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The effects of allelochemicals
...
and selected anthropogenic
substances on the diatom
Bacillaria paxillifera
Sylwia Śliwińska-Wilczewska, Zuzanna Sylwestrzak, Jakub Maculewicz, Aleksandra Zgrundo, Filip Pniewski, Adam Latała
Summary:
In aquatic ecosystems, many different substances can degrade the environment and negatively affect the func-tioning of marine, brackish and freshwater organisms. The main aim of this work was to provide a short sum-mary on the effects of allelochemicals versus herbicide Roundup®, ionic liquid [BMIM]Cl and copper chloride II on the Baltic diatom Bacillaria paxillifera. The influence of allelopathic compounds on the growth was investi-gated by addition of a cell-free filtrate of the cyanobacte-rium Nodularia spumigena cultures grown under specific light, temperature and availability of nutrients. Signifi-cant changes in diatom abundance were observed in re-sponse to all analyzed allelochemicals and anthropogenic substances. A most negative effect of allelochemicals was observed after 7th day of cyanobacterial cell-free filtrate
addition. Moreover, this study showed that after addition of copper(II) chloride, the growth of analyzed diatom was 40% lower in comparison to the control. This work showed that the tested allelopathic compounds and an-thropogenic substances have negative impact on the ana-lyzed diatom, but allelochemicals showed slightly weaker effect than the herbicide.
Key words: diatom, cyanobacteria, anthropogenic substances,
allelochemicals
Introduction
The effect of allelopathic interactions of phytoplank-ton was studied almost 100 years ago (Dakshini, 1994). However, recently researchers conducted more inten-sive analysis of this phenomenon (Kubanek et al., 2005; Granéli and Hansen, 2006; Granéli et al., 2008; Lycz-kowski and Karp-Boss, 2014). The increased interest of allelopathic interactions was caused by reports of the harmful metabolites produced by cyanobacteria and their impact on the surrounding ecosystem (Granéli et al., 2008). It was showed that the Harmful Algal Blooms (HABs) in many aquatic reservoir were significantly in-creased. This problem concerns both marine and fresh-water environments (Smayda, 1990; Van Dolah, 2000), because HABs caused enormous economic losses in aquatic ecosystems (Granéli and Hansen, 2006). HABs
affected the functioning of the whole ecosystem, caus-ing increased mortality of phytoplankton, macroalgae, zooplankton, zoobenthos and fish (Granéli et al., 2008). Moreover, cyanobacteria produce a wide range of sec-ondary metabolites with different scope of their bio-logical effects (Berry et al., 2008; Mazur-Marzec et al., 2015). Only a small part of these compounds has been identified.
Allelopathic compounds play a role in the interac-tion between donor and target organisms. Allelopathic interactions are an important factor in determining the distribution and abundance of phytoplankton species (Wardle et al., 2011; Yang et al., 2012). Primary produc-ers in aquatic ecosystems compete for the space, light and nutrients. This suggests that competition for envi-ronmental factors can strongly affect the donor species for production of allelopathic compounds. The release of active compounds is an adaptation developed by the primary producers against their competitors. Cyano-bacterial allelopathy may negatively affect the condition of the predators, which may result in their death. On the other hand, the conditions and characteristics of target organisms is strongly dependent on their sensitivity and the scope of detoxification mechanisms to allelopathic compounds (Suikkanen et al., 2004). In contrast to the terrestrial environment, the direct demonstration of the effect of allelopathic interactions in aquatic ecosystems is very difficult. Therefore, many studies showed the al-lelopathic interactions between microorganisms by per-forming a series of laboratory experiments.
