Newsletter for the Community of Duckweed Research and Applications, edited by the ISCDRADUCKWEEDFORUM

Volume 8 (4), issue 31, pages 94 - 126 (2020)

Lemna minor L., clone 9441

Cover page
Lemna minor L., clone 9441
Lemna minor turned out recently, in the genomics era, to be a very interesting species in as far as a significant
variance has been observed in genome sizes and apparent ploidy between natural populations. This is very unusual
when compared with the highly conserved genome structure observed in the well characterized genus of Spirodela.
At the same time clones of this species are top candidates for large scale cultivation to produce high amounts ofbiomass because it is a robust species for such practical purposes. Thus, potential use for nutrition and as energy
plants is ahead for this species. Clone 9441 is the oldest clone in cultivation and its fascinating story is told inside
this issue. This clone is also the preferred duckweed for phytotoxicity assays according to ISO 20079. Photo credit:
Dr. Klaus-J. Appenroth, Friedrich Schiller University of Jena, Germany.

In this issue
Letter from the Editor:..........................................................................................................................94
6th ICDRA 2022: 2nd Circular.............................................................................................................95
Acceptance of Wolffia arrhiza as Novel Food in Switzerland and China....................................96
Historical Account: Lemna minor, clone 9441 – the oldest living duckweed clone in culture............................................................................................................................................................... 100
Announcement of upcoming special issue in the journal ‘Plants”............................................102
Student Spotlight: Minako Isoda.....................................................................................................103
From the Database............................................................................................................................ 105
Instructions to Contributors for the Duckweed Forum...............................................................124
Links for Further Reading.................................................................................................................126

Disclaimer: Articles in the Duckweed Forum are published in the form sent in by the authors, except for some minor
language editing. The authors are responsible and are liable for any legal issues concerning plagiarism and/or use of
copyrighted material in any form.

The 4th International Steering Committee on Duckweed
Research and Applications Members• Chair: Prof. Eric Lam, Rutgers, The state University of NJ, New Brunswick, USA;
ericL89@hotmail.com• PD Dr. Klaus-J. Appenroth, Friedrich Schiller University of Jena, Germany; Klaus.Appenroth@unijena.de• Dr. K. Sowjanya Sree, Central University of Kerala, Periye, India; ksowsree9@cukerala.ac.in• Dr. Yubin Ma, Ocean University of China, Qingdao, China; mayubin@ouc.edu.cn• Dr. Tsipi Shoham, GreenOnyx Ltd., Tel Aviv, Israel; tsipi@greenonyx.biz

All prior Duckweed Forum issues: http://www.ruduckweed.org/

Science meets art: Wolffia cylindracea Hegelm.

The genus Wolffia contains 11 species. Especially in the section
Wolffia it is not easy to distinguish the seven different species on amorphological basis. Wolffia cylindracea does not have stomata on
the upper surface and the ratio between width and length of the fronds
is larger than in other species. Edges of the fronds are not translucent.
Christoph Friedrich Hegelmaier described this species for the firsttime in 1868. This species can be found south of the equator, e.g. in
Angola. The drawing shows a top view with the ellipsoid dorsal
surface of the frond and the widest part of the frond just beneath.
Drawing by Dr. K. Sowjanya Sree, Central University of Kerala, India.

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Letter from the Editor:
Dear Readers of the DF,
Greetings to you all on behalf of the members of the International Steering Committee for
Duckweed Research and Applications (ISCDRA). I hope this new issue of the Duckweed Forum, now
the 31st edition already, will find everyone in good health and coping alright with the global healthcrisis over the past months. As we are nearing the end of the year 2020, it is sobering to reflect on allthat has transpired in the past ten months to begin this new decade with. Our world has changed somuch in such a short time during this pandemic and the future yet remains uncertain to this day.
However, as a scientist, it is at least heartening to witness the ability of technologies that are now at
our disposal to tackle this invisible disease agent and can hope for an effective vaccine before long.
In addition, the availability of online tools allowed us to quickly adopt alternatives to keep our society
functioning in new ways and adapt to the new constraints. Indeed, as this pandemic demonstrated, I
believe the resilience of humanity to global challenges such as future health crisis and climatechange will significantly depend on technological advances that we make today. As a highly versatile
plant family, I believe duckweed-based platforms for bioproducts can have important impact in thesescenarios in the not-so-distant future. Our community will have a significant role to play in bringing
this promise to reality.
In this issue of the Duckweed Forum, a contribution from two young university students in
Switzerland strived to identify the variance in consumer preference for acceptance of duckweed intheir diet, using Wolffia arrhiza as their model. This type of marketing survey would be an important
step toward a more data-driven approach for the identification of successful strategies to bring
duckweed-based food to commercial deployment. An interesting item in this DF is the account of thehistory of Lemna minor 9441, the oldest known duckweed isolate that has been in continuous culture
since the end of WWII almost 75 years ago. It is indeed fitting the proverb of “Necessity is the Motherof Invention” that the lack of adequate space and financial resources in post-war Germany was what
prompted Prof. Pirson to isolate and culture this strain of duckweed in Marburg to enable him tocarry on biological research. In addition to a nice Student Highlight from the Oyama lab in Kyoto, we
also bring you the Database section by Klaus Appenroth to bring you abstracts for duckweed-related
papers over the past quarter. Last but not least is an important announcement in this DF issue by
Prof. Ingo Schubert (Gatersleben, Germany) to postpone the next ICDRA meeting by 1 year to May of2022 due to issues for traveling internationally during this pandemic. To coordinate with this change,
all members of the current ISCDRA agreed to serve until the ICDRA meeting in 2022, when the newly
appointed members by the community will be inaugurated.
Finally, the ISCDRA and I hope you will enjoy this issue of the DF. I especially like toacknowledge all contributors for their time and effort to share interesting ideas and knowledge withothers in our community. We look forward to hearing from you in the near future.
Sincerely,
Eric Lam
Chair, ISCDRA

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6th ICDRA 2022: 2nd Circular

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Acceptance of Wolffiaarrhiza as Novel Food in
Switzerland and China
Sophie Hofer and Yutong Fu
Dept. Health Sciences and Technology, ETH Zurich, 8092 Zurich, Switzerland(Email for correspondence: Sophie Hofer: hoferso@student.ethz.ch; Yutong Fu: yutofu@student.ethz.ch )1. Introduction and objective of the project
Wolffia (shown in Fig.1) is a promising food source. It is a fastgrowing plant with relatively low environmental impact and is also
especially attractive to consider due to its high nutritional value.
Wolffia could be a good source of the essential omega-3 fatty
acids and provides important fibers. In addition, it contains all
essential amino acids making it a valuable, alternative plant-based
protein source (Appenroth et al., 2018; van der Spiegel et al., 2013).
Wolffia has already been served as food for human in several Fig 1: Picture of fresh Wolffia arrhiza(provided by LemnaPro)
Southeast Asian countries (Saengthongpinit, 2017). For European
and Chinese consumers, however, Wolffia is a novel, unfamiliar food. According to the literature theacceptance of novel foods is influenced by various factors such as cultural habits, social norms,
general demographic factors in addition to the price, expected taste, quality and nutritional value(Barrena and Sanchez, 2013; Rozin and Vollmecke, 1986).
The general objective of our project was to investigate the acceptance of Wolffia arrhiza as a novel
food and the possible influencing factors. A cross-cultural comparison was performed between
Switzerland and China to investigate the influence of culture, positive information about Wolffia andmeal context on its acceptance, willingness to try and the expected preference.

2. Experimental set-up
A 10-minute online questionnaire was designed to investigate the acceptance of Wolffia as a novel
food in Switzerland and China. The questionnaire was peer-reviewed by 10 students of the mastercourse “Selected Topics in Sensory Science” at ETH Zurich. The final questionnaire, consisting of 5
parts, was translated into Chinese.
In Part 1, participant’s attitude towards novel foods and the importance of nutritional value andenvironmental impact was assessed for foods in general. The participants chose their familiarity
with Wolffia in Part 2, followed by Part 3 where they were asked about the willingness to try Wolffia
before and after being exposed to its positive information. In Part 4, the impact of meal contexts on
the respective expected preference was assessed. The participants were invited to an imaginary
serving of 7 dishes according to their dietary preferences (plant-based/meat-based, no allergens,etc.): Sushi with Wolffia as filling, cooked vegetables with Wolffia, Wolffia as an ingredient of a fresh
salad, pasta made from Wolffia; Burger patty with Wolffia; chocolate specialty with Wolffia filling andvol 8 (4), page 96 of 126

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a green smoothie based on Wolffia juices. The participants were asked to indicate how much their
expected preference is for the dishes in random order on the hedonic 9-point preference scale. The
dietary pattern and some demographic questions were investigated in the final part of thequestionnaire.

3. Results and discussion
Data was collected in March 2020 and consisted of 32 ETH Zurich food science bachelor students
and 35 Chinese participants. Similar to the Swiss sample cohort, most of the Chinese respondents
were also food science students in universities but living in China instead of Switzerland. The
participants’ detailed information profile is shown in the Table 1. The results were analyzed by
performing statistical analysis such as ANOVA and t-tests with the software IBM SPSS 25.0 and Rstudio. The table below summarizes the data collected using our online questionnaire from both
Swiss and Chinese cohorts.

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Influence of information: The two-way mixed ANOVA revealed a significant main effect of
information (F(1,65) = 21.3, p < 0.001). As assumed, exposing participants to advantageous
nutritional and environmental facts about Wolffia increased the acceptability of Wolffia as a novel
food. These findings were consistent with previous studies showing that positive communication
leads to a better evaluation of Wolffia by Dutch consumers (de Beukelaar et al., 2019).
Influence of culture: There was also a significant effect of country (F(1,65) = 21.0, p < 0.001), whichshowed that Chinese participants had more conservative attitude towards trying Wolffia. It wasconsistent with the results that Chinese participants had a higher food neophobia score, meaning alower willingness to try new foods. This observation was in agreement with literature (Johns et al.,2011) reporting that Asians, especially Chinese, are generally more neophobic than Europeans.
Influence of meal context: The paired-sample t-test of the Swiss sample revealed a significant effectof the addition of Wolffia regarding to cooked vegetables (p = 0.009), chocolate specialty (p < 0.001),
and fresh salad (p = 0.002). These findings indicate that the presence of Wolffia in these three typesof meal contexts significantly decreased the respondents’ expected preference. In the Chinese
cohort, there was a significant effect from the introduction of Wolffia in terms of cooked vegetables(p = 0.006) and burger (p = 0.031). These findings suggest that the Chinese participants’ expected
liking of cooked vegetables and burger were significantly lower due to the addition of Wolffia.
Therefore, smoothie, sushi, burger and pasta may be more appealing routes of application for
Wolffia in the Swiss food sector, while sushi, chocolate specialty, fresh salad and pasta could be
combinations that are better appreciated for Chinese consumers.

