Polychaeta, Nereididae) Carlos E. da Rosa
a,b
, Ma´rcio S. de Souza c, Joa˜o S. Yunes d, Luis A.O. Proenc¸a e, Luiz E. M. Nery a,b, Jose´ M. Monserrat a,b,*
a
e
Departamento de Cieˆncias Fisiolo´gicas, Fundac¸a˜o Universidade Federal do Rio Grande (FURG), R. Eng Alfredo Huch 475, 96201-+55 53900, Rio Grande, Brazil b Programa de Po´s-graduac¸a˜o em Cieˆncias Fisiolo´gicas, Fisiologia Animal Comparada (PGCF-FAC), FURG, Brazil c Laborato´rio de Fitoplaˆncton e Microorganismos Marinhos, Departamento de Oceanografia (FURG), Brazil d Unidade de Pesquisa em Cianobacte´rias, Depto. de Quı´mica, FURG, Brazil Universidade do Vale de Itajaı´ (UNIVALI), Centro de Cieˆncias Tecnolo´gicas da Terra e do Mar (CTTMar), Santa Catarina, SC, Brazil
Abstract In January of 2003, a cyanobacterial bloom in the PatosÕ Lagoon (Southern Brazil) (3205 0 S–5212 0 W) was observed. Water samples were taken to identify the composition and abundance of the bloom, as well as the occurrence of toxins. The effects of this occurrence on the estuarine worm Laeonereis acuta (Polychaeta, Nereididae) was also evaluated. Predominance of cyanobacteria, particularly Anabaena trichomes (2.5.106 individuals per liter) was observed, and low concentrations of microcystins and anticholinesterasic toxins were detected. Augmented levels of lipid hydroperoxides (LPO) and glutathione-S-transferase activity, and lowering of total protein content were also observed in organisms collected during the bloom event. Although non-toxic, the cyanobacterial bloom could augment the cycle of hyper-oxygenation and hypoxia in the water. During hyperoxia, L. acuta, an oxyconformer, should consume more oxygen, thus augmenting the rate of reactive oxygen species generation. A repeated cycle of hyperoxygenation and hypoxia would finally induce oxidative stress, as evidenced by the high levels of LPO and glutathione-S-transferase activity. 2005 Published by Elsevier Ltd. Keywords: Cyanotoxins; PatosÕ Lagoon; Laeonereis acuta; Oxidative stress; Antioxidant enzymes; Cholinesterase
1. Introduction The presence of cyanobacterial blooms in natural and artificial water bodies has been frequently reported around the world (Christoffersen, 1996). These photoautotrophic microorganisms are well represented in a wide range of habitats, where they reach an overwhelming dominance (Vance, 1965; Reynolds, 1997). The bloomforming cyanobacterial events can have potential health hazards to humans and aquatic fauna. Harmful effects *
Corresponding author. Tel.: +55 53 2338695; fax: +55 53 2338680. E-mail address:
[email protected] (J.M. Monserrat).
