Stem cells in regenerative medicine

KWARTALNIK KWARTALNIK

ISSN ISSN 1643-8779 1643-8779

(53) 14(58) 2016 2014

BIOLOGICZNA II ŚRODOWISKOWA ŚRODOWISKOWA

Stem cells in regenerative medicine Transcriptional and translational mechanisms diversifying the proteome

Dlaczego koala jest cool?

Theories explaining astronomical phenomena at children and adults

/ Współczesna estetyka przyrody / Wszystko o kortyzolu Long-lasting environmental project for classes / Questioning the author: jak modelować dialogi na lekcjach? Evolution, devolution, science – book review / Scenariusz lekcji: komórki macierzyste

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in this issue: 1/2016

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Anna Dawid

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Anita Helińska, Barbara Świerczek, Igor Meszka, Bartosz Mierzejewski, Karolina Archacka

Transcriptional and translational mechanisms diversifying the proteome

Potential and actual use of stem cells in regenerative medicine

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Sylwia Śliwińska-Wilczewska, Zuzanna Sylwestrzak, Jakub Maculewicz, Aleksandra Zgrundo, Filip Pniewski, Adam Latała

The effects of allelochemicals... and selected anthropogenic substances on the diatom Bacillaria paxillifera

28 Martyna Nowak

The reasons for current air quality in Poland and main activities to its improvement

36 Urszula Ejlak

Balance is everything – how environment changes affect farm fish

web sources SCHOOL how to draw attention

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Jan Amos Jelinek

Theories explaining astronomical phenomena at children and adults

49 Marcin Trepczyński

Reasoning in natural science against school tasks

63 Natalia Bartoszek, Eliza Rybska

Why does villagers’ giraffe feed on grass and citizens’ cats do not drink water?

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Eliza Rybska

A model for conceptualizing drawing as a teaching-learning activity in biology education

82 Sebastian Pilichowski. Agnieszka Tokarska-Osyczka

Long-lasting environmental project for classes

reviewsevents

photos informations IN SHORTrecent discoveries

90 Sebastian Pilichowski

Review of the book and discussion: M. Giertych: Evolution, devolution, science

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Ilona Żeber-Dzikowska

Review of the book: W. Stawiński: In the Stream of Life

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how to teach ideas scenarios tasks

94 Editorial Board

The new book: Campbell Biology (2nd Polish Edition)

95 Editorial Board

Review of the book: M. Kaczorowska: Guzik in trouble

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SCIENCE nature

physics environment

96 Alicja Walosik

XX National Conference of Specialists in Science Education – announcement

88 Science Section of ERI

The new task (chemistry) ? vent e t n a port l y im us: du.p n e a . e e r b e m @i Is th Infor ebis

EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA | ebis.ibe.edu.pl | [email protected] | © for the edition by Instytut Badań Edukacyjnych 2016

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geography

health biology chemistry research

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Meritorical editors: Urszula Poziomek, Jolanta Korycka-Skorupa Statistical editor: Jacek Haman Contact: [email protected] Website: ebis.ibe.edu.pl Address: ul. Górczewska 8, 01-180 Warszawa

Scientific council president of the Council: prof. zw. dr hab. Adam Kołątaj (Polish Academy of Sciences, Institute of Genetics and Animal Breeding, Jastrzębiec) vice-president of the Council: prof. dr hab. Katarzyna Potyrała (Pedagogical University in Cracow) and: prof. dr hab. Danuta Cichy, dr hab. Ondrej Hronec (University of Presov, Slovakia), prof. dr hab. Daniel Raichvarg (University of Bourgogne in Dijon, France), prof. dr hab. Valerij Rudenko (University of Chernivtsi, Ukraine), prof. zw. dr hab. Wiesław Stawiński (prof. em. of Pedagogical University in Cracow), dr Renata Jurkowska (University of Stuttgart, Germany), dr Paul Davies (University of London) Apart from the council the journal has also coucil teams and reviewers – see at: stronie: ebis.ibe.edu.pl

Publisher Publisher: Instytut Badań Edukacyjnych, ul. Górczewska 8, 01-180 Warszawa Project of the front-page: Marcin Broniszewski DTP: Marcin Trepczyński

The journal has 8 points and is indexed in: CEJSH and Index Copernicus.

Dear readers, I am pleased to present the first issue of Biological and Environmental Education quarterly magazine published in 2016. Each year, the first issue is published in English to enable wider readership, not necessarily familiar with Polish language, to analyse facts and data presented in our articles. As you probably noticed, the quarterly is divided into three parts: SCIENCE, SCHOOL, and IN SHORT. In this issue, we have two SCIENCE articles written by the authors from the University of Warsaw. The first one, Transcriptional and translational mechanisms diversifying the proteome, nicely describes the factors and mechanisms responsible for a huge diversity in protein composition in Eukaryota, though the number of genes in these cells are significantly smaller. I believe that this problem is important for not only young students just beginning their journey with

molecular biology, but also for school teachers teaching these issues in high schools. The second article, Potential and actual use of stem cells in regenerative medicine, touches very timely and important matter, widely discussed also in the mass media. The application of stem cells in regenerative medicine is described straightforwardly, so that everyone with basic knowledge of biology can make use of the information discussed in the article. SCHOOL section of the magazine contains, among other articles helpful in the daily work of school teachers, the article entitled Theories explaining astronomical phenomena at children and adults. It describes

how children and adults explain astronomical phenomena basing on the number of intellectual models. I think that main theses presented in the article are possible to be implemented in different area of teaching or research. I hope that the readers of the English issue will come back to our website (ebis.ibe.edu.pl) again next year to explore English issue of Biological and Environmental Education. I also kindly invite all of you to contact with Editorial Office (ebis@ ibe.edu.pl) to publish your review or research papers in our quarterly magazine. Sincerely yours, Takao Ishikawa

The original version is the electronical version published at the website: ebis.ibe.edu.pl This issue is published entirely in English and has a Polish copy. The English version is the original one.

EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA | ebis.ibe.edu.pl | [email protected] | © for the edition by Instytut Badań Edukacyjnych 2016

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Editorial team secretary: Marcin Trepczyński

Editorial

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Editor in chief: Takao Ishikawa

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Editorial board

Transcriptional and translational mechanisms diversifying the proteome Anna Dawid

Summary:

Introduction The more a particular scientific field is explored by an enthusiast, the more simplifications and concealments given by teachers in elementary, middle, and high schools can be noticed. One of such reductions is an extremely popular “one gene – one protein” scheme. A gene is represented there as a DNA segment flanked by a promoter from 5’ end and a terminator from 3’ end, that can be transcribed into linear single messenger RNA molecule (mRNA), which in turn poses a matrix in the transla-

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tion process that is in synthesising a protein coded by an original gene. In Eukaryota, whose genes are not continuous as prokaryotic ones but consist of both coding (exons) and non-coding (introns) regions, there is also a splicing process where introns from immature mRNA molecule (pre-mRNA) are removed and exons are put together forming final, mature mRNA. This molecule will be equipped with the 5’ cap and 3’ poly(A) tail and transported from the cell nucleus to the cytoplasm. This picture is definitely incomplete what is quite visible when taking following facts into account. First,

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Results of genome sequencing show a surprising lack of a clear dependence between the number of genes and the organisms’ level of complexity. Moreover, along with proteomic data they indicate substantial disparity between the number of proteins and genes in Eukaryota. The resolution of this conundrum, along with post-translational modifications, may lie in mechanisms diversifying the proteome. Here, along with the most often cited examples such as alternative splicing, transcription initiation and termination, less common phenomena are described: RNA recoding, RNA editing, alternative translation initiation, and STOP codon readthrough.

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Transcriptional and translational mechanisms diversifying the proteome | Anna Dawid | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016

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Key words: alternative splicing, alternative promoters, readthrough received: 16.01.2016; accepted: 4.03.2016; published: 1.04.2016

Anna Dawid: Bachelor’s degrees in Biotechnology and Chemistry (obtained accordingly in the Faculty of Biology and Faculty of Chemistry at University of Warsaw), currently a student of the second cycle of chemistry and physics in the College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences UW. Affiliation: MISMaP College UW and Faculty of Biology UW

Fig. 1. C-values for chosen groups of organisms (pg of DNA contained in haploid cell nucleus) Source: Gregory, 2005

EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA | ebis.ibe.edu.pl | [email protected] | © for the article by the Authors 2016 © for the edition by Instytut Badań Edukacyjnych 2016

From the middle of 19th and till the middle of the 20th century proteins were considered the most important components of the cell. Even the name itself indicates it, as it comes from a Greek word “proteios” meaning “primary”, “major”. In 1869 Friedrich Miescher, 25-year-old Swiss physician, when doing research on leukocytes obtained from the pus from used bandages collected in the clinique located nearby, isolated from cell nuclei a substance containing significant amounts of nitrogen and phosphor, precipitating in an acid environment, insoluble in water and sodium chloride, but soluble in sodium hydroxide and disodium phosphate. He correctly deduced that it could not be any known compound and called it “nuclein” – a term still preserved in today’s name deoxyribonucleic acid (Dahm, 2005). This discovery was followed by numerous others and as a result, DNA stopped being treated as an ordinary molecule functioning probably as a nucleus phosphor reservoir (as was hypothesised by Miescher till his death) and became a symbol of modern life sciences. DNA amount (in contrary to proteins) in cells of all tissues is similar except for germ cells that have half as

Fig. 2. The number of genes of chosen species Source: own elaboration based on Pertea and Salzberg, 2010.

phenomena on the chromosomal level e.g. duplications (Gregory, 2001). Although the C-value paradox has been partially explained (except for questions concerning the function and origin of non-coding DNA), the comparison of the number of genes between species also gives suprising results. First of all, evolutionary advanced as well as morphologically and physiologically complicated eukaryotes, especially humans, have relatively small number of genes (Cooper, 2000). Earliest estimations of the human genome in the late 1960s indicated that it may consist of over 2 milions protein-coding genes (Kauffman, 1969). Over the years this number had been decreasing. In the early 1990s geneticists’ estimations favoured 100 000 genes (Pennisi, 2003) to determine in 21st century that Homo sapiens has only around 22 000 of them (Figure 2). Proteomic research results were astonishing as well. In 2014 the paper was published that contained a summary of up-to-date results of the analysis of the hu-


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Historical outline

much DNA as others. That is one of arguments supporting the thesis of DNA as a hereditary material. Therefore, for every species so-called C-value can be determined that is DNA amount contained in haploid cell nucleus (usually expressed in pg or bp). Letter “C” (accordingly to the term’s author Hewson Swift) comes from “constant” (Gregory, 2005). It is logical to conclude that DNA amount is proportional to the number of genes that in turn should be proportional to the organism’s stage of evolutionary progress. This conclusion, however, was demolished immediately when the comparison of number of genes between more species became possible (Mirsky, 1951). It turned out there are plenty of cases of less complex organisms having significantly more DNA than more complex ones e.g. salamander’s C-value is over 70 times higher than chicken’s (Figure 1). In the 1970s term “C-value paradox” was formulated that embraced three curious facts: • Some simple organisms have significantly more DNA than more complicated ones. • Genomes, understood as all DNA contained in a single cell of the organism, accommodate more DNA than it would seem fit from the number of the organism’s genes. • Some groups of morphogically similar organisms have a huge dispersion of C-value. The conclusion can be derived that DNA amount is not linearly correlated with the number of genes, what is paradoxical in a sense that a constant amount of DNA in cells was indeed an argument for DNA as a material containing genes. It is not paradoxical for us today, as we know that DNA does not contain only genes, even more, significant majority of the eukaryotic genome is non-coding. There were many mechanisms that led to accumulation of non-coding DNA such as spreading of transposons (“selfish” DNA), accumulation of pseudogenes (“junk” DNA), occurance of introns and

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there is no perfect correlation between the number of genes and the organisms’ complexity level. Secondly, in Eukaryota there is a huge disparity between the number of genes and the number of proteins. It indicates an existence of mechanisms vastly diversifying the proteome in relation to the genome. They act on different levels of the transcription and translation, and the aim of this paper is to present them along with examples of geneprotein pathways where they occur, with limitation to Eukaryota. Moreover, this article will not describe posttranslational modifications (such as phosphorylation and glycosylation) that also diversify the proteome.

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trogen sythetase), contains tissue specific APs that are used differently accordingly to the stage of development what allows of a regulation of CYP19 expression (Kamat, 2002).

In other cases, transcripts coming from different APs encode different proteins. It is possible when APs contain separate transcription initiation sites. Resulting proteins may differ in N-termini sequence (Figure 3b,i)

Mechanisms diversifying the genetic information on the transcription level in Eukaryota

(a) APs expression (black arrows) does not result in a new protein isoform because of the second exon that is common for all translational options and that contains translation initiation site marked as ATG. Splicing (black lines) is marked in (a) and (b) only for two first exons.

Alternative transcripts are a result of mechanisms such as alternative splicing, use of alternative promoters, alternative polyadenylation and RNA editing. Alternative promoters From 40 to 50% of genes is estimated to have alternative promoters (APs), whose expression causes a production of transcripts with different 5’ ends originated from one gene (Landry, 2003). Research indicates that APs are used most abundantly in brain, heart and liver in embryonic and fetal stages and there is a strong correlation between use of APs and the organism’s development (Baek, 2007). It is important to note that translation of mRNA transcribed with use of different APs still may result in the same protein if in spite of different 5’ ends, they have a common exon with translation initiation site (determined usually by the first codon ATG on the sense DNA strand), so the reading frame remains the same (Figure 3a). Such transcripts, however, can differ in tissue specificity, expression level or secondary structure of untranslated regions (UTR), what also can influence the transcript stability or translation initiation efficiency e.g. gene CYP19, which encodes aromatase P450 (es-

(b) APs expression results in isoforms differing in (i) N-termini sequence (ii) N-termini length.

Figure 1. Types and consequences of alternative transcription initiation. Own elaboration based on Landry et al. (2003)

(c) Completely different proteins originate as a result of a reading frame change.

(a) APs expression (black arrows) does not result in a new protein isoform because of the second exon that is common for all translational options and that contains translation initiation site marked as ATG. Splicing (black lines) is marked in (a) and (b) only for two first exons (b) APs expression results in isoforms differing in (i) N-termini sequence (ii) N-termini length (c) Completely different proteins originate as a result of a reading frame change

Fig 4. APs of the dystrophin gene Source: Brown, 2009.

Figure 1. APs of the dystrophin gene. From Brown (2009)


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Source: own elaboration based on Landry et al., 2003.

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Fig. 3. Types and consequences of alternative transcription initiation

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man proteome. Investigation of 17 294 genes allowed to identify 30 057 proteins in total, built from 293 700 unique peptides (Kim et al., 2014), what gives almost 17 times more peptides than sequences that code them. To help with understanding these striking data, scientists turned to many studies conducted in lat 20-25 years that testify the existence of extraordinary mechanisms that allow to produce many proteins from one gene.

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(Keren et al., 2010). There are also a few interesting features of conserved alternative exons: shorter than constitutive ones, they have divisible by three number of base pairs what allows of maintaining the reading frame when the exon is skipped as well as when is included. Non-conserved alternative exons do not have these attributes (de Klerk and ‘t Hoen, 2015). Moreover, in humans, exons flanked by longer introns are skipped more often (Keren et al., 2010). Alternative ends of the transcript may occur when ending exons have more than one splice site. Such cases account for around 15% of AS. Intron retention takes place mainly in plants, fungi and protozoa, in H. sapiens only 2-5% of AS is retaining an intron, but with 252 243 introns in the human genome it can significantly influence the proteomic variety (Hubé and Francastel, 2015). It is still not completely clear how a spliceosome recognises alternative exons and choose between them.