In recent years, it was noted that increased use of var-ious anthropogenic compounds, such as the commonly used herbicide, ionic liquids or copper chloride, which can degrade the environment and negatively affect many aquatic organisms. Their impact on the aquatic environment and microorganisms are still insufficiently examined, therefore it is necessary to conduct intensive
Jakub Maculewicz:
Institute of Oceanography, University of Gdańsk
received: 21.11.2015; accepted: 5.01.2015; published: 1.04.2016
dr Aleksandra Zgrundo:
Institute of Oceanography, University of Gdańsk
dr Filip Pniewski:
Institute of Oceanography, University of Gdańsk
prof. Adam Latała:
Institute of Oceanography, University of Gdańsk mgr Zuzanna Sylwestrzak:
Institute of Oceanography, University of Gdańsk dr Sylwia Śliwińska-Wilczewska:
Institute of Oceanography, University of Gdańsk
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studies in this area (Kwiatkowska et al., 2013). One of the most widely used herbicide in Poland is Roundup® which contains the glyphosate (N-phosphonomethylg-lycine). Glyphosate penetrate plant cells and inhibit the EPSP (5-enolpyruvylshikimate-3-phosphate) synthase. Inactivated enzyme stops the production of aromatic amino acids such as phenylalanine, tyrosine and tryp-tophan, which are very important for plants as they are used for synthesis of many pigments (Franz et al., 1997). In addition, this compound hinder photosynthe-sis (Pieniążek et al., 2003). Another analyzed substance was 1-butyl-3-methylimidazolium chloride ([BMIM] CI) belonging to the group of imidazolium ionic liquids. Ionic liquids are widely used in “Green Chemistry”, the area considered to be environmentally friendly. These liquids, due to the low volatility, can replace previously used organic solvents. Despite the growing popular-ity of these compounds in the industry, their toxicpopular-ity was poorly studied. Moreover, it was showed that these substances are toxic to different groups of organisms, including microalgae (Latała et al., 2005, 2009a, 2009b, 2010; Kulacki and Lamberti, 2008; Samorì et al., 2011). The last analyzed substance was copper(II) chloride. Copper in enzymatic reactions is involved in the trans-fer of electrons and thus in small amounts is an essential for growth of plants. However, large quantities of these element act as algaecide and is very toxic for microalgae (Brown et Rattigan, 1979).
The aim of this study was to compare the influence of allelopathic compounds produced by cyanobacte-rium N. spumigena grown under different light inten-sity, temperature and availability of nutrients, and the effect of selected anthropogenic substances on growth of diatom B. paxillifera. This study was motivated by relatively few works that investigated the effect of alle-lochemicals and anthropogenic substances on B.
paxil-lifera in aquatic environments.
Material and methods
The experiments were conducted on the cyano-bacterium Nodularia spumigena Mertens ex Bornet & Flahault 1888: 245 (strain BA-15) and the Bacillaria
paxillifera (O.F. Müller) T. Marsson 1901: 254 (syn. Vibrio paxillifer O.F. Müller, 1786) (strain BA-14). The
strains were isolated from the coastal zone of the Gulf of Gdańsk (southern Baltic Sea) in 2003 and are main-tained as unialgal cultures in the Culture Collection of Baltic Algae (CCBA) at the Institute of Oceanography, University of Gdańsk, Poland (Latała, 2003; Latała et al., 2006). The tests on the “batch cultures” were carried out in 25 ml glass Erlenmeyer flasks containing steril-ized f/2 medium (Guillard, 1975). The media were pre-pared from Baltic water with a salinity of about 8 PSU (Practical Salinity Unit), which was filtered through glass fiber filters (Whatman GF/C) and autoclaved. The cyanobacteria and diatom cultures were acclimated for 7 days; afterwards, actively growing cultures were used as a source of inoculum (constituting the suspension of cyanobacteria and diatom cells) for the experiment.
Allelopathic interactions were determined by us-ing the modified method proposed by Suikkanen et al. (2004). Allelopathic activity was studied by adding
the cell-free filtrate obtained from cyanobacterial cul-ture to the tested diatom. The culcul-ture of N. spumigena was filtered through Whatman GF/C filters. The cell-free filtrate (V = 2 ml) was added to 25 ml Erlenmeyer flasks containing the tested diatom (V = 20 ml). In all experiments, the ratio of cyanobacteria to target species in Erlenmeyer flasks was adjusted to 1:1 based on the chlorophyll a content (final chlorophyll a concentration in the experimental cultures was 0.8 µg·ml-1). Control
samples were prepared by adding mineral medium f/2 with a volume equal to the added cell-free filtrate. The donor cyanobacteria culture was incubated under a 16:8 h light:dark cycle at 190 μmol photons·m-2·s-1,
temperature 25°C and f/2 medium (N and P). The tar-get diatom was grown in constant conditions of 20°C and f/2 medium, under a 16:8 h light:dark cycle at 50 μmol photons·m-2·s-1 and this served as control
condi-tion. Tests were conducted in triplicate and all analyzed species were obtained from early exponential growth phase. Culture density was determined by the number of cells and optical density (OD). The number of cells was counted using Bürker chamber and OD was meas-ured spectrophotometrically at 750 nm with a Thermo Scientific Multiskan GO UV-VIS spectrophotometer. The results of cell counts and respective OD
meas-Fig. 1. Cyanobacteria and microalgae strains used in this study: A) Bacillaria paxillifera BA-14 and
B) Nodularia spumigena BA-15.