0025-326X/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2005.04.004
extend up to the ecosystem level by changing species interaction and community structure (Lehtonen et al., 2003). Some cyanobacteria produce toxins as secondary metabolites. Molecular structures of about 60 toxin variants produced by brackish and freshwater cyanobacteria are known to be hepatotoxic (microcystin and nodularin), neurotoxic (anatoxin-a, anatoxin-a(s), saxitoxin), or to cause allergenic reactions or irritation (LPS) (Codd, 1995). The toxicity of hepatotoxins is exerted by effective inhibition of serine/threonine phosphatases (PP1/PP2A), resulting in a hyperphosphorylation of many kinds of hepatic functional proteins (Lehtonen et al., 2003). This hyperphosphorylation causes cyto-
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
skeletal deformation in hepatocytes, collapse of liver architecture, profuse hemorrhage and necrosis (Lyu et al., 2002). The neurotoxins are toxic through several mechanisms. The anatoxin-a is an analogue of the neurotransmitter acetylcholine, which cannot be degradated by acetylcholinesterase (Charmichael, 1994). The anatoxin-a(s) is a natural organophosphate which exerts their toxicity by the inhibition of acetylcholinesterase (Charmichael, 1994; Monserrat et al., 2001). Although the molecular targets of several cyanotoxins are well established, other alternative mechanisms of toxicity can be considered, such as oxidative stress (Ding et al., 1998a,b). Pflugmacher et al. (1998) have demonstrated the existence of a microcystin–glutathione conjugate formed enzimatically by glutathione-S-transferase activity. This process can induce a depletion of the cellular glutathione (GSH) pool, favoring oxidative stress, since GSH is the main non-enzymatic antioxidant defense and constitutes the first line of defense against reactive oxygen species (Sies, 1999). These mechanisms of conjugation have been described in various aquatic organisms, ranging from plants to fish (Pflugmacher et al., 1998; Beattie et al., 2003). Some works have demonstrated that oxidative stress and/or antioxidant responses are induced by cyanobacterial toxins (Ding et al., 1998b; Guzman and Solter, 1999; Vinagre et al., 2003). However, cyanobacterial blooms can exert other toxicological effects, related to variations in oxygen concentration. Seki et al. (1979) have observed that, during a bloom event, the profiles of dissolved oxygen in the water column augmented its amplitude, ranging from 0 (at night) to 190% (at day) of normal saturation. Under such conditions, the biota in these regions must cope with cycles of anoxia–hyperoxia resembling the well-known physiological process of ischemia-reperfusion (Halliwell and Gutteridge, 1999). Several animal species present well-developed mechanisms to avoid the deleterious effects caused by variations on oxygen availability. These known mechanisms include metabolic rate depression (Hochachka and Somero, 1984; Storey, 1996a; Hochachka and Lutz, 2001), and maintenance and/or increase of antioxidant defense systems previous to the re-oxygenation period (Hermes-Lima et al., 1998; Lushchak et al., 2001). In the PatosÕ Lagoon, the largest lagoonal system in South America, the occurrence of cyanobacterial blooms, dominated by the genus Microcystis, have been registered irregularly during the last years (Odebrecht et al., 1987; Yunes et al., 1998). A typical inhabitant of this environment is the estuarine benthonic worm Laeonereis acuta (Polychaeta, Nereididae), a selective deposit feeder, with high abundance and biomass, occurring in the Atlantic coast of South America from Recife (Northeastern Brazil) to Penı´nsula de Valdez (Southern Argentina) (Omena and Amaral, 2001). A previous study showed that this animal is susceptible
957
to oxidative stress when exposed to metals (Geracitano et al., 2002), showing different antioxidant profiles in populations sampled at unpolluted and polluted sites (Geracitano et al., 2004). The objective of the present study was to characterize the cyanobacterial bloom occurred in January 2003 in the PatosÕ Lagoon (Southern Brazil). The putative harmful effect of the bloom on Laeonereis acuta was accessed considering the pro-antioxidant balance. Also, the activity of the enzyme cholinesterase (ChE) was measured as a biomarker of the presence of anatoxin-a(s) in the water.
2. Material and methods 2.1. Samples collection Water and animals samples were collected after 7, 10 and 16 days of the beginning of the bloom. Previously to this and after the end of bloom (35 and 77 days, respectively), additional animal samples were collected. Water samples from surface layer were divided in two aliquots: one fixed in formaldehyde (10%) and other stored to 20 C for posterior chemical analysis. The samples were collected at one station located at ‘‘Saco do Justino’’ in the PatosÕ Lagoon (32C05 0 S– 52C12 0 W) (Fig. 1). 2.2. Algal identification and enumeration The phytoplanktonic organisms were observed under a transmitted and inverted light microscope (ZEISS Axiovert 135). They were mainly identified and counted at genus level using modified sedimentation chambers with 10 ml aliquots (Utermohl, 1958; Sournia, 1978) at 400· to study the community composition and abundance. For identification, specific taxonomic informations were used (Bourrelly, 1972; Drebes, 1974; Koma´rek and Anagnostidis, 1989; Round et al., 1990; Anagnostidis and Koma´rek, 1996; Hasle and Syvertsen, 1996; Koma´rek and Anagnostidis, 2000). The density of the organisms is expressed as individuals/l (ind./l). An individual was considered an isolated unit of cells, such as cyanobacteria trichomes, colonies and cenobia, of certain chlorophytes and other cyanobacteria. Unidentified centric and pennate diatoms were counted using size classes. 2.3. Microcystin detection assays Water samples were frozen, thawed three times and then centrifuged (12,000 · g) at room temperature, for 10 min. The supernatant was collected and microcystins content determined using a commercial enzyme-linked immunoassay (ELISA) with policlonal antibodies
958
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
Fig. 1. Site of Laeonereis acuta collection during the cyanobacterial bloom at Patos Lagoon estuary (Southern Brazil; 3205 0 S–5212 0 W).