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exons from pre-mRNA are joined in different order (what may result e.g. in a change of product function) and when some introns get included to mRNA or some exons – excluded (what may have impact on translation intensity or mRNA stability). Around 95% of mammalian genes encode transcripts that are a subject of AS (Shabalina, 2014). Five main events of AS can be distinguished: exon skipping (Figure 5a), use of alternative 5’ end and 3’ end exons (Figure 5b,c), introns retention (Figure 5d) and mutual exclusion of exons (Figure 5e). Also trans-AS was observed i.e. joining exons from different genes (not shown in the Figure 5). When analysing AS, exons get classified into two types: constitutive that almost always are included to mature mRNA and alternative that usually are skipped. Exon skipping accounts for almost 40% of AS cases in higher eukaryotes and is very rare in lower ones

Fig. 5. Alternative splicing Source: Keren et al., 2010. Exons are represented by rectangles (constitutive – blue, alternative – violet), introns by solid lines, dotted lines indicate splicing options

Alternative splicing Canonical splicing is removing introns (non-coding sequences) from pre-mRNA and joining exons (coding sequences). Alternative splicing (AS) occurs when Figure 1. Alternative splicing. From Keren et al. (2010) Exons are represented by rectangles (constitutive – blue, alternative – violet), introns by solid lines, EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA | ebis.ibe.edu.pl | [email protected] | © for the article by the Authors 2016 © for the edition by Instytut Badań Edukacyjnych 2016 dotted lines indicate splicing options

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or length (so-called ΔN isoform) (Figure 3b,ii) or be completely different as a result of a change of a reading frame (Figure 3c). APs expression in the gene of PPARγ protein leads to two protein isoforms differing in tissue specificity (Fajas et al., 1997). Transcription of one gene with use of different APs results in two proteins INK4A and ARF having distinct functions (Quelle et al., 1995). There is also an interesting example of ΔN isoform that comes from the AP expression, namely ΔNp73 isoform. p73 protein has a similar function to p53 i.e. takes part in stopping the cell cycle, apoptosis induction and activation of genes encoding proteins that cooperate with it. ΔNp73 isoform, however, is a negative regulator of both p73 and p53. Evolutionary conservation of this AP between species suggest there is an advantage in expression of potentially oncogenic protein. Research indicates its presence is necessary to correct induction of the apoptosis by p53 and p75 (Pozniak et al., 2000). Research conducted on single genes indicates that the choice of APs plays an important role during cell development and differentiation and mutations in these promoters lead to diseases including cancer, neuropsychiatrist and development disorders. The AP can be chosen in two ways: by changes in the chromatin and by cell regulation through tissue specific factors (de Klerk and ‘t Hoen, 2015). The typical example of the second mechanism is the dystrophin gene (Figure 4), distinctive also for being the longest known human gene (2.5 Mb, 1.5% of chromosome X length, 0.1% of the human genome). It consists of 79 exons and has many APs that are expressed depending on the tissue what results in different proteins with distinct functions.

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RNA editing RNA editing is the process of modifying the nucleotide sequence of RNA right after its synthesis. Despite the fact that it is relatively rare, it has been observed in many eukaryotic groups i.e. humans as well as protists (e.g. Trypanosoma) and plants (Brennicke et al., 1999). Phenomena described above as splicing, 5’ cap adding

Human transcriptome contains over 80 000 protein-coding transcripts, and estimated proteome size amounts between 250 000 and 1 000 000 polypeptides, excluding posttranslational modifications (de Klerk and ‘t Hoen, 2015). Therefore, in average, one transcript is a matrix for 3 – 13 proteins what suggests the significant scale of translational mechanisms that diversify the proteome i.e. alternative translation initiation and RNA recoding. The following processes count as RNA recoding: frameshifting, ribosome hopping, change of codon – amino acid recognition and readthrough.

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Use of alternative polyadenylation sites results in transcripts differing in 3’ end. If these sites lie in the exon (or in the intron included to mRNA in the process of AS), then transcripts differ in the coding region length. However, usually alternative polyadenylation accounts for different lengths of 3’-UTR what influences mRNA localization, stability, and translation efficiency but also makes alternative polyadenylation a process that does not significantly diversify the proteome (de Klerk and ‘t Hoen, 2015). The example of generating the proteome variety by alternative polyadenylation is the using of alternative polyadenylation site in the intron 9 of the luc7l2 gene transcript in mice resulting in a shorter protein with different C-termini domain that occurs among others in brain, kidneys and stomach (Howell et al., 2o07).

Translational mechanisms diversifying the proteome

Alternative translation initianion In 1987 scientists had already known that mammalian translation may begin from the codon different than AUG e.g. ACG (Peabody, 1987) or CUG (Starck et al., 2012). The mechanism of choosing alternative translation initiation sites (alternative TISs) is still unclear, nevertheless it is thought to occur in two ways: dependent on the 5’ cap and independent of it (Wan and Qian, 2013). In the capdependent mechanism, the small ribosomal subunit along with many translation initiation factors is assembled on the 5’ cap structure, creating so-called “43 S pre-initiation complex”. Then, in an ATP-dependent process, it scans 5’ UTR to find a TIS, usually AUG codon and starts the translation. This mechanism is the most common in eukaryots. In the second type of translation initiation, the more alternative one, the translation machinery starts in the site indicated by a specific mRNA secondary structure (usually, but not always, in 5’ UTR) called “internal ribosome entry site” (IRES). There are two possible consequences of choosing an alternative TIS in 5’ UTR in the context of creating the proteome diversification:


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Alternative polyadenylation

and polyadenylation do not count as RNA editing, instead the definition covers such mechanisms as insertion, deletion and nitrogenous base change within edited RNA. All types of RNA may be edited, but obviously only modifications in mRNA can have impact on the amino acid sequence of the final protein. The most popular example of this phenomenon is editing of human apolipoprotein B mRNA (Figure 6). In liver this mRNA is a matrix for translation of 4563-amino acid protein called “apolipoprotein B100” (100 states for number of kDa) which is secreted into the bloodstream and takes part in the lipid transport. In the small intestine cells, however, this mRNA is edited by deamination of cytosine into uracil, what changes CAA codon (encoding a glutamine) into UAA i.e. STOP codon. It results in a shorter protein – apolipoprotein B48 (having only 48 kDa out of original 100) responsible for binding and resorbing fatty acids in the lining of the small intestine. The best known type of RNA editing is deamination of adenosine into inosine catalysed by adenosine deaminases acting on RNA (ADAR) (Sacharczuk et al., 2004). The ADAR substrate is a double stranded RNA that can result from pairing intron and exon sequences before intron removal during splicing or from pairing mRNA with antisense RNA. Adenosine conversion into inosine was observed for the first time in the yeast’s tRNA. Inosine is interpreted by translation machinery as guanosine what is an effect of their chemical similarity. This conversion causes among others decrease of calcium flow in glutamate-gated channels because of an exchange of glutamine codon into arginine codon in mRNA encoding a subunit of glutamate receptor and drastic changes in efficiency of G protein binding by serotonine receptor. RNA editing also plays a role in achieving immunoglobulin variety by modification of antibodies’ mRNA in B lymphocytes.

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AS, among others, plays an important role in sex determination in Drosophila (Venables et al., 2012) and is the source of diversification of auditory predisposition between humans by generating a huge number of gene SLO transcripts in cochlear hair cells (Graveley, 2001). While AS is thought to be rather a rule that an exception for human genes, it is not suprising that particular genes’ splicing disorders may cause genetic diseases. Best-characterised examples are spinal muscular atrophy, myotonic dystrophy, retinitis pigmentosa, Frasier syndrome, and hemophilia A (Ghigna et al., 2008).

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RNA recoding More than 40 years ago scientists noted first indications that the genetic code is not fully universal (Weiner and Weber, 1973 and Gesteland and Atkins, 1996). Different variants of the genetic code were observed in many organisms and it this case it means codon reassignment in the entire genome of a species (or an organellum) irrespective of a codon’s mRNA context (Atkins and Baranov, 2010). Subsequent discoveries were even more mysterious. In 1980s, a shift of the reading frame during translation in E. coli was observed, and then a use of two non-canonical amino acids, selenocysteine and pyrrolysine, in

the translation was noted. These phenomena are counted as RNA recoding i.e. the mechanism in which particular nucleotide sequences influence the translational machinery what results in translational changes such as shift of the reading frame (“frameshifting”), redefinition of the codon, ribosome hopping (also: bypassing) and readthrough i.e. recognition of the STOP codon as a protein-coding one. The consequence of RNA recoding is change of amino acid sequence of a final protein. It is important to note that RNA recoding is a dynamic process regulated by many cell’s signals and in constant competition with the standard mRNA meaning (Atkins and Baranov, 2010). Frameshifting When the ribosome meets a frameshifting site, signals coming usually from distant RNA regions can make it efficiently “skip” one or two nucleotides (+1 and +2) or “jump back” by one or two nucleotides (-1 and -2), what causes the reading frame change and synthesis of a new protein with amino acid sequence completely different

Fig. 6. Human apolipoprotein B mRNA editing Source: own elaboration based on Brown, 2009.

Fig. 7. Frameshifting in translation of ODC antizyme Source: Ivanov et al., 2000.


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Use of alternative TISs is common among animals (de Klerk and ‘t Hoen, 2015) and occurs both under stress and physiological conditions. 50%-65% of mRNA is estimated to have more than one TIS, and TISs in mammalian mRNA is being catalogued in TISdb (Translation Initiation Sites database) (Wan and Qian, 2013).

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• If the translation starting from the alternative TIS ends with the same STOP codon as the canonical one, the outcome is a protein that differs in Nterminal end from a protein being a result of the canonical translation. • If an alternative TIS is in the different reading frame than a primary open reading frame (ORF), or if the alternative translation ends with the STOP codon that is upstream in terms of the primary ORF, the outcome is a completely new protein. What is more, ORFs that are entirely upstream in terms of the primary ORF (uORFs, Upstream Open Reading Frames) take part in regulating the gene expression. Recognition of a TIS in the beginning of the uORF by the 43 S pre-initiation complex may result in stopping the ribosome in the elongation or termitaion stage on the uORF, blocking the access to the primary ORF for next ribosomes or even inducing mRNA decay, influencing directly the translation efficiency of the primary ORF (Medenbach et al., 2011 and Barbosa et al., 2013).

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Incorporation of selenocysteine into polypeptyde chain An unusual example of RNA recoding is incorporation of selenocysteine (Sec) (Figure 8). Sec is called “the 21st amino acid” and is incorporated into polypeptide chain during translation in archaea, bacteria and eukaryotes (Ivanov et al., 2000). To translate the STOP codon UGA (named opal) as a selecysteine, the following elements must be present (Copeland, 2003):

Fig. 8. Complex that recognizes UGA codon as selenocysteine-coding Source: Copeland, 2003.

Rybosome hopping (also: bypassing)

Readthrough

In a translational hopping, a ribosome “bypasses” some part of mRNA without insertion of any amino acid. It is sort of a translational splicing. A bypassed region always has a number of nucleotides divisible by three, so the ribosome hopping does not cause any shift of the reading frame. If a bypassed sequence is entirely coding, then a product is just shorter than one that would result from a canonical translation. If a bypassed region contains a STOP codon, a ribosome hopping causes translation of a sequence that so far was noncoding (Ivanov et al., 2000). For many years the expression of the bacteriophage T4 gene 60 had been the only experimentally confirmed example of the ribosome hopping. Lately, however, common occurance of this phenomenon was confirmed in mitochondrial genes expression of the yeast Magnusiomyces capitatus (Lang et al., 2014).

For the first time, a term “readthrough” was used probably in 1970s (Forget et al., 1975), and it is used to a phenomenon in which a termination signal coming from a STOP codon is ignored, and translation continues in the same reading frame. It is still argued if readthrough is a translational error or a “programmed” process (Rospert et al., 2005; von der Haar and Tuite, 2007). Recognition of the opal codon as selenocysteinecoding is not counted as a readthrough because of completely different mechanism of this phenomenon. Most of what is known about the translation termination and eukaryotic readthrough mechanism comes from research on yeasts, especially on Saccharomyces cerevisiae (von der Haar and Tuite, 2007). Readthrough is an interaction between a STOP codon and tRNA loaded with an amino acid that results in continuing the translation. The tRNA that inserts


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• cis elements in 3’ UTR mRNA with a stem-loop structure, called “selenocysteine insertion sequence” (SECIS), assembled with SECIS-binding protein 2 (SBP2) into a complex, • tRNA with UCA anticodon and loaded with selenocysteine (tRNASecUCA), created as a result of serine modification in tRNASerUCA catalysed by selenocysteine synthase and consisting in selene transfer from selenophosphate to serine hydroxyl group, • elongation factor eEFSec specific for this tRNA, which determines the ability of translational binding of tRNASec to opal codon. Most selenoproteins play roles in redox reactions and in defense mechanisms against reactive oxygen species. Sec is in the active centre of such enzymes as glutathione peroxidase, iodothyronine deiodinases and thioredoxin reductase (Kryczyk and Zagrodzki, 2013).

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from the canonical one starting from the frameshifting site. +1 and -1 frameshifting occur in many species, but +2 and -2 have been described so far only in artificial test systems (Gesteland and Atkins, 1996). A well-known example of this process is a frameshifting in translation of ornithine decarboxylase (ODC) antizyme (Manteuffel-Cymborowska, 1996). For the first time it was described as an ODC inhibitor whose activity was increased by polyamines – compounds that are highly important in the organism (e.g. influence translation rate and efficiency), but have toxic properties in bigger concentrations. ODC takes part in their biosynthesis. Cloning of a gene that encodes the antizyme indicated that its ORF did not contain the START codon. Instead, the translation is initiated in an uORF, which partially overlaps the primary ORF, in such a way that +1 frameshifting gives a functional antizyme (Ivanov et al., 2000). Further analysis allowed to state that frameshifting indeed took place and was stimulated by high concentration of polyamines in a cell (Figure 7). It means that antizyme synthesis is under a negative feedback control – its synthesis leads to ODC inhibition and therefore to polyamine concetration decrease and lack of frameshifting stimulation which in turn causes an inhibition of antizyme synthesis. Frameshifting occurs widely in many species, from fungi to mammals, and is strongly evolutionarily conserved.

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Conclusion All described mechanisms increase coding potential of genomes, and with posttranslational modifications pose an answer to the question how it is possible that the proteome contains dozens of times more types of proteins than the genome has protein-coding genes. Their occurance also indicates that the number of genes and the evolutionary progress level along with physiological complexity do not have to be proportional. Discussed mechanisms could have arisen as a result of strong evolutionary pressure exerted on organisms with compact genomes such as viruses, and indeed, they occur in these organisms most commonly. In Eukaryota, except for increasing the coding potential of the genome, which seems to be mostly non-coding, these mechanisms give an opportunity to regulate a ratio between different proteins coded by the same gene on different levels of its expression. Not so long ago the molecular biology seemed to be governed by a simple scheme “one gene – one protein”. It is obvious now, however, that the gene expression is far more complicated and richer in events than it was thought. Amazing complexity of the genetic information reading process may be suprising – why did the evolution favour decrease of the number of coding se-

quences and mechanisms diversifying the proteome, instead of using bigger part of the genome to code proteins? Too little is known about these mechanisms’ evolution and the role of non-coding DNA to determine the answer, but there are some clues suggesting that mechanisms increasing coding potential have at least one advantage, namely, greater possibility of their control. They are sensitive to many regulation factors, often are tissue specific and change along with the organism’s development. We live in a splendid era when the genomes’ and transcriptomes’ sequencing allows large-scale research on various biological mechanisms and comparing them between branches of the evolutionary tree. Publications of the genomes of human, S. cerevisiae, and many others vertebrates, invertebrates, yeasts, plants and protozoa as well as their transcriptomes give great opportunities to comparative genomics and transcriptome analytics. Since the genetic code was broken in 1960s, huge progress has been made in understanding the genetic information decoding mechanisms and despite many questions left without answers, next discoveries hopefully should explain at least part of them.