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urements were then used to determine the linear cor-relation between them for each species. Determined relationships were subsequently used to estimate the number of cells in the experimental cultures after 1st, 3rd
and 7th day of the diatom exposure to the cyanobacterial
filtrate.
In this work the influence of anthropogenic sub-stances – herbicide Roundup® (containing glyphosate), ionic liquid [BMIM]Cl and copper(II) chloride was investigated using the Baltic diatom B. paxillifera. The tests were carried out in 25 ml glass Erlenmeyer flasks containing the tested diatom. The diatom was grown in 20°C under a 16:8 h light:dark cycle at 50 μmol photons·m-2·s-1 and f/2 medium. The control samples
contained 25 ml of f/2 medium with a salinity of about 8 PSU. To evaluate the toxicity of anthropogenic sub-stances, the following concentrations were used: CuCl2 – 0.001 g·l-1, herbicide containing glyphosate – 0.05 g·l -1 and [BMIM]Cl – 0.0175 g·l-1. The concentrations of
the anthropogenic substances were selected according to the literature referring to the EC50 (effective
concen-tration), i.e. the concentration that causes a particular
effect in 50% of experimental organisms, under cer-tain laboratory conditions. The reaction of organisms shown as the changes in the number of cells compared to control conditions analyzed after 1st, 3rd and 7th day
of the diatom exposure to the anthropogenic substance. In addition, a photographic documentation was done using a Nikon Eclipse 80i microscope with a Nikon DSU2 camera.
Analysis of variance (ANOVA) was used to test dif-ferences in analyzed parameters between the target algae cultures treated with cyanobacterial cell-free fil-trates, anthropogenic substances, and the control over the experimental period. A post hoc test (Tukey’s HSD) was used to show which treatments for growth signifi-cantly differed from the control and from each other.
creased the number of cells of B. paxillifera. After the 7th
day of experiment for a filtrate addition obtained from
N. spumigena grown at 190 µmol photons⋅m-2⋅s-1, 25ºC
and f/2 medium (N and P) growth inhibition expressed as a percent of culture density constituted 85%, 86% and 81%, respectively (ANOVA, p < 0.05). Furthermore, in this study, the influence of the ionic liquid [BMIM]CI, Roundup® and copper chloride II on the number of ana-lyzed diatom was investigated (Fig. 2, D-F). It was ob-served that the addition of copper(II) chloride at a con-centration of 0.001 g·l-1 was the most inhibitory effect on
the Baltic diatom B. paxillifera, wherein the number of cells on the last day of the experiment constituted 40% Data are reported as mean ± standard deviation (SD).
The statistical analyses were performed using the Statis-tica® 10 software.
Results
The effects of cyanobacterial cell-free filtrate ob-tained from N. spumigena cultures grown under 190 µmol photons·m-2·s-1, 25ºC and f/2 medium (N and P)
on the diatom B. paxillifera growth after 1st, 3rd and 7th
days of incubation are shown in Fig. 2 (A-C). On the ba-sis of the results it was found that the allelopathic effect of the cyanobacterium N. spumigena significantly
de-Fig. 2. The effect of the addition of cell-free filtrate from N. spumigena cultures grown under:
A) 190 µmol photons·m-2·s-1,
B) 25ºC,
C) f/2 medium (N and P) and anthropogenic substances: D) BMIM[Cl],
E) glyphosate and F) copper(II) chloride on the growth of B. paxillifera after 1, 3 and 7 days of exposition, expressed as a percent of the number of cells (%N)
The values refer to means (n = 3, mean ± SD).
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compared to the control. Moreover, the addition of two analyzed substances: the ionic liquid at a concentration of 0.0175 g·l-1 and glyphosate at a concentration of 0.05
g·l-1, resulted in inhibition of growth of B. paxillifera.
After 7 days of exposure yield of B. paxillifera culture was estimated as 58% and 51%, respectively, compared to control conditions (ANOVA, p < 0.05).