(EnviroLogix Inc., Portland, ME), according to Vinagre et al. (2003). 2.4. Animal tissues homogenates preparation Whole animals were homogenized (25% W/V) in cold buffer containing Tris 20 mM, EDTA 1 mM, dithiothreitol (DDT) 1 mM, sucrose 500 mM, KCl 150 mM and phenylmethylsulfonyl fluoride 0.1 mM, with pH adjusted to 7.60 (Geracitano et al., 2002). After a centrifugation at 9000 · g at 4 C during 45 min the supernatant was stored (80 C) for posterior enzymatic assays. 2.5. Biochemical measurements in tissue homogenates Total protein content of tissue extracts was determined using a commercial diagnostic kit (Doles Reagentes LTDA, Goiaˆnia, GO, Brazil) based in Biuret reagent. The determinations were done at least in duplicate, at 550 nm. All enzyme assays were conducted as previously described (Geracitano et al., 2002), except for cholinesterase (ChE). Briefly, the activity of catalase (CAT) was measured by following the initial rate of 50 mM H2O2 (Merck) decomposition at 240 nm (Beutler, 1975). The results were expressed in CAT units/mg protein and CAT units/g wet weight, were one unit is the amount of en-
zyme hydrolyzing 1 lmol of H2O2 per minute and per g of protein or wet weight (ww), at 30 C and pH 8.00. Superoxide dismutase (SOD) activity was determined according to McCord and Fridovich (1969). In this assay superoxide anion is generated by the xanthine/xanthine oxidase system and the reduction of cytochrome c monitored at 550 nm. Enzyme activity is expressed as SOD units/mg of protein and SOD units/g of ww, where one unit is defined as the amount of enzyme needed to inhibit 50% of cytochrome c reduction per minute and per g of protein or ww at 25 C and pH 7.80. Glutathione S-transferase (GST) activity was measured by monitoring the formation of a conjugate between 1 mM GSH and 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB, from Sigma) (at 340 nm) (Habig et al., 1974; Habig and Jakoby, 1981). The results are expressed in GST unit/mg of protein and GST unit/g wet weight, where one unit is defined as the amount of enzyme that conjugate 1 lmol of CDNB per minute and per mg of protein or ww at 30 C and pH 7.4. ChE activity was measured according to Ellman et al. (1961) and adapted to worms by Rao et al. (2003), using DTNB (5,5 0 -dithio-bis (2-nitrobenzoic acid); 0.5 mM, from Sigma) and acetylthiocoline iodide (7.5 mM, from Sigma) as substrate, monitoring the change of absorbance at 412 nm at 25 C and pH 7.2. Results are expressed in nmoles of acetylthiocholine iodide hydrolyzed per minute and per mg of protein or g of ww. The acet-
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
ylthiocoline iodide concentration employed in L. acuta ChE activity determination was selected after kinetic assays with different substrate concentrations in the same conditions previously described.
959
chambers, with a volume of 10 ml, containing brackish water (10 &) and different oxygen concentrations, ranging from 3.7 to 19 mg O2/l, were maintained at 20 C. Oxygen concentrations were measured with an oxymeter (Digimed). VO2 is expressed as mg O2/h/g ww.