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Summary: In Recently, a great interest in stem cells issues has risen both among the scientific community and the society. Meeting these requirements, organizations promoting knowledge about stem cells have been founded. Among them EuroStemCell – a partnership of stem cell research centres and institutions from many European countries has been established. Additionally, different types of actions devoted to stem cells issues have been initiated with UniStem Day being the great example of them. The UniStem Day is organized every year simultaneously at many European universities. The main aim of this event is to disseminate knowledge about stem cell research among high school students. However, many myths about stem cells still exist in public opinion. This article – being the follow-up of the publications focused on stem cells and published in Biological and Environmental Education in 2013 – presents the current information about stem cells with special emphasis on their potential and actual use in regenerative medicine. Key words: stem cells, regenerative medicine, cell therapy, clinical trials, dissemination of knowledge received: 15.02.2016; accepted: 18.03.2016; published: 1.04.2016

Regenerative medicine is an interdisciplinary branch of science that includes medical biology, biotechnology, and biophysics. Its main aim is to elaborate and apply methods that allow the improvement of structure and function of tissues and organs that deteriorated due to disease, injury or as a result of ageing. To this end projects focused on tissue engineering, whose main aim is to obtain organs in laboratory that may be used in transplantation, as well as research concerning stem cells that play the key role in regeneration of different tissues in physiological conditions are conducted (the characteristics of different types of stem cells was presented in the articles published in Biological and Environmental Education in 2013; Archacka 2013; Bauer et al., 2013; Świerczek et al., 2013). Despite the progress, until 2015 the only stem cell therapy that was clinically approved was the bone marrow transplant.

mgr Anita Helińska: Department of Cytology, Faculty of Biology, University of Warsaw

mgr Barbara Świerczek: Department of Cytology, Faculty of Biology, University of Warsaw

Igor Meszka: Students Scientific Association of Medical Biology „Antidotum”, Faculty of Biology, University of Warsaw; Department of Cytology, Faculty of Biology, University of Warsaw Bartosz Mierzejewski: Students Scientific Association of Medical Biology „Antidotum”, Faculty of Biology, University of Warsaw; Department of Cytology, Faculty of Biology, University of Warsaw dr Karolina Archacka: Department of Cytology, Faculty of Biology, University of Warsaw

There are several types of stem cells in bone marrow, including hematopoietic stem cells (HSC), from which all types of blood cells are derived (Bianco et al., 2001). Therefore HSC are crucial for functionality of hematopoietic and immune systems. Failure in the process of blood cell formation – hematopoiesis – is the main reason for many hematopoietic system disorders characterized by high mortality, such as acute and chronic myeloid leukemias (Linker, 2003) and cancer of the lymphatic system (Passweg et al., 2015). For example, leukemia develops as a result of uncontrolled proliferation of white blood cells (leukocytes) which prevail over healthy blood cells, leading to patient’s health deterioration. As a result of the decreased number of red blood cells (erythrocytes) responsible for the oxygen distribution throughout the body, the patient becomes weaker. Moreover, he suffers from frequent bleedings and numerous bruises on the skin resulting from the shortage of blood platelets which are crucial for blood clotting. Colonization of lymph nodes, liver, and spleen by tumor cells leads to the gradual impairment of their functioning. In order to destroy tumor cells in patients suffering from the diseases mentioned above, chemotherapy and/or radiotherapy are applied. However this procedure leads to the destruction of other blood cells, including HSC (Fig. 1). The reduced immune resistance means that even mild infection or rhinitis may lead to life-threatening complications. Transplantation of HSC gives a chance to reconstitute the destroyed bone marrow of the recipient and – in consequence – to reinitiate the production of new blood cells including the immune cells that combat pathogens – leukocytes, monocytes, eosinophils, and basophils (Fig. 1). In addition, the donor immune cells which are also present in the graft, recognize and destroy the cancer cells which can remain in the patient’s body despite the radio- or chemotherapy (Hilgendorf et al., 2015).

A. Helińska, B. Świerczek, I. Meszka, B. Mierzejewski, K. Archacka


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Anita Helińska, Barbara Świerczek, Igor Meszka, Bartosz Mierzejewski, Karolina Archacka

Introduction

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Potential and actual use of stem cells in regenerative medicine

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Potential and actual use of stem cells in regenerative medicine | A. Helińska, B. Świerczek, I. Meszka, B. Mierzejewski, K. Archacka | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016

Fig. 1. The outline of bone marrow transplantation in allogeneic conditions The production of blood cells in bone marrow of patient suffering from leukemia is impared (green frame). Radiotherapy leads to the destruction of tumor cells which cause leukemia development but also other cells that are present in the bone marrow. This results in destruction of the patient’s hematopoietic system (yellow frame). Bone marrow of the donor contains healthy hematopoietic stem cells. They can be isolated from the femur (blue frame) through the puncture. Transplantation of bone marrow stem cells obtained from a donor (black frame) leads to the reconstruction of the damaged hematopoietic system of the recipient and – in consequence – in the resumption of blood cell production (red frame).

allogeneic bone marrow transplantation in Poland was conducted by professors Cezary Szczylik and Wiesław Jędrzejczak in 1984 (Sobiak, 2011). Nowadays HSC transplantations are available in 17 clinical centers in Poland. In each of them the average number of transplantations ranges from 20 to 165 per year (Resolution of the Council of Ministers No. 164/2010, the National Programme for the Development of Transplantation Medicine). Since 1984 in Poland circa 9 000 HSC transplantations have been performed. As a result of them five thousand patients are still alive (Resolution of the Council of Ministers No. 164/2010, the National Programme for the Development of Transplantation Medicine). The average number of bone marrow transplantations conducted per every 10 million of citizens in European Union is 450 per year while in Poland – 210 per year (Resolution of the Council of Ministers No. 164/2010, the National Programme for the Development of Transplantation Medicine). In 2013 the total number of HSC transplantations conducted in Europe reached almost 40 000, with 43% of them in allogeneic while 57% – in autologous conditions (Passweg et al., 2015). There are several factors that are crucial for making a decision about transplantation performance including the type of disease, the age and condition of the patient as well as the donor availability. Reconstruction of the hematopoietic and immune system usually takes from two to six weeks. During this time the patient stays in special conditions which minimize the risk of infection. The transfusion of an additional blood portion is often necessary to supplement the number of red blood cells or platelets in the patient. Another complication which may occur after the bone marrow transplantation is graft versus host disease (GVHD). GVHD develops when the transplanted donor cells recognize the recipient’s blood cells as strange ones and destroy them (Ball and Egeler, 2008). GVHD is mostly mani-


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recipients, usually relatives. Therefore many allogeneic transplantations are performed between siblings, children and parents, or other family members. In the case of autologous transplantation the same person is both the donor and the recipient. The first successful

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Transplantation of HSC obtained from a donor is an example of the allogeneic graft, in which the donor and the recipient of transplanted cells have similar human leukocyte antigens (HLA). Perfectly matched HLA compatibility occurs between 20 to 25% of donors and

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Fig. 2. Potential stem cell based therapies for eye diseases Anatomy of the eye. Red color indicates corneal limbus, where limbal stem cells reside. The outline of LSCD therapy based on the limbal stem cells. Limbal stem cells are isolated from corneal limbus of the patient’s healthy eye and subsequently cultured in vitro. Cells are placed on the fibrin lens when sufficient number of them is available. From 80 to 300 thousands of cells are placed on a lens with 2 cm in diameter. The implant is transferred to the patient’s injured eye, what lead to the cornea reconstruction. The derivation and transplantation of retinal pigmented epithelium cells differentiated from pluripotent stem cells. Transplantation of retinal pigmented epithelium cells is considered as a potential therapy for patients suffering from AMD and Stargardt’s disease. Such cells can be obtained from pluripotent ESC. Transplantation of 50-150 thousands of these cells to the patients suffering from Stargardt’s disease or AMD leads to the restoration of retinal pigmented epithelium layer in the macula and – in consequence – to the vision improvement.

Cornea is a transparent layer covering the front of the eyeball (Fig. 2A). Apart from its protective role the cornea serves also as an element of the eye optical system. Due to cornea transparency the light can reach the retina which coats the inner part of the eyeball (Fig. 2A). Cornea contains a small pool of stem cells known as limbal stem cells which are responsible for the regeneration of the cornea after its damage (discussed in Pellegrini and De Luca, 2014). Unfortunately, the cornea regeneration becomes impossible if the injury also affects the stem cells population. Expansion of blood vessels and connective tissue on the surface of the eyeball leads to the formation of light-impermeable membrane which restricts the vision ability of the patient (Pellegrini and De Luca, 2014). For many years the transplantation of cornea obtained from deceased donors was the only available treatment for the patients suffering from diseases caused by limbal stem cells deficiency (LSCD). In 1905 the first documented transplantation of cornea isolated from a deceased donor to a patient with chemical eye burn was conducted by a medical doctor, Eduardo Zirma. Interestingly, as a result of the immune privilege of the eye nearly 90% of corneal transplantations were successful. The phenomenon of immune privilege means that the transplant is accepted despite the differences between HLA of the donor and recipient. Additionally, in order to minimize the risk of immune reaction the corticosteroids with anti-inflammatory and anti-allergic properties were administrated to the eye after the transplantation (Szaflik and Izdebska, 2003). However, the major limitation of the cornea transplantations was the reduced availability of the tissue samples. Moreover, some patients suffered from strong inflammation and ulceration of the eye. As an


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HOLOCLAR – a new chapter in the cornea damage treatment

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scientific publications describing the phenomenon of stem cell plasticity, i.e. conversion of stem cells of one tissue into cells specific for other tissues (discussed in Bauer et al., 2013), the results of current research indicate that HSC efficiently form only blood cells and does not transform into other types of cells, for example neurons or myoblasts (Flores-Guzman et al., 2013). Thus, other sources of stem cells with potential therapeutic effect are still being sought.

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fested by skin rash, mouth ulcers, erosions in the genitals, and dry eye syndrome. However, the more serious complications may also occur including necrotic lesions in the liver, intestines, and other organs, which can even lead to death of the patient (Chinen and Buckley, 2010). Although bone marrow transplantation has become a routine medical procedure, it is truly beneficial only in the case of hematologic diseases. Despite numerous

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The clinical trials based on pluripotent stem cells for eye diseases treatment Age-related macular degeneration (AMD) is another disease affecting the eye. AMD develops in older people and is the cause of 5% of all registered cases of blindness (Pascolini and Mariotti, 2012). Macula is a part of retina, which is characterized by the highest number of the eye photoreceptors (cones and rods). Cones and rods are responsible for the conversion of light into chemical signals that are transduced to the brain (Fig. 2C). The degeneration of macula leads to the vision impairment and – finally – to the vision loss. Similar symptoms occur in the case of Stargardt’s disease. This genetic disorder develops in children and is caused by the mutation in ABCA4 gene encoding the protein responsible for metabolites transport in the retina. ABCA4 gene mutation leads to the accumulation of toxic substances in the retinal pigmented epithelium cells leading to their death. Since these cells support rods and cones functions by providing them with nutrients, their loss leads to the death of photoreceptors (Glazer and Dryja, 2002). Stargardt’s disease is diagnosed in one in ten thousand children (Sharokh Kapadia, 2000) and, similarly to AMD, remains incurable. In 2015 the results of the first clinical trial based on the use of stem cells in AMD

and Stargardt’s disease treatment were published. Pluripotent stem cells were differentiated into retinal pigmented epithelium cells (Schwartz et al., 2015; Fig. 2C). Previous studies on mice proved that transplantation of retinal pigmented epithelium cells into the eye with degenerating macula prevents death of photoreceptors (Lu et al., 2009). Unfortunately, transplantation of retinal pigmented epithelium cells in autologous conditions required highly dangerous and invasive isolation of cells from patient’s retina (Binder et al., 2012). Clinical trials conducted by Schwartz and his colleagues encompassed 18 patients: nine with Stargardt’s disease and nine with AMD. Transplantation of cells obtained through the differentiation of pluripotent stem cells resulted in the restoration of retinal pigmented epithelium in 13 patients (Fig. 2C). After six and 12 months the visual acuity improvement was confirmed in three patients while other four did not show disease progression (Schwartz et al., 2015). The first clinical trials based on pluripotent stem cells were proposed for eye diseases for a reason. As mentioned above, the eye is an immune-privileged site (Medawar, 1948). Therefore, even in the case of nonmatched HLA between donor and recipient, transplantation of cells into the eye does not trigger the immunological response that would lead to the destruction of cells recognized as “strange” (Streilein, 1987). This phenomenon results from the anatomical separation of the eye from lymphatic vessels. This precludes immune cells which are responsible for the recognition and removal of strange cells from migrating to the eye (Streilein, 2003). Additionally, some structures of the eye, such as retina may block the activity of immune cells and reduce the immunological response. Due to their ability to differentiate into all cell types building mammalian body, pluripotent stem cells are considered as a potential source of cells for therapies for other diseases (reviewed in Świerczek et al., 2013;


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therapy was initially approved for use for patients with LSCD (the report of the European Medicines Agency, EMA/25273/2015). In 2010 the results of Holoclar therapy were described in detail. The transplantation of the implant resulted in a significant reconstruction of the cornea and the improvement in vision ability in 75 out of 104 patients (Rama et al., 2010). On 17th February 2015 the Holoclar therapy was finally approved by the European Commission for use in medical centers in all member countries of the European Union.

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alternative, microsurgical procedures involving the removal of the damaged cornea (known as keratectomy) were performed. However, this method had mostly an aesthetical effect and did not lead to the recovery of vision. The first transplantations of stem cells to injured cornea were performed in the 70s of the XX century, but at that time they did not result in the expected therapeutic effect. The development of effective therapy for damaged cornea required many years of collaboration between researchers, medical doctors, and patients participating in clinical trials focused on this new treatment, further supported by huge money donations. This resulted in Holoclar – the first stem cell therapy for patients with LSCD – which was eventually approved in 2015 (Fig. 2B). Holoclar is dedicated for patients with LSCD in one eye. The second healthy eye serves as a source of cells which are required for transplantation. First, doctors isolate 1-2 mm2 fragment of the limbus from the healthy eye of the patient (Fig. 2B). Next, cells obtained from the limbus biopsy are multiplied in the special culture dishes (Fig. 2B). When appropriate number of cells is available, cells are placed on a special eye lens which is then transplanted to the injured eye of the patient. Earlier, the connective tissue membrane covering the front of the eyeball is removed by microsurgical techniques. The procedure described above serves as an example of autologous transplantation which is characterised by the lack of the risk of the graft rejection. Holoclar therapy is especially valuable because it leads both to the restoration of the damaged cornea with transplanted cells as well as to the renewal of limbal stem cell population. This means that cornea regains the ability to regenerate in case of contingent damage. The most common side effect of Holoclar is the inflammation of the eyelid, while the most serious side effects are cornea perforation and ulcerative keratitis. In 2008 Holoclar

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Fig. 3. Properties of pluripotent stem cells that promotes or inhibits their potential use in regenerative medicine

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The diagram provides information about the characteristics of pluripotent stem cells that are favorable (green frame) or unfavorable (red frame) in the context of their potential clinical use.

Fig. 4. Properties of mesenchymal stem cells that promotes or inhibits their potential use in regenerative medicine The diagram provides information about the characteristics of mesenchymal stem cells that are favorable (green frame) or unfavorable (red frame) in the context of their potential clinical use.