The morphological changes of the B. paxillifera cells after the addition cyanobacterial cell-free filtrate and
anthropogenic substance – Roundup® was shown in Fig. 3 and Fig. 4. In the study, the deformation of the analyzed organism was indicated by the arrows. Ob-servations made by light microscope showed that the cells of B. paxillifera were in several stages of degenera-tion, showing restriction of pigmentation and empty cells, compared to the control culture. Furthermore, many dead cells of the former species were identified, while the cells were discoloured, deformed, and vary-ing in size. Analyzed diatom showed also an increased number of the teratological forms. B. paxillifera showed some degree of alteration when cultivated with the cell-free filtrate of N. spumigena. In this case, a large num-ber of empty cells in varying states of deformation were observed. Moreover, addition of Roundup® caused, after 1 day of exposure, the formation of a visible structures inside the diatom cells, which are classified into an
in-Fig. 3. The cells morphology of B. paxillifera for
A) control sample and in the experiments with the addition of cyanobacterial cell-free filtrate after
B) 1 day and
C) 7 days of exposure.
Source: Author’s own work.
Fig. 4. The cells morphology of B. paxillifera for A) control sample and B) in the experiments with the addition of glyphosate (as Roundup®) after 1 day of exposure.
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obtained from the Baltic cyanobacteria N. spumigena inhibited the growth of diatom B. paxillifera. Chan et al. (1980) noted that compounds released by Skeletonema
costatum (Greville) Cleve 1873: 7, Phaeodactylum tricor-nutum Bohlin 1897: 520 and Heterocapsa triquetra
(Eh-renberg) Stein 1883: 13 has stronger negative effect on diatom Cylindrotheca fusiformis Reimann & J.C.Lewin 1964: 288 than two heterotrophic bacteria
Staphylococ-cus aureus and Bacillus subtilis. Similarly, compounds
in the culture filtrate extract from P. tricornutum and
H. triquetra gave a pronounced zone of inhibition with
the algal assay and no inhibition with the bacterial one. Moreover, the filtrate extract of the dinoflagellate
Scrippsiella sweeneyae Balech ex A.R.Loeblich III 1965:
15 was inhibitory to both C. fusiformis and the two bac-terial species. Authors observed that C. fusiformis and
Nitzschia angularis W.Smith 1853: 40, two species that
are fast growing and show even distribution of cells on the agar surface, were also the most suitable assay or-ganisms (Chan et al., 1980). Furthermore, the study al-lowed to determine the effect of widely used, but less studied herbicide called Roundup® consisting of glypho-sate: the ionic liquid and copper(II) chloride on the Bal-tic diatom B. paxillifera. Ionic liquids are organic salts with a low melting point, which has been recognized as green alternatives for industrial and volatile organic compounds. The popularity of these solvents as “envi-ronmentally friendly” chemicals was based primarily on their very low vapor pressure. However, high solubility of ionic liquids in water gives a rise to concerns in rela-tion to their effects on aquatic organisms. Understand-ing the type and extent of the impact of these substances on microorganisms is extremely important, because the aquatic ecosystems are possible recipient of industrial pollution (Phama et al., 2010). In this study, the effect of the ionic liquid in a concentration of 0.0175 g·l-1 [BMIM]
Cl was investigated. It resulted in decrease in the
num-ber of cells of B. paxillifera on subsequent days of the experiment. Latała et al. (2009b), showed that ionic liquids were toxic for diatoms Cyclotella
meneghini-ana Kützing 1844: 50 and Skeletonema marinoi Sarno
& Zingone in Sarno et al. 2005: 160 and EC50 values for ionic liquids with chain contained 4–10 carbon atoms, ranged from 0.02 do 6.5 µM. In this study Roundup® was used which has a wide spectrum of biological ac-tivity. It is extremely effective herbicide, which inhibits metabolic pathways of plants. In recent years the popu-larity of this herbicide has increased with the prolifera-tion of GMO crops (Maa et al., 2006; Kwiatkowska et al., 2013). Roundup® contains not only the active substance (glyphosate), but also many secondary compounds. Therefore, it is important to study the combined effect of the active substance and other compounds (includ-ing surfactants), because it was a form to which the en-vironment was the most affected. In this study it was demonstrated that the glyphosate concentration of 0.005 g·l-1 resulted in a reduction of the number of cells
of B. paxillifera compared to the control conditions. Wong (2000) also noted that glyphosate concentration of 0.02 g·l-1 inhibited growth of green algae Scenedesmus
quadricauda (Turpin) Brébisson in Brébisson & Godey
1835: 66. Maa et al. (2006) noted that EC50 for glypho-sate for green algae Raphidocelis subcapitata (Korshi-kov) Nygaard, Komárek, J.Kristiansen & O.M.Skulberg 1987: 31 was lower than used in this study and consti-tuted 0.0055 g·l-1. Furthermore, an important element,
which is often noted in higher concentrations in the aquatic environment is copper. This element occurs in the natural environment and was responsible for the electrons transfer in the enzymatic reactions (Wells et al., 2005). In the seawater, the concentration of copper is 0.02-0.32 µg·l-1 (Kabata-Pendias and Pendias, 1999). In
an amount greater than classifying it as a microelement is one of the most toxic heavy metals (Brown and Rat-creased number of swollen gas vacuoles. What is more,
it was observed that the longer the exposure time, the stronger effect of cells degradation of the analyzed dia-tom.