4. Outlook
As summarized in the table above, the willingness to try Wolffia varied significantly among theparticipants with different dietary patterns. Vegans might be the potential target group of Wolffia
consumption. However, the specific influence of dietary pattern on the acceptability and thecorrelations of acceptability of Wolffia with other factors such as demographics, general food choiceand changing residence of country need to be further investigated in view of the limited and unevenly
distributed sample. Moreover, sensory characteristics like taste and flavor may also influence
consumers’ behavior towards Wolffia. Therefore, the combination of online questionnaire withsensory test about Wolffia should be conducted in further research. Independent of the product, we
would suggest sticking an information card with the description of the potential benefits of Wolffia
on the food packaging. Furthermore, educating potential consumers about these benefits byadvertisement can serve to increase its familiarity and consequently also its acceptance.

Acknowledgement
We would like to thank Prof. Dr. Jeannette Nuessli Guth and Prof. Dr. Sandrine Gouinguené for theirguidance through this interesting project and for the implementation of the online questionnaire in
Compusense. Also, many thanks go to our supervisors Melanie Binggeli and Cyrill Hess from
LemnaPro for introducing us to the world of Wolffia, giving us useful feedback throughout the projectand supporting us with the statistical analysis.

References
Appenroth KJ, Sowjanya Sree K, Bog M, et al. Nutritional value of the duckweed species of the Genus Wolffia(Lemnaceae) as human food. Front Chem. 2018;6(OCT):1-13. doi:10.3389/fchem.2018.00483
van der Spiegel M, Noordam MY, van der Fels-Klerx HJ. Safety of novel protein sources (insects, microalgae,
seaweed, duckweed, and rapeseed) and legislative aspects for their application in food and feed production.
Compr Rev Food Sci Food Saf. 2013;12(6):662-678. doi:10.1111/1541-4337.12032

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SaengthongpinitW.
Indigenous
CuisinewithaModernFlare.;
http://www.ruduckweed.org/uploads/1/0/8/9/10896289/iscdra-duckweedforum_issue16-2017-01.pdf.
Accessed June 10, 2020.

2017.

Barrena R, Sánchez M. Neophobia, personal consumer values and novel food acceptance. Food Qual Prefer.
2013;27(1):72-84. doi:10.1016/j.foodqual.2012.06.007
Rozin P, Vollmecke TA. FOOD LIKES AND DISLIKES. Ann rev Nutr. 1986;(6):433-456. www.annualreviews.org.
Accessed June 15, 2020.
de Beukelaar MFA, Zeinstra GG, Mes JJ, Fischer ARH. Duckweed as human food. The influence of meal
context and information on duckweed acceptability of Dutch consumers. Food Qual Prefer. 2019;71:76-86.
doi:10.1016/J.FOODQUAL.2018.06.005
Johns N, Edwards JSA, Hartwell H. Food neophobia and the adoption of new food products. Nutr Food Sci.
2011;41(3):201-209. doi:10.1108/00346651111132475

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Historical Account: Lemna
minor, clone 9441 – theoldest living duckweed
clone in culture
Klaus-J. Appenroth1 & K. Sowjanya Sree2
University of Jena, Matthias-Schleiden Institute – Plant Physiology, Jena,
Germany (Email: Klaus.Appenroth@uni-jena.de)2
Central University of Kerala, Department of Environmental Science, Periye,
Kerala, India (Email: ksowsree9@cukerala.ac.in)
1

The Lemna minor clone 9441 wasisolated more than seven decades ago
in 1946 from Marburg, Hesse, Germany
by André Pirson (1910-2004). It isnoteworthy that this was just one year
after the end of the second world war.
In a letter written by Professor Pirson
to one of us (KJA) in 2003 (Figure), lessthan a year before he passed away (7 thof February, 2004), he wrote “Whenafter 5 years of service in the war andbeing a prisoner of war I could finallyestablish myself in Marburg, there wasno opportunity to grow higher land
plants under controlled conditions in a
climate chamber. So, as a less thanideal solution, we have cultivatedduckweed indeed for the first time
under axenic conditions, howeverunder the misguided hope of “Thesimple is the seal of the true” (Pirson,2003). Pirson was the Director of the
Botanical Institute in Marburg. Lacking
sufficient space in his Institute, he
could not start his research on crop
plants and decided to work with theless space-demanding duckweeds.
Together with his PhD student, Franz
Seidel, he isolated a clone of L. minor in
May, 1946 from a pond (so-called“Mittelteich”) in the old Botanical Garden of the University of Marburg, Pilgrimstein 4, Germany. Seidel
defended his thesis in December 1950 under the title “Contribution to the cell and metabolic
characterisation of potassium and calcium deficiencies” (Seidel, 1950) and this research was
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published in the same year (Pirson and Seidel, 1950). In 1958, Pirson moved to the University of
Goettingen, Hesse, Germany, where he worked with even smaller organisms than duckweed, algae,
until his retirement (Winkler and Pirson, 1980). Most probably he passed this L. minor clone toReinhard Bornkamm at the University of Goettingen.
As clone 9441 was isolated in 1946, it is the oldest living duckweed clone in culture known to us(Cover photo). Elias Landolt, working in the ETH Zurich, started his duckweed collection much later,
during his post-doctoral stay from 1953-1955 in several Institutes in California (Laemmler and
Bogner, 2014) and published his first duckweed related research paper in 1955 (Landolt, 1955).
Moreover, with this L. minor clone, Pirson and Seidel established axenic cultures of duckweeds forthe first time. They called this clone St, most probably meaning “Sterile”. The nomenclature of theclone changed from St to 9441 when Elias Landolt agreed in 2011 to use his four-digit clone ID for all
the clones under his international duckweed stock collection (Zhao et al., 2012).
Lemna minor clone 9441 has been kept under axenic conditions in the stock collection of the
University of Jena since 1969 under the responsibility of Helmut Augsten who was the Director of
the Botanical Institute since March 1966. This clone is now available in many international duckweed
stock collections. Tracing back the travels of this clone, the manager of the duckweed stockcollection in that time at the University of Jena, Fritz Jungnickel, received this plant in 1969 from
Erich Ohmann, Institute of Biochemistry of Plants ADW, Halle/ Saale, Saxonia-Anhalt, Germany.
Ohmann communicated that he received this clone from R. Bornkamm, University of Goettingen(Jungnickel, 1978). Since then, this clone has been used for several research purposes under thename ‘St’ (Naumann et al., 2007) and more recently under the revised clone identity of 9441 (Ziegler
et al., 2015). This clone was characterised by molecular taxonomy (Bog et al., 2010), growth ratemeasurements (Ziegler et al., 2015) and was used as a model system for phytotoxicity tests
according to the standard protocol ISO20079 (Naumann et al., 2007). Meanwhile, the whole genomeof this clone has recently been sequenced but the results are yet to be published (Todd P. Michael,
The Salk Institute, California and Eric Lam, Rutgers University, New Brunswick, NJ).
Soon, in 2021, the 75-year ‘Diamond Jubilee’ can be celebrated for isolation of this clone, 9441
Lemna minor L. and all the research that it had enabled over this time.

References
Bog, M., Baumbach, H., Schween, U., Hellwig, F., Landolt, E., and Appenroth, K.J. (2010). Genetic structure of thegenus Lemna L. (Lemnaceae) as revealed by amplified fragment length polymorphism. Planta 232, 609–619.
Jungnickel, F. (1978) Phosphatbedarf und Mangelsymptome bei einigen axenisch kultivierten Lemnaceen.
Limnologica (Berlin) 11, 469-478.
Laemmler, W., and Bogner, J. (2014). Elias Landolt and the Duckweeds. Aroideana 37, 80 – 88.
Landolt, E. (1955). Über das Wachstum in der Dunkelheit bei einigen Lemnaceen. Verhandlungen der
Schweizerischen Naturforschenden Gesellschaft. 135, 135-136.
Naumann, B., Eberius, M., and Appenroth, K.J. (2007). Growth rate based dose–response relationships and ECvalues of ten heavy metals using the duckweed growth inhibition test (ISO 20079) with Lemna minor L. clone St. J.
Plant Physiol. 164, 1656—1664.
Pirson, Á. (2003). Letter to PD Dr. K-J. Appenroth. Personal communication dated 10.06.2003.
Pirson, Á., and Seidel F. (1959). Zell- und Stoffwechselphysiologische Untersuchungen an der Wurzel von Lemna
minor L. unter besonderer Beruecksichtigung von Kalium- und Kalziummangel. Planta 38, 431-473.
Seidel, F. (1950). Beitraege zur Zell- und Stoffwechselphysiologischen Kennzeichnung des Kalium- undKalziummangels. PhD thesis, Marburg 1950.
Winkler, U., and Pirson, A. (1980). Cell physiological studies on Hydrodictyon reticulatum. 1. Synchronized cell
development and main composition. Biochem. Physiol. Pflanzen 175, 460-475.
Zhao, H., Appenroth, K.J., Landesman, L., Salmean, A.A., and Lam, E. (2012). Duckweed rising at Chengdu: summaryof the 1st International Conference on Duckweed Application and Research. Plant Mol. Biol. 78, 627–632.
Ziegler, P., Adelmann, K., Zimmer, S., Schmidt, C., and Appenroth, K.J. (2015). Relative in vitro growth rates of
duckweeds (Lemnaceae), the most rapidly growing higher plants. Plant Biol. 17, 33–41.

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Announcement ofupcoming special issue inthe journal ‘Plants”
Plants: ISSN 2223-7747
Title of the special issue: "Duckweed: Research Meets Applications"
The aim of this Special Issue is to provide a comprehensive update of the current progress in duckweed
research and applications. Contributions in forms of original research papers, reviews and shortcommunications from a broad scope of disciplines related to duckweed research and applications (e.g.,morphology, taxonomy, and ecology including ecological interactions, ecotoxicology, environmental monitoring
and remediation, physiology, biochemistry, genetics, omics, biotechnology, biomass production and its uses,etc.) are welcome. We hope that this overview will be of interest to those involved in basic research or potential
applications of duckweeds, and will also attract readers from other fields.
Keywords: Duckweed, Lemnaceae, Physiology, Genetics, Omics, Phylogenetics, Ecology, Ecotoxicology,
Remediation, Biomass
This special issue is now open for submission.
Deadline for manuscript submissions: 31 May 2021
Manuscript Submission Information
Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website.
Manuscripts can be submitted until the deadline. All papers will be peer-reviewed.
Plants is an international peer-reviewed open access monthly journal published by MDPI. Please visit theInstructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for
publication in this open access journal is 1600 CHF (Swiss Francs).
For more details please visit: https://www.mdpi.com/journal/plants/special_issues/duckweed
We look forward to your article submissions.
Special Issue Editors
Dr. Viktor Oláh
Dept. of Botany
Institute of Biology and Ecology
Faculty of Science and Technology
University of DebrecenH-4032 Debrecen, Hungary

Dr. Klaus-J. Appenroth

Dr. K. Sowjanya Sree

Matthias Schleiden Institute –Plant Physiology
Friedrich Schiller University Jena
07743 Jena, Germany