2.6. Lipid peroxidation assay 2.9. Statistical analysis Lipid peroxidation was measured according to Monserrat et al. (2003). Whole L. acuta were homogenized in methanol (10% W/V) and centrifuged at 1000 · g, for 10 min. Lipid hydroperoxides were determined using FeSO4 (0.25 mM) prepared immediately before use, H2SO4 (0.25 mM), xylenol orange (1 mM, from Sigma). Samples absorbance (580 nM) were measured in microplate reader after 1 h of incubation at room temperature and quantified in terms of cumene hydroperoxide (CHP, from Sigma) equivalents, which was used as standard (5 nmol/ml). 2.7. In vitro acetylcholinesterase inhibition by bloom water samples The effect of bloom water samples on eel (Electrophorus electricus) electrogenic organ purified acetylcholinesterase (AChE) (V-S type, from Sigma) was determined in vitro in order to estimate the percentual inhibition of enzymatic activity, according to Monserrat et al. (2001). The purified enzyme (50 ll of a 0.25 U/ml solution) was dilluted in 950 ll of phosphate buffer (50 mM) containing 20% of glycerol at pH 7.4. To determine the AChE activity DTNB (0.4 mM) and acetylthiocoline iodide (0.8 mM) as substrate were employed. In order to evaluate the presence of anticholinesterasic compounds in bloom water samples, several aliquots, ranging from 1.25% to 5% of the final reaction volume (1 ml), were used. Samples were incubated for 1 h at 25 C. After that, the substrate and DTNB were added, and enzyme activity measured as previously described. In addition, eserine inhibition tests were conducted in order to evaluate the responsiveness of the purified eel acetylcholinesterase to a known inhibitor. For this purpose, the bloom water samples were substituted for eserine solutions in a final concentration of 5.6 and 1.2 · 106 M and incubated 1 h at 25 C. The acetylcholinesterase activity was determined as described above. Results are expressed as relative inhibition (percentual) in respect to the control group (AChE activity in absence of water samples). 2.8. Determination of oxygen consumption rates (VO2) by L. acuta at different water oxygen concentration Oxygen consumption rate of individuals specimens of L. acuta, collected in August 2003 in ‘‘Saco do Justino’’, weighting 83.37 ± 5.1 mg (mean wet weight ± S.E.), were measured according to Nithart et al. (1990). Respiration
Values for all enzymatic determinations were computed as means ± standard error (±SE). Statistical analysis was performed by means of analysis of variance followed by Newmann–Keuls test or polynomial contrasts (a = 0.05). Normality and variance homogeneity were previously verified (Zar, 1984).
3. Results During the bloom period, of approximately 3–4 weeks, the mean (±SE) values of pH and temperature of the water during the sampling time (10:00–14:00 h) were 9.55 ± 0.59 and 27 ± 1.53 C, respectively. Analysis of fixed water samples revealed a clear predominance of certain cyanobacterial genera during the bloom period (Table 1). The genera Anabaena, Aphanocapsa, Merismopedia and Snowella were the most predominant photoautotrophic constituents, with maximum abundance values of 2.96, 4.60, 7.74 and 2.41.106 ind./l, respectively. Specimens of Anabaena spp. were characterized by more than 20 lm long, straight or spiral filamentous (trichomes) structures, while Aphanocapsa spp., Merismopedia spp. and Snowella spp. were only observed forming smaller colonies. Cyanobacteria were characterized by frequency values between 75.9% and 83.5%, followed by chlorophytes (13.4% and 21%) and diatoms (3.1% and 4.5%) (Table 1). Cyanobacteria and chlorophytes stood out among the other phytoplanktonic groups, i.e. diatoms and euglenids, either in number of taxa or by their higher frequency values in the samples. The detection tests of microcystin in water samples reveal low levels of this toxin during the bloom period. The mean toxin value during the sampling period was 0.29 ± 0.14 lg/l, with the highest value of 0.46 lg/l recorded at day 77. The total protein content of tissue extracts of L. acuta varied during the sampling period, being highest before the beginning of the bloom (35 days) (23.57 ± 1.78 mg/ ml) returning to mean (17.17 ± 0.54 mg/ml) values in the next sampling periods. For this reason, the enzyme determinations realized in all the homogenates were expressed in terms of the total protein content and in wet weight basis. The whole body homogenates of L. acuta reveals no significant differences (p > 0.05) in ChE activity during the sampling period, remaining stable. Mean values