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Fig. 3). There are many diseases caused by the lack or improper functioning of certain cell types. Diabetes type I is an example of such diseases as it results from the insufficient number of pancreatic beta cells that produce insulin. Insulin is a hormone which is responsible for maintaining the normal level of glucose in the blood. Long-lasting high concentration of glucose in the blood, which results from untreated diabetes, may lead to vision impairment, healing deficiency, coma or even death. Every year approximately 80 thousands cases are diagnosed around the world (Chiang et al., 2014). Currently available treatment involves insulin injections and maintaining proper diet which reduces disease progression. For many years researchers have been trying to design a protocol for the conversion of pluripotent stem cells into insulin producing cells. In 2006 the method of efficient derivation of beta cells from embryonic stem cells (ESC) was described (D’Amour et al., 2006). Recently, the first clinical trials based on such cells has been started by ViaCyte company. Pancreatic beta cells, obtained through differentiation of ESC, are placed in a semi-permeable capsule, which is subsequently injected under the skin of patients. Due to the capsule properties the cells receive nutrients and oxygen from the bloodstream. Moreover, in response to the high glucose levels they can secrete insulin (described on ViaCyte. com). It is also important that the capsule protects the cells located within from the attack of host’s immune cells. ESC, as well as more specialized cells derived from them, are recognized as strange to the host and for this reason could be eliminated from the host body by the immune cells. The origin of ESC is another difficulty in the clinical application of these cells. ESC are derived from the embryos at early stages of development. Isolation of ESC may lead to the destruction of the embryo which remains controversial (Fig. 3). The promising alternatives for ESC are induced pluripotent stem cells

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Mesenchymal stem cells and their potential use in regenerative medicine Pluripotent stem cells are not the only cells that could potentially be used in regenerative medicine. Recently more and more projects are focused on mesenchymal stem cells (MSC). MSC were discovered in the 60s of the last century but their actual physiological role remained unknown for a long time. In 1980 a Russian scientist – Alexander Friedenstein – published the results of his experiments in which he transplanted cells isolated from bone marrow to other organs and tissues

possible therapies based on these cells are not as numerous as in the case of pluripotent stem cells (Fig. 3, Fig. 4). However, MSC are characterized by unique feature – the ability for immunomodulation – which means that they can influence the immune system functioning. It has been proved that MSC secrete substances that suppress the activity of immune cells such as lymphocytes T and B, macrophages, and dendritic cells (Najar et al., 2016). These substances are called anti-inflammatory cytokines. Due to their unique features MSC are currently tested as a possible tool in the therapy for different diseases. Growing popularity of MSC is indicated by the number of clinical trials based on these cells: in 2009 there were 30 of such clinical trials registered around the world, in 2013 – 300, while in February 2016 – five thousand. Most of these trials are focused on the potential application of MSC in the therapy for skeletal diseases such as children osteogenesis imperfecta which develops due to the mutation in collagen coding gene. Collagen is the main component of laminae osseae. The presence of abnormal form of the collagen is manifested by an excessive fragility of bones which can be broken even during sleep (Biggin and Munns, 2014). The other group of trials are focused on testing the immunomodulatory ability of MSC. For example, MSC are currently being tested in the treatment for Crohn’s disease, which is characterized by a chronic inflammation of gastrointestinal tract. The results of both clinical trials mentioned above have not been released yet. However, in 2014 the results of other clinical trial were published, indicating that transplantation of MSC to patients suffering from rheumatoid arthritis led to the reduction of pain caused by the strong inflammation in the joints (El-Jawhari et al., 2014). This effect was achieved due to anti-inflammatory cytokines produced by MSC which suppressed the activity of lymphocytes B and T, and – in consequence – limited the inflammation (El-Jawhari et


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of a mouse. He observed a formation of bone, cartilage, and adipose tissues in the areas engrafted by transplanted cells (Friedenstein, 1980). Currently, it is known that these tissues were derived from multipotent MSC, which together with HSC are localized in bone marrow (Pojda et al., 2013). MSC are crucial elements of the niche for HSC that differentiate into all types of blood cells. MSC also participate in bones and cartilage remodelling, and can differentiate into adipocytes as well. Apart from bone marrow MSC are also present in liver, pancreas, kidney, muscles, and adipose tissue (Nombela-Arrieta et al., 2011). Over the years several methods for isolation of MSC from different organs and tissues as well as for their further culture in vitro were developed (Bianco, 2014). The morphology of MSC is similar to fibroblasts: they are fusiform, long, and adhere to the culture dish. Lately, more and more attention is being paid to adipose derived stem cells (ADSC), i.e. MSC derived from adipose tissue. It is associated with the fact that 200 thousand times more MSC can be obtained from adipose tissue than from bone marrow, where MSC make up no more than 0,002 % of all cells (Pojda et al., 2013). The possibility of derivation of MSC from different niches (tissues and organs) in adult organisms as well as from the fetal tissues and organs such as placenta, Wharthon’s jelly or umbilical cord blood enhances the potential use of these cells in regenerative medicine (Fig. 4). In comparison to pluripotent stem cells MSC have more limited potential to differentiate (multipotency) and – in consequence – do not create teratomas after transplantation (Fig. 4). In the case of pluripotent stem cells even one inadequately differentiated cell in the graft may lead to teratoma formation (Fig. 3). Although teratomas are not malignant tumors they can develop in tissues and organs that are crucial for proper body functioning (for example lungs) leading to their destruction. On the other side, multipotency of MSC means that the

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(iPSC), which similarly to ESC are able to differentiate into all cells types and tissues in the mammalian body (described in Świerczek et al., 2013). First iPSC were derived by Takahashi and Yamanaka in 2006 (Takahashi and Yamanaka, 2006). Nowadays such cells can be obtained from nearly all fetal or adult cells through the procedure called reprogramming (reviewed in Świerczek et al., 2013). Thanks to iPSC technology cells that could serve as an universal source of various cells types for autologous transplantation became available. In July 2013 Japanese government allowed the first clinical trial based on iPSC in AMD treatment. iPSC were derived from fibroblasts which were isolated from the patients participating in the study. Next, iPSC were differentiated into retinal pigmented epithelium cells which were transplanted into the patients’ eyes (Kamao et al., 2014). The results of this clinical trial have not been published yet. However, in 2015 the trial was stopped. It is known that a mutation which could potentially cause tumor formation was found in iPSC derived from one of the patients. This indicates that there are still many challenges to overcome before iPSC are successfully and safely used in regenerative medicine (Fig. 3).

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The diagram shows the stages of implementation of a new medical product in accordance with the legal requirements in Europe. The process is preceded by a complex laboratory research (preclinical stage) in vitro (experiments using cell culture) and in vivo (experiments using animal models). The following clinical trials are divided into four phases. Their common goal is to verify the efficiency, safety and identification of possible side effects of tested medical product. Starting of the next phase of clinical trials is not possible without the completion of the previous stage.

eases had has not been verified in any scientific studies, the idea of using MSC in therapies has become popular in Italy and also in other countries. Since 2007 many MSC transplantations have been performed, but not documented in the proper way. This made the assessment of the actual number of people participating in this procedure impossible (Abbot, 2013). In 2013 in Rome, demonstrators claimed public access to stem cell transplantations, even if not properly verified. According to the demonstrators’ beliefs, such approach would be beneficial for patients suffering from chronic, life-threating diseases, which could not be efficiently cured by available methods. In response to this social phenomena, leading scientists and physicians described in details

possible side effects and danger resulting from the use of unproved treatments (Bianco et al., 2013). The gaps and shortcomings accompanying the transplantation of MSC were also indicated. Nowadays, the implementation of new treatment for human requires complex clinical trials (Fig. 5; Maciulaitis et al., 2013). Among the controversial issues of MSC transplantation there were insufficient characterization of injected cells as well as the lack of proper information on the method used for conversion of MSC into neurons. The results released by the company have not been confirmed by scientists (Carozzi et al., 2012). Moreover, it remains unclear how the cells were injected into the body of the patients. Introduction of such cells into circulatory system, which


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Despite the fact that any therapy based on MSC has not yet been approved for humans (except the Prochymal drug), the company offering the transplantation of MSC as a treatment for many diseases has existed in Italy for the past few years. It was founded by Davide Vannoni, a psychologist who was spreading the information about the possibility of curing such serious diseases as spinal muscular atrophy, cerebral palsy, amyotrophic lateral sclerosis, and Huntington’s disease. All of them develop due to the dysfunctions of nervous system and still remain incurable. The method proposed by Vannoni was based on the transplantation of neurons which were previously obtained in the laboratory from MSC, into the patients’ circulatory system. In physiological conditions, neurons are responsible for signal transmission through the body, serving as a functional basis of the nervous system. Although the therapeutic effect of MSC transplantations in the case of nervous system dis-

Fig. 5. Stages of bringing onto market new medical product

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Knowledge dissemination as a response of the scientific community to the myths about stem cells

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al., 2014). It should be noted, however, that the transplantation of MSC influenced only the symptoms of this disease, not its cause. Despite some difficulties in the clinical application of MSC, the first MSC-based drug – Prochymal – was approved in Canada, New Zealand, and USA in 2012. Prochymal is produced by Osiris Therapeutics Company and contains MSC derived from donors aged from 18 to 30 years old. The drug is currently used to prevent the development of GVHD, described earlier in the article. Its main effect relies on the secretion of both anti-inflammatory cytokines, and growth factors that suppress inflammatory cells and induce tissue regeneration, respectively (Kurtzberg et al., 2014)

Abbot A (2013). Stem-Cell ruling riles reasearchers. Nature. 495(7442):418–419. Ball LM, Egeler RM (2008). Acute GvHD: pathogenesis and classification. Bone Marrow Transplant. Suppl 2:S58-64. Bauer D, Neska J, Archacka K (2013). Stem cells. Part III – adult stem cells. Biological and Environmental Education. 4(48):3-10. Bianco P, Riminucci M, Gronthos S, Robey PG (2001). Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 19(3):180-192. Bianco P, Barker R, Brüstle O, Cattanego E, Clevers H, Daley QG, De Luca M, Goldstain L, Lindvall O, Mummery C, Robey GP, Sattler C, Smith A (2013). Regulation of stem cell therapies under attack in Europe: for whom the bell tolls. EMBO J. 32(11):1489–1495. Bianco P (2014). „Mesenchymal” stem cells. Annu Rev Cell Dev Biol. 30:677-704. Biggin A, Munns CF (2014). Osteogenesis imperfecta: diagnosis and treatment. Curr Osteroporos Rep. 12(3), 279-288. Binder S, Stolba U, Krebs I, Kellner L, Jahn C, Feichtinger H, Povelka M, Frohner U, Kruger A, Hilgers RD, Krugluger W (2012). Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration: a pilot study. Am J Ophthalmol. 133(2):215-225. Breitbach M, Bostani T, Roell W, Xia Y, Dewald O, Nygren JM, Fries JWU, Tiemann K, Bohlen H (2007). Potential risks of bone marrow cell transplantatations into infarcted hearts. Blood. 110(4):1362-1369. Carrozzi M, Biondi A, Zanus C, Monti F, Alessandro V (2012). Stem cells in severe infantile spinal muscular atrophy (SMA1). Neuromuscul Disord. 22(11):1032–1034. Chiang JL, Kirkman MS, Laffel LM, Peters AL, Type 1 Diabetes Sourcebook Authors (2014). Type 1 diabetes through the life span: a position statement of the American Diabetes Association. Diabetes Care. 37(7):2034-2054. Chinen J, Buckley RH (2010). Transplantation immunology: solid organ and bone marrow. J Allergy Clin Immunol. 125(2 Suppl 2):S324-335. D’Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE (2006). Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 24(11):13921401. El-Jawhari JJ, El-Sherbiny YM, Jones EA, McGonagle D (2014). Mesenchymal stem cells, autoimmunity and rheumatoid arthritis. QJM. 107:505-514. Flores-Guzmán P, Fernández-Sánchez V, Mayani H (2013). Concise review: ex vivo expansion of cord blood-derived hematopoietic

stem and progenitor cells: basic principles, experimental approaches, and impact in regenerative medicine. Stem Cells Transl Med. 2(11):830-838. Friedenstein AJ (1980). Stromal mechanisms of bone marrow: cloning in vitro and retransplantation in vivo. Haematol Blood Transfus. 25:19-29. Glazer LC, Dryja TP (2002). Understanding the etiology of Stargardt’s disease. Ophthalmol Clin North Am. 15(1):93-100. Haagdorens M, Van Acker SI, Van Gerwen V, Ní Dhubhghaill S, Koppen C, Tassignon MJ, Zakaria N (2016). Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies. Stem Cells Int. 9798374. Kamao H, Mandai M, Okamoto S, Sakai N, Suga A, Sugita S, Kiryu J, Takahashi M (2014). Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports. 2(2):205-218. Kapadia OD (2000). Stargardt’s macular dystrophy. Clinical Eye and Vision Care. Clin Eye Vis Care. 12(1-2):71-78. Kurtzberg J, Prockop S, Teira P, Bittencourt H, Lewis V, Chan K ,Horn B ,Yu L , Talano J, Nemecek E, Mills Ch, Chaudhury S (2014). Allogeneic Human Mesenchymal Stem Cell Therapy (Remestemcel-L, Prochymal) as a Rescue Agent for Severe Refractory Acute Graft-versus-Host Disease in Pediatric Patients. Biol Blood Marrow Transplant. 20(2):229-235. Linker CA (2003). Autologous stem cell transplantation for acute myeloid leukemia. Bone Marrow Transplant. 31:731–738. Lu B, Malcuit C, Wang S, Girman S, Francis P, Lemieux L, Lanza R, Lund R (2009). Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells. 27(9):2126-2135. Maciulaitis R, D’Apote L, Buchanan A, Pioppo L, Schneider CK (2012). Clinical development of advanced therapy medicinal products in Europe: evidence that regulators must be proactive. Molecular Therapy. 20(3), 479-482. Medawar PB (1948). Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol. 29(1):58-69. Najar M, Raicevic G, Fayyd-Kazan H, Bron D, Toungouz M, Lagneaux L (2016). Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells. Cytotherapy. 18(2):160-171. Nombela-Arrieta C, Ritz J, Silberstein LE (2011). The elusive nature and function of mesenchymal stem cells. Nat Rev Mol Cell Biol. 12(2):126-131. Pascolini D, Mariotti SP (2012). Global estimates of visual impairment: 2010. Br J Ophthalmol. 96(5):614-618. Passweg JR, Baldomero H, Bader P, Bonini C, Cesaro S, Dreger P,


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References

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is not their physiological niche, generates possible side effects such as uncontrolled migration of transplanted cells to different tissues and organs (including the brain and kidneys) which can be deleterious for their further functioning. The investigation conducted by the Italian Pharmaceutical Agency (AIFA, it. Agenzia Italiana del Farmaco) revealed that the safety requirements for transplantation in humans were not fulfilled during the procedure described above (Abbot, 2013). The response of scientific community to social needs encompasses publishing of articles but also setting up organisations focused on disseminating the knowledge about the progress in biomedical sciences. Numerous articles, movies, and workshops have been created by scientists who decided to share their knowledge. An example of such initiative is UniStem Day – the a meeting dedicated for teachers and high school students, which is organised annually on dozens of European universities. This event was initiated by a group of scientists at the University of Milan. In 2016 UniStem Day will take place for the first time in Poland, at the University of Warsaw. This day lectures, workshops, movie shows, and debates will give the teachers and students a great opportunity to learn about stem cells, and their potential and actual clinical application. Despite intensive research and numerous clinical trials bone marrow transplant, Holoclar therapy, and Prochymal are the only examples of the stem cells based treatments that have been approved for use in humans. Great attention is paid to the potential use of stem cells for improving functioning of such crucial organs as the heart and brain. Although currently any cell therapy for these organs are available, cooperation between scientists and doctors may lead to the development of new treatment based on the latest achievements in biomedical science as it has happened in the case of Holoclar therapy.