Dept. of Environmental ScienceCentral University of Kerala
Periye 671320, India

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Student Spotlight: Minako
Isoda
Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho,
Sakyo-ku, Kyoto 606-8502, Japan (Email: isoda.minako.78n@st.kyoto-u.ac.jp)
When I was a child, I lived in the countryside near Chiba. My playground was mostly rice fields,
forests, and small rivers. I often spent my free time finding wild berries in the field, catching insects
in the forests near shrines or temples, or fishing for crayfish in the river. There were so many chance
for me to be interested in nature, but I was not. Nature was too familiar to me at that time that I took
it for granted. Nature surrounded my life like air; hence, I did not really think about it.
The situation changed when I entered High School. My Biology teacher in High School was great atteaching, and her lecture made me intrigued by Biology. I became curious about what exactly
happens in nature at the molecular level, and I started developing an interest in it. When my family
moved to the urban city of Tokyo, I realized how I love being in nature and how much precious time I
had spent in there. My curiosity about Biology continued to grow; therefore, I decided to study
Biology at the University.
After I entered the University, I took a class named Biological Rhythms. I was extremely intrigued by
the knowledge that almost all organisms have a biological clock to adapt them to the 24-hour cycleof environmental changes caused by the rotation of the earth. I enquired the professor of my class,Prof. Okamura, started a biological clock experiment at the lab using mice, and it seemed as if
everything was going well. Unfortunately, two years later, I developed an allergy to mice and could
not continue the experiment. I needed to change my lab, but I wanted to continue studying biologicalclock. Luckily, I found a lab where biological clock is being studied using plants. That was the firsttime I met Prof. Tokitaka Oyama. When he showed me around his lab, I came across wonderfulplants called duckweed. I loved duckweed the minute I saw them, and my favorite one is Wolffiellahyalina.
My research theme is based on the mechanism offlowering in duckweed, especially W. hyalina. The
reason I am interested in this theme is that theflowering mechanism is controlled by a biologicalclock. Plants sense seasonal changes inenvironments to synchronize their flowering tomaximize fitness. The integration of external lightinformation into
biological clock-controlled
mechanisms enables plants to precisely measure
day length changes in the environment. When it
comes to some duckweed plants, salicylic acid(SA) is important for flowering. According toprevious studies, W. hyalina 7378 strain does notnormally flower when grown on a basal medium under either long- or short-day photoperiodic
schedule; however, flowering is induced under a short-day photoperiodic schedule when SA is added
to the medium. These results suggest that there are photoperiodic and SA-induced pathways in W.
hyalina flowering system; however, the relationship between these two pathways and their flower
induction mechanisms have not been analyzed at the molecular level.
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Therefore, we approached the photoperiodic- and SA-induced flowering pathway of W. hyalina. Next,
to investigate the relationship between the two pathways, we have created W. hyalina cDNA libraryusing de novo transcriptome assemblies derived from normalized RNA-Seq. We identified two
flowering-related genes, FLOWERING LOCUS T homolog (WhFT) and a 14-3-3 homolog (Wh14-3-3).
We analyzed the expression of WhFT and Wh14-3-3 under flowering inductive/non-inductive
conditions using W. hyalina 7378. It has been reported that FT acts as florigen and 14-3-3 protein
GF14c acts as intracellular receptors for florigen in shoot apical cells in rice. FT interacts with 14-3-3
protein and binds to the FD1 transcription factor, and the 14-3-3 - FT complex induces flowering. In
my study, WhFT mRNA was found to be upregulated by SA treatment under both long-day and shortday conditions, suggesting the involvement of WhFT only in the SA-induced pathway. Wh14-3-3mRNA was upregulated by SA treatment only under the short-day condition, suggesting the
involvement of Wh14-3-3 in the downstream flowering pathway of SA and photoperiod signals.
According to these results, the WhFT-Wh14-3-3 complex might be necessary for flowering. Threedays after the addition of SA, the flowering-related genes, including WhFT and Wh14-3-3, wereupregulated under short-day conditions, suggesting that the plants may already be in a state ofreadiness for flowering before the addition of SA. I hypothesize that biological clock may prime theplants to a state that is more likely to induce flowering. To verify this hypothesis, we are now
analyzing the differences in the variation of expressed genes when SA is added under short- andlong-day conditions. I look forward to sharing my results with the community soon.
Sometimes, aside from working in the laboratory, we go on short trips to find duckweed. When I did
not know about duckweed, rice fields were just rice fields. But now, I can find some beautiful things
there. I am so grateful that I have a chance to know duckweed, and I will continue trying to do mybest to elucidate the unclear mechanism of flowering control in duckweed.

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From the Database
Highlight
Preference, performance, and impact of the water-lily aphid on multiple
species of duckweed
Subramanian, SK; Turcotte, MM (2020) Ecological Entomology DOI: 10.1111/een.12932
The role of herbivores in driving the structure of freshwater macrophyte communities remains poorly
understood in comparison with terrestrial ecosystems. For instance, although duckweed (subfamily
Lemnoideae) are globally distributed, can be locally highly abundant and ecologically dominant, andare of growing economic importance, their interactions with herbivores remain understudied. To
address how herbivores may impact duckweed species composition, we here experimentallyquantify the preference and performance of a common duckweed herbivore, the water-lily aphid(Rhopalosiphum nymphaeae) on four widespread duckweed species. Our two-way choice
experiments reveal that aphids display a preference for Spirodela polyrhiza > Landoltia punctata =Lemna minor> > Wolffia brasiliensis. These results are rarely influenced by natal host species. By
evaluating the growth of aphid populations on each duckweed species, we find that preference may
be adaptive in certain ecological conditions. Quantifying the population growth rate of duckweed
growing in the presence and absence of aphids revealed differential tolerance of herbivory acrossduckweed species. This study shows that aphids, through preferential feeding and significant
differential effects on duckweed growth, can have a significant impact on duckweed population
dynamics and potentially community composition.

Biotechnology
Expression of LamB vaccine antigen in Wolffia globosa (Duckweed)
against fish vibriosis
Heenatigala, PPM; Sun, ZL; Yang, JJ; Zhao, XY; Hou, HW (2020) Frontiers in Immunology 11: 1857
Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline
waters. A multitude of Vibriospecies have been identified as the causative agents. LamB, a memberof outer membrane protein (OMPs) family of these bacteria is conserved among all Vibriospecies
and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed(Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as abioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in
nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was
modified to maximize the protein expression level and translocate the protein to the endoplasmic
reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S
promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation.
Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western
blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective
property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding freshtransgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent
survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia
were well-protected from Vibrioinfection. These findings suggest that Wolffia expressed LamB could
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serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing
Wolffia as bioreactor to produce edible vaccines.

Effects of reaction conditions on products and elements distribution via
hydrothermal liquefaction of duckweed for wastewater treatment
Chen, G; Yu, Y; Li, W; Yan, B; Zhao, K; Dong, X; Cheng, Z; Lin, F; Li, L; Zhao, H; Fang, Y (2020)
Bioresource Technology 317:124033
Wastewater treatment by duckweed is a naturally sustainable technology. However, its development
is limited due to the lack of a follow-up treatment of duckweed. The duckweed was proposed for thetreatment of rural domestic wastewater and agricultural wastewater, and it was further processed to
produce bio-oil via hydrothermal liquefaction at various temperatures (250°C-370°C) and residence
times (15-60min). The highest bio-oil yield of 35.6 wt% was obtained at 370°C, 45min. The higher
heating value of bio-oil was 40.85 MJ kg -1, and the H/C ratio (1.72-1.98) was similar to that of
petroleum (1.84). The gas chromatography-mass spectrometry analysis results revealed that thebio-oil mainly consisted of N-heterocycles, cyclic ketones, esters, amides, long-chain hydrocarbons,phenols, and aromatic intermediates. Valuable compounds (3-pyridinol, 2-pyrrolidinone, and its
analogues) of high concentration were identified in the water-soluble organic matter. Compared withother materials, this study produced higher-quality bio-oil and water-soluble organic matter.

Nutrient starvation and light deprivation effects on starch accumulationin Landoltia punctata cultivated on anaerobically digested dairy manure
Kruger, K; Chen, L; He, BB (2020) Journal of Environmental Quality 49:1044-1053
Duckweed has been recognized for its potential of producing biomass on nutrients from waste
streams. Our research has shown that strains of duckweed can be successfully cultivated on
anaerobically digested (AD) dairy manure under controlled levels of nitrogen (N) and phosphorus (P).
The objective of this study was to explore the maximization of starch accumulation in Landoltia
punctata (Mey.) Les & Crawford strain 0128 under different cultivation conditions using AD dairy
manure as the nutrient source. Experimental results have shown that the most influential factors forstarch accumulation in L. punctata were the nutrient concentration and the appropriate scheduling of
nutrient starvation at the right growth stages. In our study, nutrient starvation for starch
accumulation in L. punctata was achieved by incorporating nutrient addition of appropriately diluted
AD dairy manure sequentially in a controlled manner under a constant light intensity of 3,000 lx. It
was observed that a starch concentration of 30% (w/w) within the L. punctata biomass wasachieved with an initial total N of 57.1mg L -1 and a total P of 6.7mg L -1 after a 30 d cultivation. Under
the abovementioned cultivation conditions, the duckweed L. punctata recovered 16.3% (±4.0%) of
total N and 25.9% (±6.6%) of total P from AD manure into its biomass. It is concluded that L.
punctata can be successfully cultivated on nutrients from dairy manure for starch production, which
would achieve well-being for dairy farmers by producing a feedstock for biofuels while treating dairy
wastewater in an environmentally friendly manner.

Ecology
Latitudinal variation in norms of reaction of phenology in the greater
duckweed Spirodela polyrhiza
Hitsman, HW; Simons, AM (2020) Journal of Evolutionary Biology DOI: 10.1111/jeb.13678
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Variable environments may result in the evolution of adaptive phenotypic plasticity when cuesreliably indicate an appropriate phenotype-environment match. Although adaptive plasticity is wellestablished for phenological traits expressed across environments, local differentiation in norms ofreaction is less well studied. The switch from the production of regular fronds to overwintering'turions' in the greater duckweed Spirodela polyrhiza is vital to fitness and is expressed as a norm ofreaction induced by falling temperatures associated with the onset of winter. However, the optimal
norm of reaction to temperature is expected to differ across latitudes. Here, we test the hypothesisthat a gradient in the length and predictability of growing seasons across latitudes results in theevolution of reaction norms characterized by earlier turion production at higher latitudes. We test thisby collecting S. polyrhiza from replicate populations across seven latitudes from Ontario to Florida
and then assessing differentiation in thermal reaction norms of turion production along a common
temperature gradient. As predicted, northern populations produce turions at a lower birth order andearlier; a significant latitude-by-temperature interaction suggests that reaction norm differentiation
has occurred. Our results provide evidence of differentiation in reaction norms across latitudes in aphenological trait, and we discuss how the adaptive significance of this plasticity might be further
tested.