960
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
Table 1 Composition and succession of cyanobacterial blooms in ‘‘Saco do Justino’’ (Patos Lagoon, Southern Brazil; January 2003) Organisms Cyanobacteria Anabaena spp. Anabaenopsis sp. Aphanizomenon sp. Aphanocapsa spp. Cyanodictyon sp. Gomphosphaeria spp. Merismopedia spp. Planktolyngbya limnetica Pseudanabaena sp. Snowella spp. Spirulina sp. Other cyanobacteria
7
10
2.96 0.04 0.04 2.11 0.13 0.09 0.69 0.08
(34.1) (61) (61) (24.3) (610) (61) (610) (61)
1.93 0.14 0.13 4.21 0.39 0.21 0.52 0.09
16 (16.9) (610) (610) (36.8) (610) (610) (610) (61)
2.79 (11.0) 0.83 (610) 0.34 (610) 4.6 (18.1) 0.09 (61) 0.43 (610) 7.74 (30.4) 0.4 (610)
0.11 (610) 0.3 (610) 0.02 (61) 0.01 (61)
0.04 (61) 1.29 (11.3) 0.01 (61) 0.6 (610)
0.26 2.41 0.02 0.87
(61) (610) (61) (610) (610) (61) (61) (610)
– – 0.21 (610) 0.05 (61)
0.1 (61) 0.002 (61) 0.17 (610) 0.01 (61)
0.45 0.08 0.17 0.27
0.01 (61) –
– 0.08 (61)
0.1 (61) 0.07 (61)
Euglenophyceae
–
–
0.006 (61)
0.13 (610)
0.13 (610)
0.01 (61)
– 0.13 (610) 0.47 (610) – 0.21 (610)
0.002 (61) 0.09 (61) 0.39 (610) 0.13 (610) 0.04 (61)
0.01 0.21 0.82 0.13 –
0.08 0.21 0.17 0.18 – 0.13
0.12 – 0.24 0.12 0.21 0.04
0.29 (610) 0.13 (61) 0.84 (610) 0.3 (610) 0.39 (610) 0.17 (61)
(61) (610) (610) (610) (610)
0.11 (610)
(61) (610) (61) (610) (61)
0.02 (61)
nmols/(min x mg of protein) nmols/(min x g of wet weight)
0.3
Diatomophyceae Aulacoseira spp. Cyclotella sp. >20 lm Cyclotella spp. 0.05). Data is expressed as mean ± 1 S.E. of at least four samples per sampling time. Left axis indicates enzyme activity per mg of proteins. Right axis represents enzyme activity per g of wet weight.
(±SE) varied between 0.096 ± 0.013 and 0.156 ± 0.003 nmoles/min/mg protein (751.17 ± 15.15 and 1135.55 ± 323.78 nmoles/min/g ww) (Fig. 2). The inhibition of purified eel AChE was very low, about 4.85 ± 6.84 % (mean values ± SE), and do not follow a dose response relationship for the dilutions of all water samples. The inhibition registered with a known AChE inhibitor like eserine was higher than 95% and confirmed the responsiveness of the purified eel AChE (Table 2). Also, during all the sampling period CAT and SOD activities remained almost constant (p > 0.05), ranging from (mean values ± SE) 2.06 ± 0.17 to 3.46 ± 0.97 U CAT/mg of protein (132.15 ± 10.82–257.41 ± 45.78 U CAT/g ww) and 17.70 ± 1.90 to 30.183 ± 5.26 U SOD/ mg of protein (1354.71 ± 363.83–1852.55 ± 320.6 U SOD/g ww), respectively (Fig. 3). GST activity fitted to a second-order function, showing a peak in sampling day 10 (p < 0.05). At the end of the sampling period (77 days), GST activity was similar
Table 2 Inhibition of purified eel acetylcholinesterase (AChE, V–S type) activity after 1 h exposure to different dilution of aqueous extracts of the water bloom samples Final dilution or concentration
5% 3.75% 2.5% 1.25% 5.6 · 106 M 1.2 · 106 M
Samples 7
10
16
77
Eserine
Ni Ni 19.7 ± 7.8 14.8 ± 1.8 – –
7.15 ± 7.5 3.92 ± 2.18 3.22 ± 5.89 7.45 ± 6.59 – –
3.36 ± 5.02 Ni 2.20 ± 6.25 12.40 ± 6.66 – –
Ni Ni Ni 4.06 ± 2.54 – –
– – – – 97.29 ± 0.09 96.05 ± 0.78
The values are expressed in % of activity inhibition ± S.E. compared to control activity. Seven, 10, 16 and 77 are the four different sampling periods during the bloom (see the text for definitions). The eserine concentrations employed were 5.6 and 1.2 · 106 M. Ni: no inhibition registered.