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Abbot A (2013). Italian stem-cell trial based on flawed data. Nature. The article available on: http://www.nature.com/news/italian-stem-cell-trial-based-on-flawed-data-1.13329. The characteristics of the VC-01 therapy by ViaCyte company. The product description available on: http://viacyte.com/products/vc-01-diabetes-therapy/ Mesenchymal stem cell therapy for the treatment of severe or refractory inflammatory and/or autoimmune disorders. The information on clinical trial available on: https://clinicaltrials.gov/ct2/show/NCT01540292 Repeated infusions of mesenchymal stromal cell in children with osteogenesis imperfecta (STOD3). The information on clinical trial available on: https://clinicaltrials.gov/ct2/show/NCT01061099?term=Osteoge nesis+Imperfecta+mesenchymal&rank=1 Report of the European Medicines Agency, EMA/25273/2015 (2014). Assessment report. Holoclar. International non-proprietary name: Ex vivo expanded autologous human corneal epithelial cells containing stem cells. European Medicines Agency. Information on clinical trial available on: www.ema.europa.eu/docs/en_GB/document_library/EPAR_ Public_assessment_report/human/002450/WC500183405.pdf Resolution of the Council of Ministers No. 164/2010, “The National Programme for the Development of Transplantation Medicine” (consolidated text). The resolution available on: http://www2.mz.gov.pl/wwwfiles/ma_struktura/docs/program_ wieloletni_04032011.pdf

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Websites

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Duarte RF, Dufour C, Falkenburg JH, Farge-Bancel D, Gennery A, Kröger N, Lanza F, Nagler A, Sureda A, Mohty M, European Society for Blood and Marrow Transplantation (EBMT) (2015). Hematopoietic SCT in Europe 2013: recent trends in the use of alternative donors showing more haploidentical donors but fewer cord blood transplants. Bone Marrow Transplant. 50(4):476-482. Pellegrini G, De Luca M (2014). Eyes on the prize: limbal stem cells and corneal restoration. Cell Stem Cell. 15(2):121-122. Pojda Z, Macha E, Kurzyk A, Mazur S, Dębski T, Gilewicz J, Wysocki J (2013). Mesenchymal Stem Cells. Advanced in Biochemistry. 59(2):187-197. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G (2010). Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 363(2):147-155. Schwartz SD, Regillo CD, Lam BL, Eliott D, Rosenfeld PJ, Gregori NZ, Hubschman JP, Davis JL, Heilwell G, Spirn M, Maguire J, Gay R, Bateman J, Ostrick RM, Morris D, Vincent M, Anglade E, Del Priore LV, Lanza R (2015). Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet. 385(9967):509-516. Sobiak J (2011). Organ and hematopoietic stem cell transplantations – historical background. JMS. 80(2):157–161. Streilein JW (1987). Immune regulation and the eye: a dangerous compromise. FASEB J. 1(3):199-208. Streilein JW (2003). Ocular immune privilege: the eye takes a dim but practical view of immunity and inflammation. J Leukoc Biol. 74(2):179-85. Szaflik J, Izdebska J (2003). Corneal transplantation. Guide for GPs. 6(6):69-72. Świerczek B, Dudka D, Archacka K (2013). Stem cells. Part II – pluripotent stem cells. Biological and Environmental Education. 2:213. Takahashi K, Yamanaka S (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126(4):663-676. Yoshida M, Takeuchi M, Streilein JW (2000). Participation of pigment epithelium of iris and ciliary body in ocular immune privilege. 1. Inhibition of T-cell activation in vitro by direct cell-to-cell contact. Invest Ophthalmol Vis Sci. 41(3):811-821.

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Summary: In aquatic ecosystems, many different substances can degrade the environment and negatively affect the functioning of marine, brackish and freshwater organisms. The main aim of this work was to provide a short summary 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 investigated by addition of a cell-free filtrate of the cyanobacterium Nodularia spumigena cultures grown under specific light, temperature and availability of nutrients. Significant changes in diatom abundance were observed in response 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 anthropogenic substances have negative impact on the analyzed diatom, but allelochemicals showed slightly weaker effect than the herbicide.

The effect of allelopathic interactions of phytoplankton was studied almost 100 years ago (Dakshini, 1994). However, recently researchers conducted more intensive analysis of this phenomenon (Kubanek et al., 2005; Granéli and Hansen, 2006; Granéli et al., 2008; Lyczkowski 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 increased. This problem concerns both marine and freshwater environments (Smayda, 1990; Van Dolah, 2000), because HABs caused enormous economic losses in aquatic ecosystems (Granéli and Hansen, 2006). HABs

Key words: diatom, cyanobacteria, anthropogenic substances, allelochemicals received: 21.11.2015; accepted: 5.01.2015; published: 1.04.2016

dr Sylwia Śliwińska-Wilczewska: Institute of Oceanography, University of Gdańsk

mgr Zuzanna Sylwestrzak: Institute of Oceanography, University of Gdańsk

Jakub Maculewicz: Institute of Oceanography, University of Gdańsk

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

affected the functioning of the whole ecosystem, causing increased mortality of phytoplankton, macroalgae, zooplankton, zoobenthos and fish (Granéli et al., 2008). Moreover, cyanobacteria produce a wide range of secondary metabolites with different scope of their biological 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 interaction 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 producers in aquatic ecosystems compete for the space, light and nutrients. This suggests that competition for environmental 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. Cyanobacterial 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 allelopathic interactions between microorganisms by performing a series of laboratory experiments. In recent years, it was noted that increased use of various 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

S. Śliwińska-Wilczewska, Z. Sylwestrzak, J. Maculewicz, A. Zgrundo, F. Pniewski, A. Latała EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA | ebis.ibe.edu.pl | [email protected] | © for the article by the Authors 2016 © for the edition by Instytut Badań Edukacyjnych 2016

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Sylwia Śliwińska-Wilczewska, Zuzanna Sylwestrzak, Jakub Maculewicz, Aleksandra Zgrundo, Filip Pniewski, Adam Latała

Introduction

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The effects of allelochemicals... and selected anthropogenic substances on the diatom Bacillaria paxillifera

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The effects of allelochemicals... | S. Śliwińska-Wilczewska, Z. Sylwestrzak, J. Maculewicz, A. Zgrundo, F. Pniewski, A. Latała | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016

The experiments were conducted on the cyanobacterium 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 maintained 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 sterilized f/2 medium (Guillard, 1975). The media were prepared 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 using the modified method proposed by Suikkanen et al. (2004). Allelopathic activity was studied by adding

the cell-free filtrate obtained from cyanobacterial culture to the tested diatom. The culture of N. spumigena was filtered through Whatman GF/C filters. The cellfree 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 target 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 condition. 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 measured 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. Source: Author’s own work.


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Material and methods

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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-phosphonomethylglycine). 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 tryptophan, which are very important for plants as they are used for synthesis of many pigments (Franz et al., 1997). In addition, this compound hinder photosynthesis (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 popularity of these compounds in the industry, their toxicity 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 transfer 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 cyanobacterium N. spumigena grown under different light intensity, 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 allelochemicals and anthropogenic substances on B. paxillifera in aquatic environments.

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Results The effects of cyanobacterial cell-free filtrate obtained 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 basis of the results it was found that the allelopathic effect of the cyanobacterium N. spumigena significantly de-

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 analyzed diatom was investigated (Fig. 2, D-F). It was observed that the addition of copper(II) chloride at a concentration 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%

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). Source: Author’s own work.


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Data are reported as mean ± standard deviation (SD). The statistical analyses were performed using the Statistica® 10 software.

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urements were then used to determine the linear correlation 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 substances – 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 substances, the following concentrations were used: CuCl2 – 0.001 g·l-1, herbicide containing glyphosate – 0.05 g·l1 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 concentration), i.e. the concentration that causes a particular effect in 50% of experimental organisms, under certain 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 differences in analyzed parameters between the target algae cultures treated with cyanobacterial cell-free filtrates, anthropogenic substances, and the control over the experimental period. A post hoc test (Tukey’s HSD) was used to show which treatments for growth significantly differed from the control and from each other.

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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.

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.

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. Observations made by light microscope showed that the cells of B. paxillifera were in several stages of degeneration, 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 varying in size. Analyzed diatom showed also an increased number of the teratological forms. B. paxillifera showed some degree of alteration when cultivated with the cellfree filtrate of N. spumigena. In this case, a large number 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-

Source: Author’s own work.


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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

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Source: Author’s own work.

In the aquatic environment there are many different substances, both allelopathic compounds and anthropogenic substances that negatively affect the functioning of many microalgae. However, knowledge of this subject is still insufficient. The study compared the influence of allelopathic compounds and anthropogenic substances such as ionic liquids, glyphosate and copper chloride in relation to the diatoms B. paxillifera. In marine, brackish and freshwater ecosystems, mode of action of the allelopathic compounds is highly diverse and donor species may influence on the target organisms in different ways. Additionally, in natural phytoplankton 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 target organisms (Valdor and Aboal, 2007; Gantar et al., 2008). Inhibition of growth of the target organism by production allelopathic compounds are relatively widespread 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 commonly used procedure which was intended to demonstrate the effect of allelopathic interactions between donor 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

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 meneghiniana 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 activity. It is extremely effective herbicide, which inhibits metabolic pathways of plants. In recent years the popularity of this herbicide has increased with the proliferation 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 (including surfactants), because it was a form to which the environment 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 glyphosate for green algae Raphidocelis subcapitata (Korshikov) Nygaard, Komárek, J.Kristiansen & O.M.Skulberg 1987: 31 was lower than used in this study and constituted 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-


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Discussion

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 tricornutum Bohlin 1897: 520 and Heterocapsa triquetra (Ehrenberg) Stein 1883: 13 has stronger negative effect on diatom Cylindrotheca fusiformis Reimann & J.C.Lewin 1964: 288 than two heterotrophic bacteria Staphylococcus 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 bacterial 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 organisms (Chan et al., 1980). Furthermore, the study allowed to determine the effect of widely used, but less studied herbicide called Roundup® consisting of glyphosate: the ionic liquid and copper(II) chloride on the Baltic 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 “environmentally 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 relation to their effects on aquatic organisms. Understanding 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-

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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 diatom.

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Conclusion Experiments described in this article showed the influence of allelochemicals secreted by cyanobacterium N. spumigena and the impact of various anthropogenic substances, on the number of cells of diatom B. paxillifera in culture. The article focuses on important aspects of the impact of pollution from commonly used chemicals as well as allelopathic compounds that will soon need to be considered as another kind of natural pollution. 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 allelopathic compounds produced by cyanobacterium N. spumigena grown under different light intensity, temperature 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 allelopathic 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 understand their role in the marine and freshwater ecosystems compared to other chemical compounds.

References Antunes JT, Leão PN, Vasconcelos VM (2012). Influence of Biotic and Abiotic Factors on the Allelopathic Activity of the Cyanobacterium Cylindrospermopsis raciborskii Strain LEGE 99043. Microbial Ecology. 64:584-592. Berry JP, Gantar M, Perez MH, Berry G, Noriega FG (2008). Cyanobacterial toxins as allelochemicals with potential applications as algaecides, herbicides and insecticides. Marine Drugs. 6:117-146. Brown BT, Rattigan BM (1979). Toxicity of Copper and Other Metal Ions to Laminaria digitata. Enviromental Pollution. 20:303-314. Chan AT, Andersen RJ, Le Blanc MJ, Harrison PJ (1980). Algal Plating as a Tool for Investigating Allelopathy Among Marine Microalgae. Marine Biology. 59:7-13. Dakshini KMM (1994). Algal allelopathy. Botanical Reviews. 60:182196. Fisher NS, Jones GJ, Nelson DM (1981). Effects of copper and zinc on growth, morphology, and metabolism of Asterionella japonica (Cleve). Journal of Experimental Marine Biology and Ecology. 51:37-56. Fistarol GO, Legrand C, Granéli E (2003). Allelopathic effect of Prymnesium parvum on a natural plankton community. Marine Ecology Progress Series. 255:115-125. Fistarol GO, Legrand C, Selander E, Hummert C, Stolte W, Granéli E (2004). Allelopathy in Alexandrium spp.: effect on a natural plankton community and on algal monocultures. Aquatic Microbial Ecology. 35:45-56. Fistarol GO, Legrand C, Granéli E (2005). Allelopathic effect on a nutrient-limited phytoplankton species. Aquatic Microbial Ecology. 41:153-161. Franz JE, Mao MK, Sikorski JA (1997). Glyphosate A Unique Glo-


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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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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 morphological and structural changes. Authors noted that the electron microscopy revealed degeneration of thylakoids into a system of irregularly arranged membranes. In addition, disappearance of other cell structures including the nucleus was apparent. This results suggested that allelopathic compounds produced by cyanobacteria and anthropogenic substances can cause physiological changes in the target organisms. This would mean that, in the long term, low concentrations of harmful substances may affect microalgae, altering the composition and structure of phytoplankton communities.

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tigan, 1979). Concentration of 0.001 g·l-1 of copper(II) chloride used in this work inhibited growth of B. paxillifera. Direct exposure to a high concentration of copper ions can be very toxic for many microorganisms (Serra et al., 2009). Copper chloride may cause negative and long-term changes in phytoplankton community in the aquatic environment (Stauber and Florence, 1985). It was observed that the presence of toxic quantity of copper decreases the rate of cell division and increases cell size of diatom Asterionella japonica Cleve in Cleve & Möller 1882: 3 (Fisher et al., 1981). Diatoms consumed organic substances faster than other marine microalgae and may therefore be more sensitive to different substances present in the environment, which inhibited their growth (Chan et al., 1980). Such properties can explain the high sensitivity of diatom B. paxillifera on analyzed allelopathic compounds and anthropogenic substances. There are several reports indicating that the chemical compounds can cause structural and morphological changes in target cells (Valdor and Aboal, 2007; Gantar et al., 2008). In the present study it was showed that the tested allelopathic compounds and anthropogenic substances caused restriction of pigmentation and cell lysis of B. paxillifera compared to the control culture. Valdor and Aboal (2007) noted that in colony-forming Pseudocapsa sp. and Nostoc sp. there was a tendency for cells to disaggregate into isolated cells. Furthermore, many dead cells of the former species were identified, while the trichomes and filaments were discoloured, fragmented, and deformed. Observations made by an electron microscope showed that the filaments and cells of Scytonema sp. were in several stages of degeneration showing, for example, swollen sheaths, degraded or dilated thylakoids with or without dense granules inside and increased cytoplasmic inclusions. What is more, Pseudocapsa sp. showed isolated, collapsed or dead

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lutionary contexts of allelopathy. Trends in Ecology & Evolution. 26:655-662. Wells ML, Trick CG, Cochlan WP, Hughes MP, Trainer VL (2005). Domoic acid: The synergy of iron, copper, and the toxicity of diatoms. Limnology and Oceanography. 50:1908-1917. Wong PK (2000). Effects of 2,4-D, glyphosate and paraquat on growth, photosynthesis and chlorophyll a synthesis of Scenedesmus quadricauda Berb 614. Chemosphere. 41:177-182. Yamasaki Y, Nagasoe S, Matsubara T, Shikata T, Shimasaki Y, Oshima Y, Honjo T (2007). Allelopathic interactions between the bacillariophyte Skeletonema costatum and the raphidophyte Heterosigma akashiwo. Marine Ecology Progress Series. 339:83-92. Yang X, Deng S, De Philippis R, Chen L, Hu C, Zhang W (2012). Chemical composition of volatile oil from Artemisia ordosica and its allelopathic effects on desert soil microalgae, Palmellococcus miniatus. Plant Physiology and Biochemistry. 51:153-158. Żak A, Musiewicz M, Kosakowska A (2012). Allelopathic activity of the Baltic cyanobacteria against microalgae. Estuarine, Coastal and Shelf Science. 112:4-10.