Greenhouse gases emissions from duckweed pond system treating
polyester resin wastewater containing 1,4-dioxane and heavy metals.
Osama, R; Awad, H M; Zha, S; Meng, F; Tawfik, A (2020) Ecotoxicology and Environmental safety
207:111253
Phytoremediation of polyester resin wastewater containing 1,4-dioxane and heavy metals using
Lemna gibba (L. gibba) was enhanced by incorporation of perforated polyethylene carrier materials(PCM) onto the duckweed pond (DWP) system. The DWP module was operated at a hydraulicretention times (HRTs) of 2, 4 and 6 days and as well as 1,4-dioxane loading rate of 16, 25 and 48g
m-3.d-1. The maximum removal efficiency of 54±2.5% was achieved for 1,4-dioxane at an HRT of 6days and loading rate of 16 g 1,4-dioxane m -3.d-1. Similarly, the DWP system provided removal
efficiencies of 28.3±2.1, 93.2±7.6, 95.7±8.9 and 93.6±4.9% for Cd 2+, Cu2+, Zn2+ and Ni2+ at influent
concentration of 0.037±0.01, 1.2±0.9, 27.2±4.7 and 4.6±1.2 mg L -1 respectively. The structural
analysis by Fourier-transform infrared spectroscopy (FTIR) clearly displayed a reduction of 1,4dioxane in the treated effluent. A strong peak was detected for L. gibba plants at frequency of3417.71 cm-1 due to N-H stretching, which confirm the proposed mechanism of partially conversionof 1,4-dioxane into amino acids. Glycine, serine, aspartic, threonine and alanine content wereincreased in L. gibba by values of 35±2.2, 40±3.2, 48±3.7, 31±2.8, and 56±4.1%, respectively. The
contribution of DWP unit as a greenhouse gases (GHG) emissions were relatively low (1.65 g CO 2 kg1
BOD removed d-1, and 18.3 g CO2 kg-1 biomass d-1) due to photosynthesis process, low excess
sludge production and consumption of CO 2 for nitrification process (1.4 g CO 2 kgN removed.d-1).
Based on these results, it is recommended to apply such a technology for treatment of polyester
resin wastewater containing 1,4-dioxane and heavy metals at a HRT not exceeding 6 days.

Modifying effects of leaf litter extracts from invasive versus native tree
species on copper-induced responses in Lemna minor
Karitonas, R; Jurkoniene, S; Sadauskas, K; Vaiciuniene, J; Manusadzianas, L (2020) PEERJ 8: e9444
Invasive plant species tend to migrate from their native habitats under favourable climaticconditions; therefore, trophic and other relationships in ecosystems are changing. To investigate theeffect of natural organic matter derived from native Alnus glutinosa tree species and from invasive invol 8 (4), page 107 of 126

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Lithuania Acer negundo tree species on copper toxicity in Lemna minor, we analysed the dynamics of
Cu binding in aqueous leaf litter extracts (LLE) and plant accumulation, morphophysiologicalparameters, and antioxidative response. The results revealed that A. glutinosa LLE contained
polyphenols (49 mg pyrogallol acid equivalent (PAE)/g DM) and tannins (7.5 mg PAE/g DM), while A.
negundo LLE contained only polyphenols (23 mg PAE/g DM). The ability of LLE to bind Cu increased
rapidly over 1.5-3 h to 61% and 49% of the total Cu concentration (6.0 ± 0.9 mg/L), respectively for A.
glutinosa (AG) and A. negundo (AN), then remained relatively stable until 48 h. At the same time, L.
minor accumulated 384, 241 or 188 µg Cu/g FW when plants were exposed to Cu (100 µM CuSO 4),
Cu with 100 mg/L dissolved organic carbon (DOC) from either AG LLE or AN LLE, accordingly.
Catalase (CAT) and guaiacol peroxidase (POD) played a dominant role in hydrogen peroxide
scavenging when plants were exposed to Cu and 10 or 100 mg/L DOCAG mixtures in both the first(up to 6h) and the second (6-48 h) response phases. Due to functioning of oxidative stress enzymes,
the levels of the lipid peroxidation product malondialdehyde (MDA) reduced in concentrationdependent manner, compared to Cu treatment. When combining Cu and DOCAN treatments, themost sensitive enzymes were POD, ascorbate peroxidase and glutathione reductase. Their activities
collectively with CAT were sufficient to reduce MDA levels to Cu induced in the initial, but not thesecond response phase. These data suggest that leaf litter extracts of different phenolic
compositions elicited different antioxidant response profiles resulting in different reductions of Cu
stress, thus effecting L. minor frond and root development observed after seven days. The complex
data from this study may be useful in modelling the response of the aquatic ecosystem to achanging environment.

Assessing the effects of tropical wood leachate to Desmodesmus
subspicatus, Lemna minor and Daphnia magna
Sackey, LNA; Koci, V (2020) HELIYON 6: e04268
Ghana has a long history as a major supplier of high-value hardwood timber and wood products to
many countries. The research seeks to assess the effects of tropical wood leachates to aquaticorganisms. Hence, five wood samples were selected; Mahogany (Khaya ivorensis), Cedrela (Cedrela
odorata), Emire (Terminalia ivorensis), Wawa (Triplochiton scleroxylon) and Ceiba (Ceiba pendandra)
from Oboyow forest reserve in Eastern Region Ghana to assess their toxicity to aquatic organisms.
Toxicity tests: Algal (Desmodesmus subspicatus) Duckweed (Lemna minor) and crustacean (Daphnia
magna) were carried out using exposures to concentrations of 20, 30, 45, 67 and 100% v/v woodleachate in control media. The high levels of phenols measured in the various wood leachates was
the main cause of toxicity. The percentage median Inhibition Concentration (%IC50) of the various
wood leachate, ranged from 21.5-55.6% with mahogany exhibiting the highest toxicity and wawa thelowest. All the wood leachates were toxic to the aquatic organisms. The %IC50 showed bothconfirmed and potential toxicity among the various wood leachates and established that there wassignificant difference between various wood leachate toxicity.

Native Growth and Conservation of Duckweed (Lemnaceae) in Jordan
Shammout MW (2020) Pakistan Journal of Biological Sciences 23: 1055-1059
Duckweeds are the world's smallest flowering plants. Its existence is affected by the water quality
and availability. The increased water demand and water scarcity in most of the Mediterraneancountries as Jordan have caused remarkable lowering in the water-table which reduced floods anddisappearance of the seasonal lakes. Due to this, aquatic plants that grow on good quality of fresh
water have disappeared totally. Therefore, this paper highlights the native growth and conservationof duckweed (Lemnaceae) in Jordan. It also focuses on the identification of water sources forvol 8 (4), page 108 of 126

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duckweed's movement. Field surveys were conducted to identify water source for water bodies that
contain duckweeds. These surveys included visits to the Zarqa river, dams, irrigation ponds andJordan valley. The source of water for duckweed movement and availability was determined. Water
samples from the targeted areas were analyzed for selected parameters as pH, EC, NO 3, PO4, BOD5,Zn, Pb, Cd and Cu. The results showed that King Abdullah Canal and Zarqa river are the water
sources that encourage the duckweeds growth. In Sukhnah, Jerash and Jordan valley areas, water
analysis ranges (mg L-1) are: NO3 0.7-38, PO4 0.3-7.6 and BOD5 0-20. The pH range value is 7.1-8.1
and EC range value is 1.62-2.5 mS cm-1.The heavy metals values are, Zn is <0.02 mg L -1, Pb is <0.01
mg L-1, Cd is <0.002 mg L-1 and Cu is <0.01 mg L -1. It can be concluded that mitigation measures arehighly needed to overcome and altering the quality of the natural water flowing and participation of
governmental institutions is also required for the success of applying mitigation measures topreserve the aquatic plants.

Feed & FoodUse of Lemna minuta Kunth. for composition of sustainable diets andinfluence on hydrochemical, technological and blood biochemicalparameters in common carp (Cyprinus carpio L.) cultivated inaquaponics
Velichkova, K; Sirakov, I; Veleva, P (2020) Bulgarian Journal of Agricultural Science 26: 674-679
The purpose of this study was to investigate the influence of aquaponically grown L. minuta used asa part of biofilter in recirculation aquaculture systems, with its subsequent inclusion in thecomposition (50 and 100% content of daily feed ratio) in sustainable diets on hydrochemical,
biochemical blood and technological parameters in common carp (Cyprinus carpio L.) fingerlings
cultivated in aquaponics recirculation systems. At the end of the experiment were calculated average
final weight, specific growth rates, feed conversion ratio, meat quality and blood biochemicalparameters. The inclusion of up to 100% duckweed (L. minuta ) in feed for common carp fingerlings
decreases the quantity of ammonium, nitrite, nitrate and phosphate accordingly with 66%, 71.4%,
38.8% and 44.1% compared with the quantities of these parameters found for experimental groups
fed with feed without inclusion of L. minuta . The carps fed with inclusion of up to 50% duckweedshowed better growth of fish, SGR and FCR compared with fish fed with a diet without substitutionof duckweed.

Interaction with other organisms
Effect of treated sewage characteristics on duckweed biomass
production and microbial communities
Iwano, H; Hatohara, S; Tagawa, T; Tamaki, H; Li, YY; Kubota, K (2020) Water Science and Technology
82:292-302
Duckweed biomass production in a duckweed pond fed with three differently treated sewage (i.e.
sewage treated by primary sedimentation (PS); conventional activated sludge process (CAS); anddownflow hanging sponge process (DHS)) was evaluated in order to assess the effects of water
quality on biomass yield. Higher and stable biomass production was observed when the duckweed
pond was fed with PS or DHS-effluent than with CAS-effluent, evidently due to the difference invol 8 (4), page 109 of 126

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nutrient loads. Availability of nutrients, especially phosphorus, affected the biomass production rate:
higher the nutrient, faster the production. Microbial community analysis revealed that the membersof Rhizobiales were likely to contribute to stable and high biomass growth. From the results of thestudy, a sewage treatment system consisting of a primary sedimentation followed by a duckweed
pond and a tertiary treatment unit can be proposed to maximize biomass production withoutcompromising treatment objectives. Size and operational parameters of the duckweed pond should
be determined primarily based on the nutrient availability in the influent water to maximize duckweed
growth.

Comparative analysis of microbial communities in fronds and roots ofthree duckweed species: Spirodela polyrhiza, Lemna minor, and Lemna
aequinoctialis
Iwashita, T; Tanaka, Y; Tamaki, H; Yoneda, Y; Makino, A; Tateno, Y; Li, Y; Toyama, T; Kamagata, Y;
Mori, K (2020) Microbes and Environments 35: DOI:10.1264/jsme2.ME20081
The microbial communities inhabiting the fronds of duckweeds have not been investigated in asmuch detail as those on the roots. We herein examined the microbial communities in threeduckweed species using 16S rRNA amplicon sequencing and compared them to those on the roots.
The microbial compositions of the fronds were distinct from those of the roots in the three species.
Various types of taxonomic bacteria, including rarely cultivated phyla, Acidobacteria,
Armatimonadetes, and Verrucomicrobia, were also isolated from the fronds, but at a slightly lower
abundance than those from the roots. These results suggest that duckweed fronds are analternative source for isolating rare and novel microbes, which may otherwise be recalcitrant tocultivation using conventional strategies.

Molecular Biology
Estimation of the SNP mutation rate in two vegetatively propagating
species of duckweed
Sandler, G; Bartkowska, M; Agrawal, AF; Wright, S (2020) G3 (Bethesda, Md.).
DOI:10.1534/g3.120.401704
Mutation rate estimates for vegetatively reproducing organisms are rare, despite their frequent
occurrence across the tree of life. Here we report mutation rate estimates in two vegetatively
reproducing duckweed species, Lemna minor and Spirodela polyrhiza. We use a modified approachto estimating mutation rates by taking into account the reduction in mutation detection power that
occurs when new individuals are produced from multiple cell lineages. We estimate an extremely low
per generation mutation rate in both species of duckweed and note that allelic coverage at de novo
mutation sites is very skewed. We also find no substantial difference in mutation rate between
mutation accumulation lines propagated under benign conditions and those grown under salt stress.
Finally, we discuss the implications of interpreting mutation rate estimates in vegetatively
propagating organisms.