Discussion
In the aquatic environment there are many different substances, both allelopathic compounds and anthro-pogenic substances that negatively affect the function-ing of many microalgae. However, knowledge of this subject is still insufficient. The study compared the in-fluence of allelopathic compounds and anthropogenic substances such as ionic liquids, glyphosate and copper chloride in relation to the diatoms B. paxillifera. In ma-rine, brackish and freshwater ecosystems, mode of ac-tion of the allelopathic compounds is highly diverse and donor species may influence on the target organisms in different ways. Additionally, in natural phytoplank-ton community it is difficult to prove a direct impact of allelopathic interactions. Therefore it is important to characterize allelopathic interactions in controlled laboratory conditions, in order to investigate the nature of released substances and their mode of actions of tar-get organisms (Valdor and Aboal, 2007; Gantar et al., 2008).
Inhibition of growth of the target organism by pro-duction allelopathic compounds are relatively wide-spread and the most frequently reported mode of action of cyanobacteria (Gross, 2003; Żak et al., 2012). In the laboratory experiments algal assay was the most com-monly used procedure which was intended to demon-strate the effect of allelopathic interactions between do-nor and target organisms (e.g. Fistarol et al., 2003; 2004; 2005; Suikkanen et al., 2004; Yamasaki et al., 2007; Gantar et al., 2008; Antunes, et al., 2012; Żak et al., 2012). In this study it was demonstrated that the filtrate
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cells. Gantar et al. (2008) also showed that after being exposed to the crude extract from Fischerella sp. for 24 h, cells of Chlamydomonas sp. showed distinctive mor-phological and structural changes. Authors noted that the electron microscopy revealed degeneration of thyla-koids into a system of irregularly arranged membranes. In addition, disappearance of other cell structures in-cluding the nucleus was apparent. This results suggested that allelopathic compounds produced by cyanobacte-ria and anthropogenic substances can cause physiologi-cal changes in the target organisms. This would mean that, in the long term, low concentrations of harmful substances may affect microalgae, altering the composi-tion and structure of phytoplankton communities.
Conclusion
Experiments described in this article showed the in-fluence of allelochemicals secreted by cyanobacterium
N. spumigena and the impact of various anthropogenic
substances, on the number of cells of diatom B.
paxilli-fera in culture. The article focuses on important aspects
of the impact of pollution from commonly used chemi-cals as well as allelopathic compounds that will soon need to be considered as another kind of natural pollu-tion. Perform such studies was motivated by relatively few works that compared the impact of allelopathic compounds and anthropogenic substances on selected Baltic microalgae. The study compared the effect of al-lelopathic compounds produced by cyanobacterium N.
spumigena grown under different light intensity,
tem-perature and availability of nutrients, and the impact of selected anthropogenic substances such as ionic liquid [BMIM]Cl, herbicide Roundup® containing glyphosate
and copper chloride on the number of cells of diatom
B. paxillifera in culture. The results showed that
allelo-pathic compounds have a significant negative impact on
the growth of tested diatom and interact similar than the anthropogenic substances. Comparing the effect of known concentrations of anthropogenic substances and the allelopathic compounds, which amount in the aquatic environment are still unknown, enabled to un-derstand their role in the marine and freshwater ecosys-tems compared to other chemical compounds.
Acknowledgements: The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. This study was supported by The National Science Centre (NCN) grants, Poland, no. DEC-2013/09/N/ST10/01929.
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