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
Catalase
Superoxide dismutase U /mg of protein
U/g of protein U/g of wet weight 5
400
A A
A a
4 a
a
A a
3
320
80
7
10
a
A
A a
16
0
77
2000 1500
A
1000
160
1
- 35
a
a
20
a
2500
A a
30
240
2
U /g of wet weight
40
A
A
0
961
10
0
500
-35
7
10
16
77
0
Time (days)
Time (days) Glutathione S-transferase
Lipid peroxidation
U/mg of protein U/g of wet weight
0.03
1.5
b b
B
0.02
b
a
b 500
0.9 a A
0.6
A
a 250
0.3 0.00
- 35
7
10
nmoles CHP/g of wet weight
1.2
B B
0.01
750
16
77
0.0
0
a
a
- 35
7
a
10
16
77
Time (days)
Time (days)
Fig. 3. Activities of catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferase (GST) and lipid peroxidation (LPO) in the worm Laeonereis acuta during the bloom period. Enzymatic activities were normalized in both, tissue extract total protein content and wet weight basis. Equal letters indicate absence of significant difference between means (p > 0.05). Data is expressed as mean 1 S.E. of at least four samples per sampling time. Left axis indicates enzyme activities per mg of proteins. Right axis represents enzyme activities per g of wet weight.
to that registered at the beginning (35 days). Mean values ± SE varied between 0.006 ± 0.001 and 0.016 ± 0.004 U GST/mg of protein (0.51 ± 0.11 and 1.12 ± 0.2 U GST/g ww) (Fig. 3). The lipid hydroperoxides content in L. acuta reveals a similar pattern to that of GST activity A peak (p < 0.01) after 10 days of the beginning of the bloom respect to the other periods was registered. The values varied between 87.24 ± 14.75 (35 days) and 508.33 ± 76.65 (10 days) nmol CHP/g ww (Fig. 3). Finally, the oxygen consumption (VO2) profile of L. acuta under different [O2] in water, reveals that this animal is an oxyconformer. The VO2 varies from 0.0460.006 mg O2/(h · g ww) under 3.7 mg O2/l to 0.85 ± 0.11 mg O2/(h · g ww) under 19.16 mg O2/l (Fig. 4).
O2 consumption (mgO2 x h x g) 1.2
0.8
0.4
0.0 0
5
10
15
20
25
[O2] in water (mg/l)
4. Discussion
Fig. 4. Rates of oxygen consumption [mg O2/(h · g ww)] versus oxygen concentration in the water (mg O2/l) at 20 C and 10& salinity by Laeonereis acuta. Data are the mean ± 1 S.E. of oxygen consumption of five animals per [O2] sampled. The line indicates the linearregression curve (R2 = 0.96).
The community composition and abundance observed in this study are different from others carried
out in the same, or close, areas during previous summer periods (Jesus and Odebrecht, 2002; Bergesch and
962
C.E. da Rosa et al. / Marine Pollution Bulletin 50 (2005) 956–964
Odebrecht, 1997; Persich et al., 1996; Yunes et al., 1994, 1998). These authors cited high density (up to 106 cells l1) of small-unidentified cyanobacteria (