Transcriptional analysis reveals potential genes and regulatory networks
involved in salicylic acid-induced flowering in duckweed (Lemna gibba)
Fu, L; Tan, D; Sun, X; Ding, Z; Zhang, J (2020) Plant Physiology and Biochemistry 155: 512-522.
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Duckweed is a simple aquatic floating plant having great potential in sewage treatment andbioenergy production. Duckweed rarely flowers in nature, which greatly limits its germplasm
collection, conservation, and heterosis usage. Salicylic acid (SA) can efficiently induce flowering of
duckweed (e.g., Lemna gibba); however, the related genes and regulatory networks remain unclear. In
this work, we demonstrated that L. gibba flowering induced by SA was photoperiod-dependent,
stress-involved, and abscisic acid (ABA)-disrupted. Totally 202, 78, and 413 differentially expressed(DE) genes were up-regulated, while 429, 72, and 307 were down-regulated at flower induction,flower initiation, and flowering stages, respectively. At the flower induction stage, the down-regulated
genes were mainly involved in cell wall, auxin and ABA, light reaction, and abiotic stress, while the upregulated genes were involved in development, brassinosteroid, major CHO metabolism, and redox.
At the flower initiation stage, the down-regulated genes were enriched in light reaction and lipid
metabolism, whereas the up-regulated genes were enriched in starch degradation and Ca 2+ signaling.
At the flowering stage, the down-regulated genes were significantly enriched in photosynthesis,
gibberellic acid, starch synthesis, nitrogen metabolism, and redox, while the up-regulated genes wereenriched in cell wall, jasmonic acid, secondary metabolism, and Ca 2+ signaling. Besides, 46transcription factors and 13 flowering-related DE genes were identified. Finally, a possible floral
pathway, where LgTEM1, LgSVP, and LgFT1 might play critical roles in SA-induced flowering in L.
gibba, was discussed. These findings provide a useful foundation for further investigation of genes
and regulatory networks of SA-induced flowering in duckweed.

Physiology & Stress
Effect of the growth medium composition on nitrate accumulation in thenovel protein crop Lemna minor
Devlamynck, R; Fernandes de Souza, M; Bog, M; Leenknegt, J; Eeckhout, M; Meers, E (2020) Ecology
and Environmental Safety 206:111380
Duckweed is a potential alternative protein source for food and feed. However, little is known about
the nitrate accumulation in this plant. A high nitrate level in vegetables can indirectly lead to anelevated intake of nitrites and N-nitroso compounds, increasing the risk of diseases for humans andanimals. This research hypothesizes that the nitrate accumulation of Lemna minor differs between
growing media. Additionally, it evaluates whether legal safety levels of nitrate for human and animal
intake are exceeded. The duckweed was grown on (i) rainwater, and (ii) three synthetic media
containing different nutrient levels. Furthermore, (iii) biological effluent of swine manure treatmentand (iv) aquaculture effluent from pikeperch production were used, as these are potential media forclosing nutrient loops in the agriculture sector. It was found that nitrate levels increased with theincreasing availability of macronutrients in the water, and pH showed a particularly strong negative
correlation with the nitrate levels in the plant. Nevertheless, nitrate content never exceeded 530 mg
NO3 kg-1 fresh weight. To conclude, L. minor's nitrate content was below safety limits for humanconsumption in all tested growing media; however, a potential risk for ruminants was observed asthese are more sensitive to nitrate conversions in their gastro-intestinal track.

Enhanced biomass production and pollutant removal by duckweed in
mixotrophic conditions
Sun, Z; Guo, W; Yang, J; Zhao, X; Chen, Y; Yao, L; Hou, HW (2020) Bioresource Technology 317:
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Duckweed is a potential biomass source for alternative energy production. This work reports theeffects of trophic modes on growth rates, biomass accumulation, and removal rates of pollutant by
duckweed. Glucose, fructose, galactose, sucrose, and maltose all supported heterotrophic andmixotrophic growth of duckweed. The mixotrophic growth rate was 4.98 and 6.22 times higher thanthose in heterotrophic and photoautotrophic conditions, respectively. Notably, mixotrophy produced
more biomass than the simple sum of the biomass accumulation during heterotrophy andphotoautotrophy. Mixotrophy was also superior in starch and protein production, as well as inremoval rates of nutrients and organic carbon from the growth medium. However, the starch content
of duckweed grown heterotrophically was 2.06 times higher than in mixotrophy, suggesting a
combination of mixotrophy and heterotrophy as an effective strategy for starch-rich biomass
production. This study thus provides a paradigm for future studies supporting duckweed-based
biomass production and organic wastewater treatment.

Phytoremediation
Microalgal and duckweed based constructed wetlands for swine
wastewater treatment: A review
Li, X; Wu, S; Yang, C; Zeng, G (2020) Bioresource Technology 123858
Constructed wetlands for swine wastewater treatment have been one of the most exciting research
topics. Usually hydrophytes based constructed wetlands could not adapt well to high concentrationof ammonia nitrogen in swine wastewater, while microalgal and duckweed based constructedwetlands are promising for the nutrient removal. In this critical review, the important roles of
microalgae and duckweeds played in wastewater treatment in constructed wetlands were firstsummarized. Performances including biomass growth, nutrient removal capacities and mechanismsof microalgal and duckweed based constructed wetlands were reviewed for swine wastewater
treatment. Challenges for the applications of constructed wetlands including microalgal andduckweed based ones were discussed which includes a better understanding and utilization ofsynergistic effects among microalgae and duckweeds, difficulty and costs in harvesting biomass,
applications in various field conditions including low temperatures, and selections of various types of
microalgal and duckweed species. Future research needs were also proposed accordingly.

Lemna minor bioassay evaluation using computer image analysis
Haffner, O; Kucera, E; Drahos, P; Ciganek, J; Kozakova, A; Urminska, B (2020) Water 12: 2207
This article deals with using computer vision in the evaluation of the Lemna minor bioassay.
According to the conventional method, the growth of Lemna minor mass is determined from thenumber of leaves grown. In this work, instead of counting individual leaves, we propose measuring
the area occupied by the leaves using computer vision and compare the new approach with theconventional one. The bioassay is performed according to the ISO 20079 standard as a 168 h
growth inhibition test; the aim of the experiment was to quantify the negative effects on thevegetative growth using two parameters - the number of leaves and the area occupied by the leaves.
The method based on image processing was faster and also more precise since it enabled us todetect the negative effect of the tested substance on leave size, not only on their number. It can beconcluded that the toxic effect has shown to be more significant when considering the leaves arearather than the number of leaves. Moreover, mistakes caused by human factor during leaves
counting are eliminated using the computer vision based method.

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Tolerance of Landoltia punctata to arsenate: an evaluation of thepotential use in phytoremediation programs
Canatto, RA; de Oliveira, JA; da-Silva, CJ; Albino, BES (2020) International Journal of
Phytoremediation DOI: 10.1080/15226514.2020.1797630
Plants used in phytoremediation should accumulate and tolerate a specific pollutant. Here, we aimed
at evaluating possible arsenic (As) accumulation and mechanisms of tolerance against As-induced
damage in Landoltia punctata to explore this species for phytoremediation. Plants were subjected toincreasing As levels. As absorption was higher with increasing As levels. The activity of superoxide
dismutase and glutathione reductase as well as anthocyanin levels increased with As levels.
Catalase and peroxidase activities increased in plants subjected to As levels up to 1.0 mg L -1 anddecreased at higher levels. Due to the antioxidant system, higher levels of reactive oxygen species
were restrained in plants under low levels of As. However, the levels of superoxide anion, hydrogen
peroxide, and lipid peroxidation increased in response to the impaired antioxidant system induced by
the highest As levels. Biomass decreased in plants exposed to As and scanning electron microscopy
revealed root structural damage in the root cap of plants under 3.0 mg L -1 As. This work highlightsthat L. punctata can be considered a hyperaccumulator species and has potential for As
phytoremediation when levels are lower than 1.0 mg L -1 - a concentration 100-fold higher than thatrecommended for drinking water.

Improving the efficiency of wastewater treatment plants: Bio-removal ofheavy-metals and pharmaceuticals by Azolla filiculoides and Lemna
minutaBianchi, E; Biancalani, A; Berardi, C; Antal, A; Fibbi, D; Coppi, A; Lastrucci, L; Bussotti, N; Colzi, I; Renai,
L; Scordo, C; Del Bubba, M; Gonnelli, C (2020) The Science of the Total Environment 746: 141219
In this study, we investigated the removal of Fe(III), Cr(VI), Al(III), diclofenac, and levofloxacin fromtreated wastewater in the presence of the free-floating plants Azolla filiculoides and Lemna minuta, tounderstand whether these species can be effectively used in a surface flow constructed wetland as
wastewater refining treatment. Fe and Al were selected owing to their wide use as coagulant agentsin wastewater treatment plants for promoting clariflocculation processes, whilst Cr was chosen due
to its common use in industry. Diclofenac and levofloxacin, two molecules belonging to the mostwidely used pharmaceutical classes in the world, were studied as representative anti-inflammatory
drugs and antibiotics, respectively. The study was performed at laboratory scale, exposing the plants
separately to each individual contaminant at the concentrations of 5 mg L -1 for the metals (i.e. 2.5-5
times higher than the European limits concerning discharge into surface water), and 1 µg L -1 for thepharmaceuticals (concentration levels commonly found in wastewater). Depending on the plant
species and contaminant tested, the range of different effects observed included low toxicity (i.e. Cr,
Fe and diclofenac in L. minuta) and even a stimulatory effect on plant growth (i.e. for A. filiculoides
with Al and for L. minuta with Al and levofloxacin). Moreover, both species proved to be very effectivein the removal of Fe, Al and levofloxacin, with A. filiculoides showing the best performance (removal
efficiency of 92%, 96%, and 60%, respectively), whereas for Cr and diclofenac the removal wasalways less than 10%. The higher removal capacity of A. filiculoides compared to L. minuta can be
attributed to its superior tolerance of the contaminants, probably in turn related to the presence ofnitrogen-fixing microorganism in its fronds.