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and pyridinium based ionic liquids towards algae. Chlorella vulgaris, Oocystis submarina (green algae) and Cyclotella meneghiniana, Skeletonema marinoi (diatoms). Green Chemistry. 11:580588. Latała A, Nędzi M, Stepnowski P (2010). Toxicity of imidazolium ionic liquids towards algae. Influence of salinity variations. Green Chemistry. 12:60-64. Lyczkowski ER, Karp-Boss L (2014). Allelopathic effects of Alexandrium fundyense (Dinophyceae) on Thalassiosira cf. gravida (Bacillariophyceae): a matter of size. Journal of Phycology. 50:376-387. Maa J, Wangc S, Wangb P, Mab L, Chena X, Xua R (2006). Toxicity assessment of 40 herbicides to the green alga Raphidocelis subcapitata. Ecotoxicology and Environmental Safety. 63:456-462. Mazur-Marzec H, Błaszczyk A, Felczykowska A, Hohlfeld N, Kobos J, Toruńska-Sitarz A, Devi P, Montalvão S, D’souza L, Tammela P, Mikosik A, Bloch S, Nejman-Faleńczyk B, Węgrzyn G (2015). Baltic cyanobacteria–a source of biologically active compounds. European Journal of Phycology. 50:343-360. Phama TPT, Choa CW, Yuna Y (2010). Environmental fate and toxicity of ionic liquids: A review. Water Research. 44:352-372. Pieniążek D, Bukowska B, Duda W (2003). Glifosat – nietoksyczny pestycyd? Medycyna Pracy. 54:579-583. Samorì C, Sciutto G, Pezzolesi L, Galletti P, Guerrini F, Mazzeo R, Tagliavini E. (2011). Effects of imidazolium ionic liquids on growth, photosynthetic efficiency, and cellular components of the diatoms Skeletonema marinoi and Phaeodactylum tricornutum. Chemical research in toxicology. 24: 392-401. Serra A, Corcoll N, Guasch H (2009). Copper accumulation and toxicity in fluvial periphyton: The influence of exposure history. Chemosphere. 74:633-641. Smayda TJ (1990). Novel and nuisance phytoplankton blooms in the sea: evidence for a global epidemic. In: Granéli S, Sundström B, Edler L, Anderson DM, eds. Toxic Marine Phytoplankton. Elsevier Science, New York, NY, USA, pp. 29-40. Stauber JL, Florence TM (1985). The influence of iron on copper toxicity to the marine diatom, Nitzschia closterium (ehrenberg) W. Smith. Aquatic Toxicology. 6:297-305. Suikkanen S, Fistarol GO, Granéli E (2004). Allelopathic effects of the Baltic cyanobacteria Nodularia spumigena, Aphanizomenon flos-aquae and Anabaena lemmermannii on algal monocultures. Journal of Experimental Marine Biology and Ecology. 308:85-101. Valdor R, Aboal M (2007). Effects of living cyanobacteria, cyanobacterial extracts and pure microcystins on growth and ultrastructure of microalgae and bacteria. Toxicon. 49:769-779. Van Dolah FM (2000). Marine algal toxins: origins, health effects, and their increased occurrence. Environmental Health Perspectives. 108:133-141. Wardle DA, Karban R, Callaway RM (2011). The ecosystem and evo-

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bal Herbicyde. ACS Monograph 189. American Chemical Society, Washington CD. Gantar M, Berry JP, Thomas S, Wang M, Perez R, Rein KS, King G (2008). Allelopathic activity among cyanobacteria and microalgae isolated from Florida freshwater habitats. FEMS Microbiology Letters. 64:55-64. Granéli E, Hansen PJ (2006). Allelopathy in harmful algae: a mechanism to compete for resources? In: Granéli E, Turner JT, eds. Ecology of Harmful Algae, Ecological Studies. 189. Springer-Verlag, Berlin Heidelberg, Germany: 189-201. Granéli E, Weberg M, Salomon PS (2008). Harmful algal blooms of allelopathic microalgal species: The role of eutrophication. Harmful Algae. 8:94-102. Gross EM (2003). Allelopathy of Aquatic Autotrophs. Critical Reviews in Plant Sciences. 22:313-339. Guillard RRL (1975). Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH, eds. Culture of Marine Invertebrate Animals. Plenum Press, New York, USA, pp. 26-60. Kabata-Pendias A, Pendias H (1999). Biogeochemia pierwiastków śladowych. Wydawnictwo Naukowe PWN, Warszawa. 1.4:111126. Kubanek J, Hicks MK, Naar J, Villareal TA (2005). Does the red tide dinoflagellate Karenia brevis use allelopathy to outcompete other phytoplankton? Limnology and Oceanography. 50:883-895. Kulacki KJ, Lamberti GA (2008). Toxicity of imidazolium ionic liquids to freshwater algae. Green Chemistry. 10:104-110. Kwiatkowska, M, Jarosiewicz P, Bukowska B (2013). Glifosat i jego preparaty – toksyczność, narażenie zawodowe i środowiskowe. Medycyna Pracy. 64:717-729. Latała A (2003). Autecological characteristic of some algal strains from Culture Collection of Baltic Algae (CCBA). In: Lima N, Smith D, eds. Biological Resource Centers and the Use of Microbes, Micoteca da Universidade do Minho, Braga, Portugal, pp. 323345. Latała A, Stepnowski P, Nędzi M, Mrozik W (2005). Marine toxicity assessment of imidazolium ionic liquids: acute effects on the Baltic algae Oocystis submarina and Cyclotella meneghiniana. Aquatic Toxicology. 73:91-98. Latała A, Jodłowska S, Pniewski F (2006). Culture Collection of Baltic Algae (CCBA) and characteristic of some strains by factorial experiment approach. Archiv für Hydrobiologie 165, Algological Studies. 122:137-154. Latała A, Nędzi M, Stepnowski P (2009a). Toxicity of imidazolium and pyridinium based ionic liquids towards algae. Bacillaria paxillifer (a microphytobenthic diatom) and Geitlerinema amphibium (a microphytobenthic blue green alga). Green Chemistry. 11:1371-1376. Latała A, Nędzi M, Stepnowski P (2009b). Toxicity of imidazolium

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Summary: Human activity has an impact on environment, especially on air quality changes. In the article there are presented main pollutants emission sources in Poland, including emissions of particulate matter, SO2, CO, NOX, non-methane volatile organic compounds, ammonia, dioxins and furans, PCB, PAH, and heavy metals. There are also shown reasons for present air quality and means of reduction of various compound emissions. An attention has been paid to health problems, which can occur during long-time exposure on pollutions. Key words: air quality, emissions of pollutants, low-stack emission received: 30.11.2015; accepted: 1.03.2016; published: 1.04.2016

mgr eng. Martyna Nowak: Institute for Chemical Processing of Coal, ul. Zamkowa 1, 41-803 Zabrze; e-mail: [email protected]

Proper state of natural environment guarantees safe human functioning and the quality of particular environment elements strongly affects human health. A significant part of human activities has an impact on the environment, its resources and stability of ecosystems (Albiniak, 2014). Human activity results in air emissions of various gaseous and particulate pollutants. These compounds can react with each other and solar radiations, high temperature and high humidity favor such reactions. Accumulation of negative activities can cause non-reversible changes in the surrounding environment (Toczko, 2011). Air pollutants have also a direct impact on human health – their presence may cause health problems, related to respiratory and circulatory systems (Özkan et al., 2016), (Nurul et al., 2014). Some of the compounds, like benzo[a]pyrene show carcinogenic activity (Trojanowska and Świetlik, 2013).

Air quality assessment systems in Poland According to article 89 (1) of Act of 27 April 2001 Environmental law (Journal of Laws of 2001 No 62 item 627, as amended) Regional Inspectorate for Environmental Protection (RIEP) evaluates levels of substances in the air, based on data from State Environmental Monitoring stations. Regional reports, which contain the assessment results are published on the RIEP websites. According to article 90 of the Environmental Law, assessment results are transferred to the voivodship boards, which in case of exceeding the substance permissible levels, elaborate an air protection program (Journal of Laws of 2001 No 62 item 627, as amended). Based on regional annual assessment, the Chief Inspector for Environmental Protection elaborates cumulative assessment of air quality, which is available on the

Air Quality Portal website http://powietrze.gios.gov.pl. There is also available a free application for smartphones with Android and iOS systems, in which there are current measurements results, a map of measurement stations, information about high pollutants concentration and news from Chief Inspector for Environmental Protection (AQP, 2016). In terms of human health protection, the annual air quality assessment includes following substances: • Sulfur dioxide (SO2), • Nitrogen dioxide (NO2), • Carbon monoxide (CO), • Benzene (C6H6), • Ozone (O3), • Particulate matter PM10 (with diameter up to 10 μm), • Particulate matter PM2.5 (very small particles, with diameter up to 2.5 μm), • Lead (Pb), • Arsenic (As), • Nickel (Ni), • Cadmium (Cd), • Benzo[a]pyrene. In terms of plant protection, the annual air quality assessment includes three substances: • Sulfur dioxide (SO2), • Nitrogen oxides (NOX) – the sum of nitrogen oxide (NO) and nitrogen dioxide (NO2) expressed as nitrogen dioxide, • Ozone (O3).

Main air pollutant sources in Poland Figure 1 presents the total emissions of the most important air pollutants in Poland. In the 2000–2013 carbon monoxide was a substance which was produced in the highest amount (over 2.6 million tons in 2000 to


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Martyna Nowak

Introduction

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The reasons for current air quality in Poland and main activities to its improvement

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The reasons for current air quality in Poland and main activities to its improvement | Martyna Nowak | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016

Total emissions of selected air pollutans, thousand tons

3500 3000 2500 2000 1500 1000 500 0 Sulfur dioxide Nitrogen oxide

2000

Carbon monoxide 2005

Non-methane volatile organic compounds 2010

Ammonia

Particulate matter

2012

Fig. 1. Total air emissions of SO2, NO2, CO, NMVOC, NH3 and particulate matter in 2000-2013, Fig. 1. Total air emissions of SO2, NO2, CO, NMVOC, NH3 and particulate matter in 2000-2013, thousand tons thousand tons (Source: Bochenek 2015). Source: Bochenek, 2015. 1600

and is applied during an inventory of pollutant emissions (Bochenek, 2015). Presented in Table 1 processes of combustion in energy production and transformation industries (SNAP 01) include public power plants and thermal power plants (SNAP 0101), heating plants (0102), refineries (0103), solid fuels transformation (0104) and mining of power raw materials (0105). Nonindustrial combustion plants involves commercial and institutional plants (0201), households (0202), agriculture and forestry (0203). Road transport (07) includes combustion fuels in passenger cars (0701), light duty vehicles, heavy duty vehicles (0702 and 0703), mopeds and motorcycles (0704 and 0705), gasoline evaporation from vehicles (0706), automobile tire and brake wear and road abrasion (0707). Waste management (09) is mainly waste incineration (also agricultural wastes) (0902 and 0907) and agriculture (10) are cultures with fertilizers (1001), on-field burning of stubble and straw (1003) and manure management (1005). According to data shown in the Table 1, the biggest source of pollutants emissions are non-industrial combustion plants, mainly households. Emissions of carbon monoxide, polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAHs) (including benzo[a]pyrene) and particulate matter are much higher in this sector than in the other ones. The share of PAHs emission from non-industrial combustion plants is over 92%. Emissions of dioxins and furans (PCDD/F) is equal to 155.3 g I-TEQ (toxic equivalence) and therefore the emissions are much higher in this non-industrial combustion plants than in other activities. The second largest source of pollutants emissions is road transport. In this sector, the highest share of pollutants has carbon monoxide (581.16 thousand tons). The emission of organic pollutants is also significant (both PCB and PAHs – 2521.3 kg).


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Table 1 shows the total emissions of main and organic air pollutants, by kids of activity in 2013. In the following table there are presented kinds of activities with its classification code in SNAP nomenclature (Selected Nomenclature for Air Pollution). SNAP nomenclature is an European classification of activities

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over 2.8 million tons in 2013). The highest reduction of pollutant is related to sulfur dioxide – from almost 1.5 million tons in 2000 to about 0.8 million tons in 2013. The emissions of nitrogen oxides, non-methane volatile organic compounds, ammonia and particulate matter are rather stable.

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2000

200520002010 2000 20052000 2012 2005 20102005 2010 2000 20122010 2012 2005 20122010

2012

30 Fig. 1. Total Fig. air emissions 1.Fig. Total 1. Total Fig. airofemissions 1. SO air Total NOFig. air CO, emissions 1.SOTotal NMVOC, of SOair NO CO, emissions NH SO ,and NONMVOC, ,ofCO, NH SONMVOC, NO NH23matter particulate ,and CO, NHparticulate NMVOC, and 2000-2013, matter particulate NH matter and 2000-2013, matter particulate in 2000-2013, in 2000-201 matter 2,emissions 2,of 2, NO 2,of 2,NMVOC, 32CO, 2particulate 23, and 3in 3 in thousand tons thousand (Source: thousand tons thousand Bochenek (Source: tons (Source: tons 2015). thousand Bochenek (Source: Bochenek tons 2015). Bochenek (Source: 2015). 2015). Bochenek 2015).


1600

PAH kg 1600 1600

1600

1600

12.4 1400

135.9 1400215.6 1400

1400

1400

1200 155.3

1200 1200 511.4 1200 135167.3

52.5 1000

15.7 1000 1000 6.9

1000

14.4

31.1

-

-

7195.9 800 800 -

800

-

600 600 4.3

600

0.7400

62.2

4002459.1 400

400

-

200530.5 200

200

PCDD/F

PCB

g I-TEQ

Combustion in energy production and transformation industries (01)

400.42

243.58

60.65

19.76

-

35.89

Non-industrial combustion plants (02)

284.15

93.17

1843.22

123.17

0.53

163.64

Combustion in industry (03)

149.56

69.21

256.23

10.53

-

32.12

Production processes (04)

11.20

23.61

31.86

75.10

1.14

34.12

Extraction and distribution of fossil fuels (05)

-

-

-

38.57

-

14.54

Solvent and other products use (06)

-

0.00

0.01

208.85

0.01

1.67

Road transport (07)

1.20

255.08

581.16

139.89

0.71

75.88

Other vehicles and machinery (08)

0.23

100.08

80.51

16.72

0.01

9.49

0.1200

Waste management (09)

0.09

1.66

20.50

2.94

2.55

20.13

1.5

Agriculture (10)

-

11.85

2.26

0.25

258.46

19.88

-

Other sources of pollutant emission and absorption (11)

-

-

-

282.99

-

0.22

14.3

Table 1. Total emissions of main and organic air pollutants by kinds of activity in 2013

800 600

0.0

0

1.0

1000 800 600 400 200

-

Arsenic

0 0 0 - Arsenic Chromium Arsenic Chromium Zinc Arsenic Chromium Cadmium Chromium Zinc Arsenic ZincCopper Cadmium Chromium Zinc Cadmium Nickel Copper Cadmium Zinc CopperLead Nickel Cadmium Copper Nickel Mercury Lead Nickel Copper LeadMerc Le N

-

-

-

0

1200

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PM

2000

200520002010 2000 20052000 2012 2005 20102005 2010 2000 20122010 2012 2005 20122010

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NH3

Total emission of heavy metals, tons

NVOC


CO

thousand tons


NO2


2012

Fig. 2. Total Fig. emission 2.Fig. Total 2. ofTotal Fig. emission heavy 2.Fig. emission Total metals, Fig. emission heavy tons of2. heavy Total metals, (Source: of emission metals, heavy Bochenek metals, (Source: tons of heavy (Source: tons 2015) Bochenek metals, (Source: Bochenek tons 2015) Bochenek (Source: 2015) 2015) Bochenek 2.ofTotal emission of tons heavy metals, tons

Source: Bochenek, 2015.