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Dual function of Lemna minor and Azolla pinnata as phytoremediator forpalm oil mill effluent and as feedstock
Abd Kadir, A; Abdullah, SRS; Othman, BA; Abu Hasan, H; Othman, AR; Imron, MF; Ismail, NI;
Kurniawan, SB (2020) CHEMOSPHERE 259: 127468.
In this study, two native duckweeds (Lemna minor and Azolla pinnata) were cultivated in Palm Oil Mill
Effluent (POME) to extract nutrients from the effluent. Five grams of A. pinnata and 2 g of L. minor
were transferred to 2 L POME (Initial concentrations: 198 mg/L COD, 4.3 mg/L nitrates, pH 9.53, 4
mg/L phosphate, 2.98 mg/L ammonia) with four different dilutions (2.5%, 5%, 10%, 15%) undergreenhouse conditions. Samples of POME were taken every two days up to 10 days. Growthparameter, phosphate, ammonia, nitrates, pH, and COD were monitored within 10 days to select themost suitable growth medium for both plants. Results showed that 2.5% POME dilution had positiveeffect on L. minor growth and A. pinnata (wet weight increased by 8.7 g and 9.8 g, respectively), withall plants able to survive until the final day of exposure. The highest removal of ammonia wasaccomplished in 5% POME dilution by A. pinnata (98%) and L. minor (95.5%). The maximum
phosphate removal was obtained in 10% POME dilution with 93.3% removal by A. pinnata and 86.7%
by L. minor. Significant COD removal in 15% POME was obtained by L. minor (78%) and A. pinnata(66%). Both plants responded positively to the phytoremediation process, especially for A. pinnata
which showed significant decreases in all parameters. The nutrient extraction by both plants fromPOME showed a positive effect on growth parameter, which has further promising potential to be
used as animal feedstock.
DF comment: Azolla pinnata does not belong to the family of duckweed.

Municipal wastewater treatment by combining in series microalgae
Chlorella sorokiniana and macrophyte Lemna minor: Preliminary results
Kotoula, D; Iliopoulou, A; Irakleous-Palaiologou, E; Gatidou, G; Aloupi, M; Antonopoulou, P;
Fountoulakis, MS; Stasinakis, AS (2020) Journal of Cleaner Production 271: 122704
Conventional treatment of municipal wastewater requires large inputs of energy, and high operationand maintenance costs. On the other hand, the use of microalgae and macrophytes on wastewater
treatment offers an alternative approach that combines removal of pollutants with production ofvaluable biomass. An integrated sequencing batch reactor system that combines in series
microalgae Chlorella sorokiniana UTEX 1230 and macrophyte Lemna minor was tested for municipal
wastewater treatment in the current study. The average removal of COD, TKN, NH 4-N and PO4-P was
99%, 88%, 90% and 91%, respectively, while the concentrations of major pollutants in the effluentscomplied with European Directive 91/271 for wastewater discharge to the aquatic environment aswell as with national legislation for wastewater reuse for limited irrigation. The microalgae reactor
was capable of fully removing COD and partially removing N and P nutrients, while Lemna minor
reactor contributed mainly to nitrogen removal. Batch experiments with Chlorella sorokiniana anddifferent types of municipal wastewater (raw sewage, anaerobically treated wastewater andaerobically treated wastewater) with no pH adjustment and CO 2 addition showed that the optimal
microalgae growth and the highest pollutants removal were achieved when raw sewage were used.
Comparison of mixotrophic and heterotrophic conditions showed highest biomass growth undermixotrophic conditions. Except for nitrates, the higher removal of other pollutants was also achieved
under mixotrophic conditions. Further experiments should be conducted to examine thecharacteristics of produced microalgae and macrophyte biomass and the alternative ways of theirvalorization under the frame of circular economy.
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Lemna minor influence in the treatment of organic pollution of theindustrial effluents
Alvarado, K; Esenarro, D; Rodriguez, C; Vasquez, W (2020) 3C Tecnologia 9: 77-96
The purpose of the research was to determine the influence of industrial wastewater treatmentusing the Lemna Minor aquatic plant. Certain varieties of macrophyte plants can absorb or retain
various contaminants. Thanks to this, it has been determined that the variety known as Lemna Minor
presents this type of property. Three treatment trials were carried out varying the amounts of Lemna
Minor (100, 200, and 300g). They are keeping constant the retention time of 10 days that wereanalyzed at 3, 6, and 10 days after the treatment and with a constant volume of the residual effluent.
The results indicate that in terms of the parameters that determine organic contamination, BOD was
reduced by (61 %); COD was reduced by (68 %) and the concentration of total suspended solids by(61 %).

Uncoupling growth from phosphorus uptake in Lemna: Implications foruse of duckweed in wastewater remediation and P recovery in temperate
climates
Paterson, JB; Camargo-Valero, MA; Baker, A (2020) Food and Energy Security Article Number: e244
Phosphorus (P) is an essential nutrient for crop growth and the second most limiting after N. Current
supplies rely on P-rich rocks that are unevenly distributed globally and exploited unsustainably,leading to concerns about future availability and therefore food security. Duckweeds (Lemnaceae)
are aquatic macrophytes used in wastewater remediation with the potential for nutrient recycling as
feed or fertilizer. The use of duckweeds in this way is confined to tropical regions as it has previously
been assumed that growth in the colder seasons of the temperate regions would be insufficient. In
this study, the combined effects of cool temperatures and short photoperiods on growth and P
uptake and accumulation in Lemna were investigated under controlled laboratory conditions. Growthand P accumulation in Lemna can be uncoupled, with significant P removal from the medium andaccumulation within the plants occurring even at 8 degrees C and 6-hr photoperiods. Direct
measurement of radiolabeled phosphate uptake confirmed that while transport is strongly
temperature dependent, uptake can still be measured at 5 degrees C. Prior phosphate starvation of
the duckweed and use of nitrate as the nitrogen (N) source also greatly increased the rate of P
removal and in-cell accumulation. These results form the basis for further examination of thefeasibility of duckweed-based systems for wastewater treatment and P recapture in temperate
climates, particularly in small, rural treatment works.

Exploring the efficacy of powered guar gum (Cyamopsis tetragonoloba)
seeds, duckweed (Spirodela polyrhiza), and Indian plum (Ziziphus
mauritiana) leaves in urban wastewater treatment
Pandey, N; Gusain, R; Suthar, S (2020) Journal of Cleaner Production 264: 121680
The efficiency of aqueous extract of powdered guar gum seed (Cyamopsis tetragonoloba), duckweed
fronds (Spirodela polyrhiza), and Indian plum leaves (Ziziphus mauritiana) was tested to remove
nitrate, sulphate, phosphate, and chemical oxygen demand from urban wastewater in this study.
Coagulants were applied in four different doses (1, 2, 3, and 5 mL L -1) without adjusting the pH of it inwastewater treatment and changes in wastewater parameters were measured. The crude extract ofguar gum seed, Indian plum leaves, and duckweed exhibited the removal activities 71.7-95.03%
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DUCKWEED FORUM(nitrate), 78.3-95.9% (phosphate), and 83.1-99.6% (sulphate). Nitrate removal was highest (93.5%),
followed by phosphate (83.4%) and sulphate (77.7%). The 3 mL L -1 dose of coagulant showed thebest results of wastewater treatment, followed by 2 mL L -1, 1 mL L-1, and 5 mL L-1. Chemical oxygen
demand reduction was also in a good range (73.2-79.08%) with 3 mL L -1 dose for all studied
coagulants. pH reduced significantly in all setups of wastewater spiked with plant coagulants. The
chemical analysis of Fourier transforms infrared (FTIR) spectroscopy of aqueous extract and plantpowder indicates the presence of functions groups like - OH, -COOH, -NH, C=O, C-C R-CHO, C=C-COOH, C-H, and R-NH2, in raw coagulants. Overall, results indicate the possible utility of guar gum seed,
duckweed, and Indian plum leaf powder as low-cost natural materials for treatment of urban
wastewater. However, few high doses of coagulants showed a slight increase in chemical oxygen
demand in wastewater, which can be moderated by purifying the active compounds responsible forcoagulation activities.

Bioremediation of Landoltia punctata to Microcystis aeruginosacontaminated waters
Li, S; Le, SX; Li, GL; Luo, M; Wang, R; Zhao, Y (2020) Water 12: 1764
Microcystis aeruginosa is one of the dominant algae in the "phytoplankton bloom" phenomenon. A
high density of microcystins (MCs) is produced when algae have explosive growth, which candamage the water environment and pose a great threat to aquatic animals, plants, and humanhealth. Duckweed (Landoltia punctata) is a morphologically highly degraded flowering plant with ashort growth cycle and wide environmental adaptability. Importantly, duckweed can grow ineutrophic water and has great potential in water remediation. The present study aims to analyze thephysiological and biochemical changes of L. punctata when co-culturing with M. aeruginosa in thelaboratory. Our results showed that all the biomass, chlorophyll content, antioxidant enzyme
activities, and amylase activity of L. punctata increased in 2 x 10 8 cells/L and 4 x 10 8 cells/L for M.
aeruginosa, and also significantly reduced in 1.6 x 10 9 cells/L for M. aeruginosa, while cytotoxic
substance (malondialdehyde (MDA)) content showed a completely opposite trend. After co-culturing,
it was found that the MC content in L. punctata reduced to 138.87 g/g, and the MC removal rate was
29.48%. These results indicate that L. punctata can grow normally in high-density M. aeruginosa,
which paves the way for L. punctata's bioremediation of water polluted by M. aeruginosa.

Phytoremediation efficiencies of Spirodela polyrhiza and Brassica
oleracea in removing nutrients from treated sewage effluent
Mun, NK; Mohamed, RMSR; Miswan, MS; Al-Gheethi, A; Kassim, AHM; Mishima, Y (2020)
Desalination and Water Treatment 187: 87-92
The study investigates the capacity of phytoremediation as a post-treatment step for the nutrientrich-treated sewage effluent from Saga City sewage treatment plant, Saga, Japan. Phytoremediationin the context of this study is the removal of nutrients such as ammoniacal nitrogen, nitrate nitrogenand phosphorus from the nutrient-rich-treated sewage effluent by plants. In this study, Spirodela
polyrhiza (S. polyrhiza) and Brassica oleracea (B. oleracea) were used to phytoremediate the treatedsewage effluent collected from the Saga City Sewage Treatment Plant under laboratory scale. Plants
were grown in polypropylene planter box supplied with 8 L treated sewage effluent under indoor
environment and full water retention throughout the experimental studies. The removal efficiency
and daily absorption of nutrients by phytoremediation plants were determined. It was found that themost optimal removal efficiency and average daily nutrient removal rate by S. polyrhiza throughout
the experiment were 92.42% ±1.29% or 15.4 mg/L/d for ammoniacal nitrogen achieved in day 1,
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78.69% ± 10.31% or 2.68 mg/L/d for nitrate-nitrogen achieved in day 4, and 93.45% ± 3.26% or 0.51
mg/L/d for phosphorus in day 3 of an experiment. On the other hand, the removal efficiency andaverage daily nutrient removal rate by B. oleracea throughout the experiment gave a total of 8 d
where 76.07% ± 10.38% or 1.68 mg/L/d for ammoniacal nitrogen, 78.38% ± 0.40% or 1.19 mg/L/d fornitrate-nitrogen and 67.40% ± 10.91% or 0.10 mg/L/d for phosphorus. The overall findings
demonstrated that phytoremediation by S. polyrhiza was far more effective in removing nutrientsfrom the nutrient-rich-treated sewage effluent compared to B. oleracea. The significance of the study
includes reducing the possibility of eutrophication outbreak caused by the disposal of treatedsewage effluent, advocating less dependency on global demand for non-renewable phosphorus
resources in the agriculture sector, and solving food demand due to the increasing world population.