Source: Bochenek, 2015.

The lowest amount of emissions was generated in the following sectors: waste management, where the total amount of generated pollutants was 47.87 thousand tons and extraction and distribution of fossil fuels, where the total amount of generated pollutants was 53.11 thousand tons. Data presented in the Table 1 indicates also, that the highest emissions of sulfur dioxide are generated in processes of combustion in energy production and transformation industries (mainly public power plants and thermal power plants). The biggest source of nitrogen oxides emissions is road transport. The highest NMVOCs emissions are generated during solvent

and other products use and other sources of pollutant emissions and absorption. Agriculture, mainly animal production, is the source of the highest ammonia emissions. Figure 2 presents the total amount of heavy metals – arsenic, chromium, zinc, cadmium, copper, nickel, lead and mercury. From the presented data, we can conclude, that over 2000-2013, the highest levels of pollutions were related to zinc (about 1400-1600 tons), while the lowest emissions were related to mercury (10.1-10.7 tons). Emissions of zinc in 2010 and 2013 were higher in respect to 2000 and 2005, while

the emission level of other pollutants did not change significantly. Table 2 shows emissions of air pollutants by types of road transport facilities in 2013. Passenger cars generated the highest amount of pollutants – jointly over 25 million tons. However, the smallest amount of emissions comes from mopeds. The differences between emissions from particular road transport facilities are related to amount of existing vehicles. In 2014 there were almost 26.5 million vehicles, from which about 20 million were passenger cars (Bochenek, 2015). The highest emissions of CO2, CH4, N2O, CO, NMVOC, NOX, SO2, Pb and PM were related to passenger cars. The only sources of lead


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SO2


Kinds of activity (SNAP code)


CO

NVMOC

NOX

PM

SO2

Pb

thousand tons

Passenger cars

25 231.7

2.72

1.07

456.1

43.63

98.27

7.43

0.56

0.014

Cars other than passenger cars with total mass up to 3.5 t

7 634.8

0.43

0.36

61.94

8.94

31.23

2.49

0.18

0.001

Lorries with total mass over 3.5 t

6 450.8

0.53

0.27

26.66

25.22

93.61

4.76

0.37

-

Buses with total mass over 3.5 t

1 745

0.09

0.03

7.11

3.49

15.59

1.04

0.05

-

77

0.11

0

11.72

3.9

0.14

0

0

0

Mopeds

33.9

0.05

0

5.37

2.34

0.02

0

0

0

Agricultural tractors

831.8

0.05

0.05

12.26

2.18

14.18

1.38

0.03

-

Motocycles

Table 2. Air pollutants emission by types of road transport facilities in 2013, thousand tons Source: Bochenek, 2015.

1% 5%

4%

Hard coal Lignite

14%

Crude oil

40%

Natural gas Peat and fuel wood Renewables

24%

Solid waste fuels and other sources

12% Fig. 3. Structure of consumption of primary energy commodities in Poland in 2014 Source: Bochenek, 2015.

Fig. 3. Structure of consumption of primary energy commodities in Poland in 2014 (Source: Bochenek 2015)

emissions are passenger cars and cars other than passenger one with total mass up to 3.5 t.

Analysis of the reasons of current air quality in Poland Structure of pollution in Poland depends on kind of used fuel, its quality and consumption structure. These factors has a huge influence on the emissions of most air pollutants. Technologies used in public power plants and combustion techniques used in municipal sector also have a huge impact on the emissions structure (Albiniak, 2014). Figure 3 shows the structure of primary energy commodities in Poland in 2014. The basic energy source in Poland is hard coal (40% of all energy carriers). The consumption of renewable sources of energy (i.e. hydro, wind, solar, geothermal energy and heat pumps) is the smallest in the national economy and is equal to 1% of all energy carriers. The basic problem related to air quality in Poland is the failure to meet the allowable amount of days with exceeded allowable level of particulate matter PM2.5, PM10 and exceeding the target level of benzo[a]pyrene (KPOP 2015). The excess of PM10 emissions concerns both daily (especially in winter season) and annual standards. The excess of PM2.5 emissions concerns annual standards. Increased particulate matter emissions are related to all big cities and urban areas (Albiniak, 2014). The analysis of annual assessments, conducted by Environmental Protection Inspection, showed that lowstack emissions have the main impact on current air quality (KPOP, 2015). Low-stack emission is an emission of pollutants from emitters (stacks), located at the height up to 40 m. In fact, the pollutants are emitted mainly from emitters with height about 10 m and are distributed at the nearby surroundings. Low-stack


S7

S16

S23

S1

S2

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CH4

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CO2

Types of road transport facilities

31

Crude oil

32

Natural gas

emission is a problem which considers introduction of average concentration) levels and share of particular secPeat and (i.e. fuelnon-industrial wood hazardous particulates and gases into the atmosphere, tors combustion plants, cokemaking which are generated during ineffective combustion in plants, road transport, agriculture, forestry, fishing, aluhouseholds, cars and thermal plants (Sadlok, 2014). minum production, industrial combustion plants and Renewables 24% The low-stack emission mainly comes from municiconstruction and production of electricity and heat) in pal sector and include (KPOP, 2015): Data presented in fig. 4 and Solidbenzo[a]pyrene waste fuels andemissions. other • individual heat generation sources and hot water fig. 5 are related to the reasons of exceeding the allowable sources 12% preparation (non-industrial combustion plants), levels of PM10 and PM2.5 in the air, which are determined • local thermal plants (e.g. district plants), by the air quality monitoring stations under the State • transport. Environmental Monitoring. The monitoring stations are Figures 4, 5 and 6 show the reasons of exceeding the localized mainly in the urban areas. The main reason of ucture of consumption of primary energy commodities in Poland in 2014 (Source: Bochenek exceeded levels of PM10 and PM2.5 is the emission related allowable PM10 (24 h concentration) and PM2.5 (annual

S7 0,03%

S16 1,80%

S23 0,87%

S1 5,83%

S2 3,54%

S3 2,14% S4 0,58%

S1 2,6%

S2 5,3%

to individual heating (S5). Other, significant reasons are emissions related to traffic (S1+S2) and emissions related to industrial plants, heat and power plants localized near the measuring station (S3). Data presented in fig. 6 shows, that the biggest share in benzo[a]pyrene emission have non-industrial combustion plants – 77%. Polycyclic aromatic hydrocarbons (including benzo[a]pyrene) are emitted mainly during combustion of solid fuels in households (Albiniak, 2014). A significant share in emission of this pollutant have also cokemaking plants – 15%. The share of transport in benzo[a]pyrene emissions are 6%.

S3 2,6%

Legend to fig. 4 and fig. 5: S1 – emissions related to intensive traffic in the cities’ centers S2 – emissions related to traffic on a main roads near a measuring station

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40%


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14%

S4 – emissions from mines and quarries located near the measuring station S5 – emissions related to individual heating S7 – emergency emission from sources other than industrial

S5 89,5% S5 85,21%

S16 – secondary emissions of particulate matter from uncovered surfaces, e.g. roads, pavements, courts S23 – emissions related to periodical construction and road works

Fig. 5. Reason the allowable level of PM2,5 emissions Fig. 4. Reasons for exceeding the allowable level of PM10 emissionsfor (24exceeding h Fig. 5. Reason for exceeding the allowable level of(average PM2.5 annual concentration) concentration) in 2014 – percentage level in the in the national scale emissions (average annual concentration) in 2014 in 2014 (Source: Kobus et al. 2015)

Source: Kobus al., 2015. Source: Kobus easons for exceeding theet allowable level of PM10 emissions (24h concentration) in 2014 – et al., 2015. percentage level in the in the national scale (Source: Kobus et al. 2015) Cokemaking plants

15% | ebis.ibe.edu.pl | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA Aluminium

Agriculture, forestry, fishing, [email protected] | © for the article by the Authors 2016 working machines 1%

© for the edition by Instytut Badań Edukacyjnych 2016

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S3 – emissions related to industrial plants, heat and power plants localized near the measuring station

Road transport 6% Production of electricity and heat 0,005% Industrial combustion plants and construction 0,01%

Non-industrial combustion plants 77%

Fig. 6. Share of particular sectors involved in benzo[a]pyrene emissions in Poland in 2012 Source: Kobus et al., 2015.

Fig. 6. Share analysis of particular sectors involvedofinlowbenzo(a)pyrene emissions in Poland in 2012 (Source:and eleConducted of the possibilities • demand related to passengers transport et al. 2015) ments dependent of policies in the field of: econostack emission reduction shows, that in 12.9Kobus million

households there are 7.5 million ceramic furnaces and 1.5 million steel furnaces. In most cases, the furnaces are coal fired and many of them have very low efficiency (Schönfelder, 2011). In municipal sector wastes are also a common source of heat. Wastes, which are fired, have various composition, which can have a negative impact on human health, life and the environment. In the transport sector following issues have an impact on the emissions:

my, agriculture, tourism, finances, etc. • organization of transport services (e.g. application of logistics and smart technologies), • technical solutions applied in vehicles (drive and fuel) and infrastructure, • average length of everyday rides, • uneconomical, often aggressive driving style (KPOP, 2015). In cities organization and traffic management have a large impact on the pollutants emission. Intensive traffic, its inadequate organization and improper drive

technique result in traffic jams, thereby energy losses related to frequent stops and acceleration and increased emissions (KPOP, 2015). Also the adverse weather conditions, such as windless conditions, low temperature and mist, have an impact on air quality. The meteorological conditions have a significant meaning in following emission sources: household’s furnaces, local thermal plants and car transport. Furthermore, in some Polish cities, topography conditions (i.e. emission sources placed in valleys or river basins) have a significant impact on pollutants level – such localization impedes pollutants dispersion. Another problem is related to industry concentration in urban areas or their direct neighborhood. Cracow and Upper Silesia Urban Areas are the examples of such regions (KPOP, 2015). Besides the emission reduction during last decades, the air quality in Europe still does not meet the adequate standards. It is estimated, that about 90% of European citizens are exposed to air pollutants, such as particulate matter, nitrogen dioxide, ozone or benzo[a] pyrene. These compounds have the biggest impact on humans health (KPOP, 2015).

The impact of pollutions on human health Environment, lifestyle, genes and level of medical care are the basic factors, which affect the human health. Relations between environmental factors and health are very complex. Human organism is affected simultaneously by many harmful factors, which occur in relative low concentration in air, water, soil and food. Very often their character is lingering, lasting for whole or most of the lifetime. The health effects related to exposure on harmful environment factors are various and causes temporary or permanent functional disorders, rather than obvious diseases (Albiniak, 2014).


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Cokemaking plants 15% Aluminium production 0,3% Others 0,1%

Agriculture, forestry, fishing, working machines 1%

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in 2014 (Source: Kobus et al. 2015)

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The reasons for current quality in Poland and main activities to PM2,5 its improvement | Martyna Nowak | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016 Fig. 5. Reason forair exceeding the allowable level of emissions (average annual concentration)

Activities aiming at air quality improvement In order to protect human health and natural environment in Poland, a number of instruments of air

Substance

Substance’s characteristics

Impact on human health

Ozone

Colorless gas, slightly sweet odor

Irritates respiratory system, increases symptoms of lung diseases, increases asthma symptoms, causes damage in the respiratory system cells, causes cough, discomfort in the chest, eye irritation

Particulate matter

Very small particles

Small particles can be deposited in lungs and can cause pneumonia and increases heart and lungs diseases. Infants, children and teens, elderly people over 65 years, people suffering from respiratory system or heart diseases, diabetic and people working outdoors are in the most at risk group

Carbon monoxide

Colorless, odorless gas

Decreases the oxygen absorption in the blood, which results in significant reduction of oxygen supplied to the heart. Causes dizziness, fainting, nausea

Nitrogen dioxide

NO is an odorless gas, NO2 is a gas with red-brown color

Can increase the respiratory system diseases (sore throat, cough, nasal congestion, fever), increases the risk of bronchitis and pneumonia in children

Sulfur dioxide

Colorless, odorless gas

Increases the risk of suffering from chronic respiratory system diseases, causes shortness of breath, airway narrowing in asthmatics

Lead

Heavy metal

Children are the most at risk group; can cause brain damage, neurotic disorders, digestive problems

Table 3. Pollutants impact on humans health Source: Nurul et al., 2014.

emission reduction were established. This instruments are intended to help reaching the adequate air quality. The most important instruments are: • permit for gas and particles discharge into the air, • integrated permit, • emission standards form installations, • fuel quality standards, • air protection programs in the areas, where air quality standards were exceeded (Albiniak, 2014). In the following years, activities established in air protection programs, realized in specific regions, should also improve the air quality. The aim of the National Program for Air Protection is to improve the life quality of inhabitants, especially protection of their health and living conditions, taking into account environmental protection with the principles of sustainable development (KPOP, 2015). The National Program of Air Protection is a strategic document, setting the objectives and actions’ directions, which should be taken into account in the air protection programs, especially on the local level (KPOP, 2015). Due to the current structure of fuel consumption in Poland (fig. 3) and high consumption of solid fuels (including wastes) in heating sector, actions which aim at emission (especially particulate matter and benzo[a] pyrene) reduction are necessary. These actions includes (Albiniak 2014; KPOP, 2015): • replacement of individual heating systems for more efficient and low emission systems (replacement for modern boilers with high efficiency or electric heating), • connection to district heating, gaseous network, • complex thermomodernization, which aim is to reduce the heat demand, • rational modification of production technologies in the sectors, from which the most burdensome and dangerous for humans and ecosystems pollutants are emitted,


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on very small benzo[a]pyrene concentration for a long time can cause cancer. Epidemiological studies showed a strong relation between benzo[a]pyrene concentration in air and occurrence of lung cancer. In Silesian voivodship increased incidence of lung cancer was found, especially in a group of man, exposed to PAH compounds (Trojanowska and Świetlik, 2013). Harmful substances, which are present in the air can be very dangerous to human health, so conducting activities related to air protection is necessary. Activities aiming at air quality improvement are presented in the following chapter.

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World Health Organization (WHO) estimated, that air pollutions, which occur both in cities and rural areas, were the reason for 3.7 million premature deaths in 2012. Epidemiological studies showed relation between the air pollution levels and mortality, related to respiratory and cardiovascular systems diseases in various parts of the world (Özkan et al., 2016). Table 3 shows the impact of high concentration of selected pollutants on humans health. Benzo[a]pyrene is another compound, which has a negative impact on health. Like the particulate matter, benzo[a]pyrene has a negative impact not only on human health, but also on flora, soil and water. It also has a possibility to accumulate in the organism and shows a high permanent toxicity. Like other polycyclic aromatic hydrocarbons, benzo[a]pyrene is a carcinogen and affects DNA changes (KPOP, 2015). Exposure even

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Conclusion In EU countries, including Poland, there is an air quality assessment and control system. The air quality assessment related to health and plant protection involves following substances: sulfur dioxide, nitrogen dioxide, carbon monoxide, benzene, particulate matter PM10, particulate matter PM2.5, heavy metals (lead, arsenic, nickel, cadmium), benzo[a]pyrene and nitrogen oxides.