Degradation studies of selected bisphenols in the presence of betacyclodextrin and/ or duckweed water plant
Kaleniecka, A; Zarzycki, PK (2020) Journal of AOAC International 103: 439-448
This research reports a multivariate experiment enabling observation of the potential application ofmacrocyclic compound [beta-cyclodextrin (beta-CD)] and/or duckweed organisms as the active
factors for elimination of selected bisphenols A, B, and S from water samples. Target bisphenols
selection was based on observation that such components can be present in food or environmental
samples (e.g., vegetable/fruit juices, milk, drinking water, or treated wastewater). Biological research
was carried out using aquatic organisms containing chlorophyll, particularly duckweed (Lemna
minor L), that may work as an active biomass for the elimination or extraction of bisphenols
micropollutants from water. Using such a system, we studied the potential encapsulation effect andremoval efficiency of nontoxic macrocyclic oligosaccharide (beta-cyclodextrin) acting as an
encapsulation reagent to promote the removal of selected bisphenols from liquid phase both withand without the presence of duckweed biomass. Experimental data have revealed that beta-CD or
combined beta-CD/duckweed system has an effect on bisphenols elimination from water. The initial
data set obtained from this preliminary experiment (and combined with supramolecular complex
formation data calculated from chromatographic experiments, published previously) enables
designing of further experiments focusing on the development of green chemistry technology. It is
hoped that this may be used for the efficient removal of low-molecular-mass micropollutants usingclassical technological wastewater treatment processes modified by biomass and macrocyclic
additives. This process needs to be optimized, but the results presented have revealed that suchgreen chemistry technology, if successful, may be an interesting alternative for the selective removalof the micropollutants investigated from wastewater using classical adsorbents (e.g., carbons andcarbon-related nanomaterials), particularly in terms of the worldwide problem with microplastic
pollutants in the environment and food products.

Phytotoxicity
Ecotoxicological effects of the azole antifungal agent clotrimazole on
the macrophyte species Lemna minor and Lemna gibba
de Alkimin, GD; Santos, J; Soares, AMVM; Nunes, B (2020) Comparative Biochemistry and Phyiology
C-Toxicology and Pharmacology 237: 108835
Pharmaceuticals are a large and diverse group of compounds used to treat, prevent and diagnosedisease. Among these, a group that has been recently detected in the aquatic environment is that of
the azole compounds, commonly used as antifungals. Clotrimazole (CLO) is a nonbiodegradable
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persistent azole compound, with broad-spectrum antifungal activity for which virtually no
toxicological data are available, especially towards aquatic plants. The few existent data point to a
documented interference with cytochrome P450 system of exposed organisms. Therefore, the aimof this paper was to evaluate the ecotoxicological effects of the fungicide CLO on two aquatic
macrophyte species, namely, Lemna minor and Lemna gibba. To attain this purpose, an acute assay(96 h) was performed with both species being exposed to CLO, in a concentration range of 0 to 5 µg
L-1. The analyzed endpoints were levels of chlorophyll a and b, total, carotenoids, catalase (CAT) andglutathione-transferases activities (GSTs). In general, CLO exposure caused some minor alterations
in L. minor and L. gibba pigment contents. Antioxidant enzymes exhibited a different pattern in both
species, since the highest concentrations of CLO caused an increase on CAT activity, and a decreaseon GSTs activity in L. minor, and the opposite in L. gibba, reflected by a decrease on CAT activity andan increase on GSTs activity in all tested concentrations. These results demonstrate that CLO
exposure resulted in potential deleterious effects on macrophytes, namely with the involvement of
the antioxidant defense mechanisms that were likely deployed to cope with pro-oxidative conditions
established by CLO.

Individual and combined effects of amoxicillin, enrofloxacin, andoxytetracycline on Lemna minor physiology
Gomes, MP; Brito, JCM; Rocha, DC; Navarro-Silva, MA; Juneau, P (2020) Ecotoxicology andEnvironmental Safety 203: 111025
We investigated individual and combined effects of environmentally representative concentrations ofamoxicillin (AMX; 2 µg L-1), enrofloxacin (ENR; 2 µg L-1), and oxytetracycline (OXY; 1 µg L-1) on theaquatic macrophyte Lemna minor. While the concentrations of AMX and ENR tested were not toxic,
OXY decreased plant growth and cell division. OXY induced hydrogen peroxide (H 2O2) accumulation
and related oxidative stress through its interference with the activities of mitochondria electrontransport chain enzymes, although those deleterious effects could be ameliorated by the presence of
AMX and/or ENR, which prevented the overaccumulation of ROS by increasing catalase enzyme
activity. L. minor plants accumulated significant quantities of AMX, ENR and OXY from the media,
although competitive uptakes were observed when plants were submitted to binary or tertiary
mixtures of those antibiotics. Our results therefore indicate L. minor as a candidate forphytoremediation of service waters contaminated by AMX, ENR, and/or OXY.

Copper and mercury induced oxidative stresses and antioxidant
responses of Spirodela polyrhiza (L.) Schleid.
Singh, H; Kumar, D; Soni, V (2020) Biochemistry and Biophysics Reports 23:100781
Duckweed is recognized as a phytoremediation aquatic plant due to the production of large biomass
and a high level of tolerance in stressed conditions. A laboratory experiment was conducted toinvestigate antioxidant response and mechanism of copper and mercury tolerance of S. polyrhiza(L.) Schleid. To understand the changes in chlorophyll content, MDA, proline, and activities of ROSscavenging enzymes (SOD, CAT, GPOD) during the accumulation of Cu +2 and Hg+2, S. polyrhiza were
exposed to various concentrations of Cu +2 (0.0-40 µM) and Hg+2 (0.0-0.4 µM). antioxidant activity
initially indicated enhancing trend with application of 10 µM Cu +2; 0.2 µM Hg+2 (SOD), of 20 µM Cu+2;
0.2 µM Hg+2 (CAT) and of 10 µM Cu+2;0.2 µM Hg+2 (GPOD) and then decreased consistently up to 40µM Cu+2 and 0.4 µM Hg+2. In the experiment chlorophyll and frond multiplication initially showed
increasing tendency and decreased gradually with the application of increased metal concentration.
Application of heavy metal has constantly enhanced proline and MDA content while the maximum
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increase was observed with the application of 40 µM Cu; 0.4 µM Hg for proline and MDA
respectively. The upregulation of antioxidant enzymes and proline reveals that S. polyrhiza has
strong biochemical strategies to deal with the heavy metal toxicity induced by the accumulation of
Cu+2 and Hg+2.

Growth inhibition and recovery patterns of common duckweed Lemna
minor L. after repeated exposure to isoproturon
Varga, M; Zurga, P; Brusic, I; Horvatic, J; Moslavac, M (2020) Ecotoxicology
DOI: 10.1007/s10646-020-02262-9
Aquatic non-targeted organisms are more likely to be exposed to herbicides in multiple pulse events
then long continuous exposure. The potential of an organism to recover between exposures has animportant role in the overall effects of the toxicant. Common duckweeds show high potential forrecovery after a single exposure to isoproturon. To evaluate the growth patterns and recovery
potential between multiple exposures, L. minor plants were exposed to isoproturon in three repetitive
7-day treatment cycles in three time-variable exposure scenarios with equivalent time-weighted
average concentrations. The growth was significantly inhibited during each exposure phase withsignificant cumulative effects in every subsequent treatment cycle resulting in a cumulative
decrease in biomass production. However, inhibitory effects were reversible upon transferring plants
to a herbicide-free nutrient solution. These results indicate that L. minor plants have a high recovery
potential even after multiple exposures to isoproturon. Observed cumulative decrease in biomass
production, as well as the potential for fast and efficient recovery from repeated herbicide exposure,might affect the competitiveness of L. minor in surface water communities. The observations madeduring each exposure period, recovery patterns, and the resulting cumulative effects over time may
contribute to further development, calibration and validation of mechanistic toxicokinetic/
toxicodynamic models for simulating the effects of pesticides on aquatic plants populations in thelaboratory and environmental conditions.

Assessing potential aquatic toxicity of airport runoff using
physicochemical parameters and Lemna gibba and Aliivibrio fischeribioassays
Calvo, OC; Quaglia, G; Mohiley, A; Cesarini, M; Fangmeier, A (2020) Environmental Science and
Pollution Research DOI: 10.1007/s11356-020-09848-0
A critical problem derived from airport operations is the environmental impact of runoff water.
Airport runoff includes a complex mixture of pollutants, e.g., from deicing agents, that may affectnegatively natural water bodies. This study assesses the spatial and temporal aquatic ecotoxicity ofrunoff water and possible aeroplane drift in a German airport. Over winter 2012-2013, from
November to May, water samples were collected within the airport and surrounding area. Thesesamples were analyzed using traditional physicochemical analysis and biotests with two aquaticorganisms from different trophic levels, Lemna gibba and Aliivibrio fischeri. Overall, the samples
examined in this study were relatively non-toxic to the tested organisms. The physicochemicalparameters were mainly influenced by the sampling period being higher in colder months. In
contrast, the ecotoxicity was influenced by the sampling site. For sites within the airport, a highcorrelation between the physicochemical parameters (EC and TOC) and toxicity in L. gibba wasfound. These correlations were not evident in samples taken outside the airport or when A. fischeri
was used as a bioindicator. However, a pronounced seasonality has been observed, linked to thecoldest months with average inhibition values of 50% in L. gibba and 25% in A. fischeri, particularly invol 8 (4), page 119 of 126

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January. Both biotests yielded differing results; therefore, more biotests should be included.
However, L. gibba showed a good response with this type of water samples to be included in futurestudies together with detailed chemical analysis. The present study provides data to assess thepotential ecotoxicological effects of airport runoff affected by winter operations.

The toxicity of BDE-47 to the photosystem of Lemna minor fronds
Qiu, NW; Zhang, WR; Yan, XH; Wang, RJ; Tian, L; Han, GL; Zhou, F (2020) Biologia Plantarum 64: 591597
To elucidate the toxicity of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on photosynthetic primaryprocesses, in vivo and in vitro treatments of BDE-47 were performed. The 20-d treatment in vivo (5 20 µg dm-3) suppressed the reproduction of duckweed (Lemna minor) and led to decline inchlorophyll (Chl) content of fronds. The most obvious features of BDE-47-treated fronds included a
Chl a fluorescence rise at the J phase and a depression at the G phase, whereas significant
fluorescence rises at the L, K, and J phases were found on the Delta V-t curve of thylakoid
membranes treated with 10 - 15 mg dm-3 BDE-47 for 4 h (in vitro). In both in vivo and in vitro
experiments, the BDE-47 treatments significantly reduced the density of the active reaction centers(RC/CSo), affected the efficiency and speed of photosynthetic electron transfer [the maximum
quantum yield of photosystem (PS) II photochemistry - F-v/F-m, quantum yield for electron transport(at t = 0) - phi(Eo), electron transport flux per excited cross section - ETo/CSo, and net rate ofreaction centers closure at 300 and 100 mu s - dV/dt(o) and dVG/dt(o), respectively], and increased
energy dissipation [quantum yield for energy dissipation (at t = 0) - phi(Do), dissipated energy flux per
reaction center - DIo/RC, and dissipated energy flux per excited cross section - DIo/CSo]. The BDE-47
at 5 - 15 mg dm-3 had no impact on the minimum (initial) fluorescence (Fo) and total electron carriers
per reaction center (S-m) of the thylakoid membranes, but PS II units were less tightly grouped (apositive L-band). On the contrary, there was no positive L-band on the difference between relative
fluorescence intensities of the normalized induction curves from 50 µs to 300 µs (W-K) of each BDE47 treatment and control (Delta W-K), and F-o and S-m increased after the treatment with BDE-47.
The above results indicate that BDE-47 not only affected the permeability of thylakoid membranes,
but also relaxed the structure of PS II, thereby affecting the function of PS II. In addition, BDE-47
could induce secondary damage to the PSs in duckweed fronds.