2015). Significant potential can be also seen in renewable sources of energy application.

Albiniak B. ed. (2014). Stan Środowiska w Polsce Raport 2014. Biblioteka Monitoringu Środowiska. Warszawa Bochenek D. ed. (2015). Ochrona Środowiska 2015. Główny Urząd Statystyczny. Warszawa. Available at http://www.stat.gov.pl, accessed 29.02.2016 Kobus D., Iwanek J., Kostrzewa J., Mitosek G., Parvi R. (2015). Ocena jakości powietrza w strefach w Polsce za rok 2014. Państwowy Monitoring Środowiska – Inspekcja Ochrony Środowiska. Warszawa Nurul A. B. M., Oliver L. H. L., Dasimah O. (2014). Human Health and Wellbeing: Human health effect of air pollution. Procedia – Social and Behavioral Sciences 153: 221-229 Özkan C., Bahtiyar E., Ali D. (2016). Study on the association between air pollution and mortality in Istanbul, 2007-2012. Atmospheric Pollution Research 7: 147-154 Sadlok R., ed. (2014). Przeciwdziałanie niskiej emisji na terenach zwartej zabudowy mieszkalnej. Stowarzyszenie na rzecz efektywności energetycznej i rozwoju odnawialnych źródeł energii „HELIOS”. Bochnia. Schönfelder T. ed. (2011). Analiza możliwości ograniczania niskiej emisji ze szczególnym uwzględnieniem sektora bytowo-komunalnego. Opole Toczko B. ed. (2011). Zanieczyszczenie powietrza w Polsce w 2009 roku na tle wielolecia. Biblioteka Monitoringu Środowiska. Warszawa Trojanowska M., Świetlik R. (2013). Ocena ryzyka nowotworowego związanego z narażeniem inhalacyjnym na benzo(a)piren w wybranych miastach Polski. Medycyna Środowiskowa. vol. 16, No. 2: 14-22 Act of 27 April 2001 Environmental law (Journal of Laws of 2001 No 62 item. 627 as amended) Air quality portal (AQP 2016) http://powietrze.gios.gov.pl/, accessed 29.02.2016 Krajowy Program Ochrony Powietrza do roku 2020 (z perspektywą do roku 2030) (KPOP 2015). Ministerstwo Środowiska – Departament Ochrony Powietrza. Warszawa Regulation of the Minister of Environment of 24 August 2012 on levels of some substances in the air (Journal of Laws of 2012 item. 1031) Regulation of the Minister of Environment of 4 November 2014 on emission standards from various installations, combustion sources and waste incineration or co-incineration devices (Journal of Laws of 2014, item. 1546).


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References

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Between 2000 and 2012 there was observed a significant decrease of sulfur dioxide emissions – from about 1500 thousand tons to about 800 thousand tons (Bochenek, 2014). Emissions of nitrogen oxides, nonmethane volatile organic compounds, ammonia and particulate matter are rather stable. Data analysis showed that the biggest emission source are non-industrial combustion plants, mainly households. The second biggest emission source is a road transport – passenger cars. Increased emissions of particulate matter occur mainly in big cities and urban areas. Analysis conducted by Environmental Protection Inspectorate showed, that low-stack emissions have the main impact on current air quality (Kobus et al., 2015). Particulate matter and benzo[a]pyrene have a negative impact on human health and environment. Children, pregnant women, older people and people with respiratory and circulatory system diseases are the most sensible group exposed to the pollution. Accumulation of particulate matter in the organism can cause asthma severity, harsh reactions of respiratory system and weakening of lungs functions (Albiniak, 2014). Benzo[a] pyrene also has the ability of accumulation in the organism and shows a high permanent toxicity. Like other polycyclic aromatic hydrocarbons, benzo[a]pyrene is a carcinogen (KPOP, 2015). In order to protect human health and natural environment in Poland, a number of instruments of air emission reduction were established. This are i.e. permit for discharge and particles into the air, integrated permit, emission standards and air protection programs (Albiniak, 2014). The activities aimed at reducing the particulate matter and benzo[a]pyrene emissions are related to i.e. replacement of current heating system for modern and low emission systems, thermomodernization and modification of technologies in particular sectors and activities increasing social awareness (KPOP,

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• standardization of combusted fuel quality, related to its particle size, higher heating value, water, ash and sulfur contents. Keeping the allowable levels of pollutants in the environment is an effective protection of humans and environment against the negative impact of contaminants. The levels are established in Regulation of the Minister of Environment of 24 August 2012 on levels of some substances in the air (Journal of Laws of 2012 item. 1031). It is important to identify the areas, in which the pollutants levels are exceeded and then take actions, which cause the emission reduction to the allowable level, which will not affect human health and ecosystems (Albiniak, 2014). As it was mentioned before, part of the society treats wastes as a fuel substitute and do not realize the health consequences related to their combustion in household’s furnaces. Therefore social awareness related to negative impact of pollutants on human health and environment is a very important element, which influence the improvement of air quality process (KPOP, 2015). The previous reduction of air pollutants was caused by emission reduction from industrial sources, including energy sector. It means, that legislation and requirements, established on its basis, are effective. Is also means, that significant reduction potential includes actions and regulations related to municipal sector and transport (KPOP, 2015).

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Summary: Many fish diseases do not start with presence of highly infectious factors, but in small environmental imbalances. They tend to handicap the immunological response, and, in consequence, sentisize to infections. On the other hand, severe pH, temperature and water-soluble compound level changes may be lethal itselves. This article presents some most common cases in fish farming in Poland. Key words: fish, environment, disease, hygiene, toxicity, oxygen, ammonia received: 15.04.2015; accepted: 25.03.2015; published: 1.04.2016

Urszula Ejlak: DVM-graduated from Warsaw University of Life Sciences. Intrested in farm fish diseases, was an intern at University of Warmia and Mazury in Olsztyn, Inland Fisheries Institute, Norwegian School of Veterinary Science and worked with fish veterinarians in Haugesund (Norway). Presently working as a small animal internist in a veterinary clinic in Warsaw

There are few organisms such susceptible to their environment as fish. They literally soak in their surrounding, no matter how nutritious or poisonous it may be. It is important for fish farmers and veterinarians to know how to control water parameters, so they can provide their customers healthy fish products. For example, we all know about dangers of diluted pesticides flowing around, but we rarely think of hyperoxygenated water as a toxic factor. This article highlights some most common environmental illnesses in farm fish in Poland, which we can quite easily avoid by paying attention to proper farm hygiene. To properly understand the fish struggle, we need to focus on their immune system for a moment. In intensive breeding, immunocompetence might be broken by: • high density, • poor nutrition, • low water quality, • contaminated facilities or • new fish coming to the stock. It may be also influenced by genetic predispositions (both innate and acquired immunity), age and species of the fish, seasons and photoperiod. For example, cold water fish do not need to produce immunoglobulins, as bacterial growth in such temperatures is very slow. It may prolong the immune response up to 28 days as well. As in other animals, the vital role in fighting infection is played by leukocytes. They wander around the body, differentiating cells and molecules between „self” and „non-self” and trigger immune response to destroy the „non-self” ones. The first thing to realize is that fish have no bone marrow nor lymph nodes, so blood cells (including leukocytes) are produced in other tissues than in mammals. Some of them may surpise us, for example soft

tissue of orbit and skull (Holocephali fish) or intestinal spiral fold in young lamprey. In Elasmobranchii (e.g. sharks), there is a noticeable Leydig’s organ under the esophageal mucosa. The most important blood-producing organs in bony fish are spleen and anterior kidney, where immunity and hematopoesis take place. Those viscera are highly influenced by cortisol, „the stress hormone”. During manipulation (vaccination, transport etc.) we can detect involution of kidney and spleen, cortisol also blocks forming leukocytes and their adhesion to damaged tissue, activity of kidney phagocytes is lowered, in blood smear we can see leukopenia (especially lymphopenia T). This effects in decreased immune response, so even weak pathogens can induce disease. When we talk about fighting pathogens, we may not forget about the first barrier-skin covered with mucus. Mucous glands, present in the medium layer of epidermis, produce substance full of lysozyme (which damages bacterial cell walls and is also present in our tears and saliva), complement compounds (essential for triggering the non-specific and specific immune response) and natural antibodies and immunoglobulins. It prevents bacteria, fungi and parasites from adhesion to skin. It also separates the skin from irritating factors (i.e. ammonia, detergents in water), so we can notice an overproduction of mucus in such cases.

Temperature In Poland, freshwater temperature oscilates between 0 °C and 30 °C. In summer, heated water flows to the surface and the lowest temperature is found in the bottom. On the contrary, during winter there is a cold ice surface and warmer water below. Water in reservoirs deeper than 1 m is permanently separated into three layers. The bottom layer is called hypolimnion and pre-


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Urszula Ejlak

Introduction

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Balance is everything – how environment changes affect farm fish

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Balance is everything – how environment changes affect farm fish | Urszula Ejlak | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016

Temperature [°C] Growth [mm]

1 1.0

2 3.0

4 6.0

6 8.0

Appropiate pH range for rainbow trout is 6.5–8.2; for carp it is 5.0–9.0. For fisheries, it is the best to keep neutral (7.0) or weak alkaline (7.0–8.5) water. Daily pH fluctuations in ponds are normal and fish tolerate it well. However, the basic rule is not to change it over 0.2–0.5 pH units per day. There are simple, portable pH meters avaliable and they are easy to use. Colorimetric tests are more expensive, but more reliable. Calcium carbonate is a natural water buffer, preventing sudden pH changes. Similar buffers exist widely in nature, for example in blood, to maintain the stable environment for chemical reactions. While the calcium carbonate level is down, more calcium hydroxide is formed, which results in increased water basicity. This may induce fish alkalosis. At pH over 9.0 we can observe skin loss, frayed fins, dilated pupils and turbid lenses. Gill tissue is swollen, reddish and covered with mucus. This leads to lower oxygen intake and hypoxia. When partial pressure of CO2 in water is lower than it is in fish blood, carbon dioxide is removed from tissues into water. This imbalance of gases in blood end up in suffocation despite the proper amount of oxygen (hypercarbia). Carbon dioxide exists in water in molecular (90%) and conjugated form (as CaCO3). High CO2 levels are typical for water from underground sources. It may also occur during transport or cloudy days. In deep reservoirs (e.g. lakes) we can observe stratification of carbon

8 9.7

10 12.8

12 16.9

14 20.9

16 25.0

dioxide concentration. Maximum level of free CO2 is 25 mg/dm-3 for carp and 20 mg/dm-3 for trout (Własow and Guziur, 2008). Transgression causes fish acidosis, with such symptoms as tremors, hyperactivity, troubled transpiration, excessive mucus production, pale gills and skin (even skin loss) and death. Chronic exposure to low pH leads to fertility problems, slower growth, spinal deformities and accumulation of heavy metals in tissues. Most commonly, too acidic pH is present after heavy rains. Humic acids are flushed from soil, especially in ponds near coniferous forests, moors, coal mines. Metabolic activity of water flora and fauna are considered a secondary source of low water pH. Photosynthesis processed by water flora has a major influence on water pH. Assimilation of carbon dioxide decreases the level of CaCO3 in water. One may not forget about increased toxicity of metals (e.g. aluminium, chrome), ammonia and other factors in low pH.

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pH

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sents a stable temperature of about 4 °C. Water at this temperature has the highest density and can dissolve the most oxygen. Fish, which are familiar with this law of physics, can survive winter staying in hypolimnion. While sudden temperature drop usually does not affect fish much, extraordinary rise may be lethal. Obviously it depends on season, age of the fish (there is zero tolerance for temperature change during fry transportation, for adult fish it is 4°C), their accommodation et caetera. For example for carpio 37 °C is fatal (28.1 °C – 40.6 °C according to Wolny, 1976), for brown bullhead it is 36 °C in summer and 29 °C in winter. For adult rainbow trout, there is singnificantly increased mortality over 21 °C. To minimalize loss, it is necessary to stop feeding. Optimal water temperature (14–18 °C) during summer can be maintained by introducing a twosource water collecting system (recruintment from surface waters as well as from hypolimnion). Temperature rise causes respiratory stress, faster breathing (shown by excessive movement of operculum) and swimming near surface with open mouth (called piping). Faster metabolism requires more nourishment, but eventually fish stop feeding. In such circumstances leukocytosis and erythropaenia occur. Dissociation and dissolution happen faster in warmer water (unfortunately, it does not apply to oxygen). Combined with increased fish metabolism in such cases, we may observe severe intoxications (especially with heavy metals and phenols) that would not happen if water was colder.

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Balance is everything – how environment changes affect farm fish | Urszula Ejlak | EDUKACJA BIOLOGICZNA I ŚRODOWISKOWA 1/2016

18 20.9

Table 1. Average monthly growth of rainbow trout (fry to 2-year-old fish), depending on average monthly water temperature

Fig. 1. Water carbonate buffer

Source: Goryczko, Grudniewska, 2015.

Source: Noga, 2010.


Animal oxygen intake is variable and hard to predict. As far as we know, the most important factors increasing oxygen demand are: young age, metabolic rate (much higher in warmer water), good nutritional status, activity and excitability. Rainbow trout is capable of using 40% of oxygen dissolved in water. Level of oxygen adaptation, as well as oxygen solubility, depend on water temperature. 60% saturation was established as a full oxygen adaptation level. Amount of avaliable oxygen is described as a subtraction between saturation level at inflow and adaptation threshold value (below this level fish are unable to uptake oxygen). According to Wieniawski (1969), it is 5 mg O2/l for rainbow trout. Hypoxia We can divide sources of hypoxia in two groups: too low oxygen delivery into water and too high use by fish and macrophytes, which are main oxygen consumers in water. First condition often happens in late winter, when ice cover is a barrier between water and atmosphere, and snow layer ceases underwater photosynthesis. It Temp. [°C]

Max. O2 saturation [mg/l]

Adaptation treshold [mg/l]

1 2 5 10 15 20 25

14.2 13.8 12.8 11.3 10.2 9.2 8.4

8.5 8.3 7.6 6.8 6.1 5.5 5.0

Table 2. Oxygen demand according to water temperature (for fish over 25 g) Source: Goryczko i Grudniewska, 2015.

Oxygen demand for 1 kg of fish [mg/min] 0.3 0.4 0.7 1.4 2.3 3.6 5.5

quite comfortable for carps (Własow and Guziur, 2008). If oxygen saturation changes slowly, carps can even survive in 1.8 mgDO/l (Prost, 1989). To diagnose environmental hypoxia, we need to measure oxygen level in situ, as there is no way to collect water samples without disturbing gas levels. In most cases, we must rely on clinical observations and history, however, TDS meters are increasing in popularity. Fish gathering near the inflow might be the first sign of hypoxia. They tend to jump out of water, pipe near the surface, stop feeding, show increased operculum movements and finally die with flared opercula and open mouth. Smaller specimens are more likely to survive than bigger ones. Hypoxia must be differentiated from gill parasitosis and nitrite poisoning. Average yearly precipitation in Poland is 600 mm. It is not too much and leads to draughts during summer and significantly lowers the water level in ponds, lakes and rivers. Combined with high temperatures and light exposure, it causes oxygen deprivation. The most common way to deal with the problem (besides aeration and oxygenation) is to stop feeding for a few days to prevent additional oxygen loss from decayingfodder (as said before, fish will not eat anyway). Amount of oxygen, used by aerobic bacteria to oxidate organic compounds is described as Biochemical Oxygen Demand (BOD). This parameter is a widely-used surrogate of the degree of organic pollution in water. BOD5(measured for five days) for rainbow trout is