Image analysis of Spirodela polyrhiza for the semiquantitative detectionof copper
Nesan, D; Chieh, DCJ (2020) Journal of Environmental Chemical Engineering 8: UNSP 103043
Digital image analysis is a processing technique that allows users to extract quantifiable data fromdigital images. In this study, digital camera photography was used in the determination of leaf
chlorophyll content. By analyzing the degree of color change, image analysis served as a method for
fast, inexpensive and non-destructive measurement of overall plant health. This study applied image
analysis methods on Spirodela polyrhiza plantlets which were exposed to copper, to determine if therate and degree of leaf color change is proportional to the concentration of copper present in thegrowth medium. Within 1 day, chlorophyll concentrations of plantlets grown in 2.5 mg/L and 5mg/L
CuSO4 were 0.52 and 0.47 mg/g compared to a control of 0.64 mg/g. Additionally, higher copper
concentrations in the growing medium resulted in higher measured mean colour distance, Delta
E*ab. Plantlets grown in 2.5 mg/L and 5 mg/L CuSO4 solutions showed a Delta E*ab divergence of
0.2 and 0.25 from the control. It was concluded that the leaf color change can be used as a measureof copper concentration within the range of 1.25 mg/L and 5 mg/L. Lower concentrations of copper
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did not produce a consistent measurable effect on the plantlets, while higher concentrations
exceeded the uptake ability of the plant and could not be accurately distinguished from one another.

Selenate and selenite uptake, accumulation and toxicity in Lemna minuta
Li, J; Loi, G; Otero-Gonzalez, L; Du Laing, G; Ferrer, I; Lens, PNL (2020) Water Science and Technology
81: 1852-1862
The kinetics of Se uptake and toxicity to Lemna were studied over a period of 14 days of exposure toSe(IV) or Se(VI). The growth of Lemna stopped immediately after exposure to 5.0 mg/L of Se(IV) or
Se(VI). The content of chlorophyll and phaeopigments of Lemna exposed to 5.0 mg/L of Se(IV) was
two to three times less than in the control after 3 d exposure. Lemna took up Se rapidly within thefirst 3 d. The Se content in Lemna along with the exposure time fitted well the two-compartment andthe hyperbolic model, which demonstrates that the mechanism of Se(IV) and Se(VI) uptake in Lemna
is not only through passive diffusion, but also through other processes such as ion channel proteinsor transporters. The kinetic bioconcentration factors (BCFs) were 231 and 42 for 0.5 mg/L Se(IV)and Se(VI) exposure, respectively. The uptake rate of Lemna reached 263 mg/kg/d and 28 mg/kg/din the Se(IV) and Se(VI) treatments, respectively. This study showed that Se(IV) has a fasteraccumulation rate than Se(VI), but a higher toxicity, indicating Lemna could be a good candidate toremove Se(IV) from water, producing Se-enriched biomass which may eventually also be considered
for use as Se-enriched feed supplement or fertilizer.

Toxicity of the herbicide flurochloridone to the aquatic plants
Ceratophyllum demersum and Lemna minor
Zhou, JA; Wu, ZH; Yu, D; Yang, L (2020) Environmental Science and Pollution Research 27: 39233932
As a new and efficient selective pre-emergence herbicide, flurochloridone (FLC) has been widely
promoted in recent years but readily results in residues in nature. As the primary producers andrestorers of the water environment, aquatic plants are at risk of FLC exposure. In the present
research, we studied the phytotoxicity of FLC in Lemna minor and Ceratophyllum demersum. The
physiological and growth responses of these two aquatic plants exposed to different concentrationsof FLC (0, 20, 100, 300, 1000, and 2000 µg/L) were measured. The results showed that FLC (≥ 20 µg/L) could cause serious photosynthesis pigment damage and bleaching in C. demersum and L. minor.
Significant oxidative damage was observed in L. minor at 20 µg/L FLC, while there was no severeoxidative damage in C. demersum. At 100-300 µg/L FLC, peroxidase (POD) and superoxide
dismutase (SOD) were activated to scavenge free radicals in L. minor, while POD acted as aprotective enzyme in C. demersum. At higher concentrations of FLC ≥ 1000-2000 µg/L ), L. minor
reached less than healthy stability through the regulation of the antioxidant enzyme system and thechlorophyll a/b value. POD, SOD, and protein content returned to normal levels, and the growthparameters increased. However, in C. demersum, the enzymes POD and SOD and soluble protein
were damaged, and oxidative stress reached the highest level at 1000-2000 µg/L FLC. Taken
together, our results suggested that when treated with FLC, L. minor was more sensitive at lower
doses (20 µg/L) and more adaptive at higher doses (1000-2000 µg/L) than C. demersum.

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Phytoremediation processes of domestic and textile effluents: evaluationof the efficacy and toxicological effects in Lemna minor and Daphniamagna
de Alkimin, GD; Paisio, C; Agostini, E; Nunes, B (2020) Environmental Science and Pollution Research
27: 4423-4441
Phytoremediation has been proposed as a potential biotechnological strategy to remediate effluents
before their release into the environment. The use of common aquatic plant species, such as
macrophytes (e.g., Lemna spp.) as a clean-up solution has been proposed decades ago. However,
the effectiveness of such processes must be assessed by analyzing the toxicity of resulting
effluents, for the monitoring of wastewater quality. To attain this purpose, this work intended toquantify the efficacy of a Lemna-based wastewater phytoremediation process, by analyzing
toxicological effects of domestic and textile effluents. The toxic effects were measured in Lemna
minor (same organisms used in the phytoremediation process, by quantifying toxicological
endpoints such as root length, pigment content, and catalase activity) and by quantifying individual
parameters of Daphnia magna (immobilization, reproduction, and behavior analysis).
Phytoremediation process resulted in a decrease of chemical oxygen demand in both effluents andin an increase in root length of exposed plants. Moreover, textile effluent decreased pigments
content and increased catalase activity, while domestic effluent increased the anthocyanin content
of exposed plants. D. magna acute tests allowed calculating a EC 50 and Toxic Units interval of 53.8266.89%/1.85-1.49, respectively, to raw textile effluent; however, it was not possible to calculate theseparameters for raw and treated domestic effluent (RDE and TDE). Therefore, in general, the acutetoxicity of effluent toward D. magna was null for RDE, and mild for the treated textile effluent (TTE),
probably due to the effect of phytoremediation. Exposure to textile effluents (raw and treated)
increased the total number of neonates of D. magna and, in general, both textile effluents decreased
D. magna distance swim. Moreover, although both effluents were capable of causing morphologicaland physiological/biochemical alterations in L. minor plants, organisms of this species were able tosurvive in the presence of both effluents and to remediate them.

Genotoxicity and repeated-dose toxicity evaluation of dried Wolffia
globosa Mankai
Kawamata, Y; Shibui, Y; Takumi, A; Seki, T; Shimada, T; Hashimoto, M; Inoue, N; Kobayashi, H; Narita,
T (2020) Toxicology Reports 7:1233-1241
Wolffia is a genus of protein-rich aquatic plants. Mankai, a cultivated strain of Wolffia globosa,contains more than 40 % protein based on dry matter evaluation. Furthermore, Mankai is nutritionally
excellent as a food material, and is expected to be applicable to various products as a substitute foranimal protein. A battery of toxicological studies was conducted on the dried product of Mankai (DryMankai), with the expectation to utilize it as a raw material for food applications. Dry Mankai was not
genotoxic in a bacterial reverse mutation test and in vitro micronucleus assay. In the subchronic
toxicity study, rats were provided Dry Mankai in the diet at levels of 0 %, 5 %, 10 %, or 20 % (w/w),
equivalent to 0, 3.18, 6.49, and 13.16 g/kg/day for males and 0, 3.58, 7.42, and 15.03 g/kg/day forfemales, respectively. No adverse effects that could be attributable to treatment were observed inclinical observations, body weight, food consumption, ophthalmology, hematology and blood
chemistry, urinalysis, and macroscopic and microscopic findings. According to the repeated-dosestudy in rats, the no observed adverse effect level of Dry Mankai was 20 % (w/w) for both sexes(13.16 and 15.03 g/kg/day for males and females, respectively).

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Taxonomy
Key to the determination of taxa of Lemnaceae: an update
Bog, M; Appenroth, KJ; Sree, KS (2020) Nordic Journal of Botany 38: e02658
Species of Lemnaceae have a high potential for fast biomass production, and this is increasingly
gaining attention among researchers in basic plant sciences as well as among entrepreneurs forfeed, food and energy production. Hence, the correct identification of the species being used for
different duckweed research and applications is becoming indispensable. Here, we present an
updated identification key based on morphological markers to the currently accepted 36 species of
duckweeds, considering all taxonomic revisions since the publication of the previous key by E.
Landolt in 1986. We also provide supplementary morphological characterization and thegeographical occurrence of each species of Lemnaceae.

Taxonomic identity of Landoltia punctata (Araceae, Lemnoideae) in
Korea
Lee, Y; Choi, HJ; Shiga, T (2020) Journal of Asia-Pacific Biodiversity 13: 494-498
The duckweed species, Landoltia punctata (G. Meyer) D. H. Les & D. J. Crawford (family, Araceae;
subfamily, Lemnoideae; common name, dotted duckmeat) was found in artificial wetlands in Jeju
Special Self-Governing Province, Republic of Korea; however, it has no formal record in Korea. We
identified this species based on morphological characteristics and DNA sequences. We also
checked Lemnoideae specimens in the Korean and Japanese herbaria but found no specimens ofLa. punctata collected in Korea previously. The chloroplast DNA sequence (atpF-atpH) of the species
collected from Jejudo was identical to the other La. punctata sequences in the DNA Data Bank ofJapan (DDBJ)/The European Molecular Biology Laboratory (EMBL)/GenBank. Their habitat
environments and our herbarium research suggest that this species has recently become distributedin Korea. This study also provides a key to closely related taxa in the subfamily Lemnoideae, withdescriptions of these species

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Instructions to Contributors
for the Duckweed ForumThe Duckweed Forum (DF) is an electronic publication that is dedicated to serve the Duckweed
Research and Applications community by disseminating pertinent information related to community
standards, current and future events, as well as other commentaries that could benefit this field. As
such, involvement of the community is essential and the DF can provide a convenient platform formembers in the field to exchange ideas and observations. While we would invite everyone tocontribute, we do have to establish clear guidelines for interested contributors to follow in order tostandardize the workflow for their review and publication by the Duckweed Steering Committee
members.
Contributions to DF must be written in English, although they may be submitted by authors from any
country. Authors who are not native English speakers may appreciate assistance with grammar,
vocabulary, and style when submitting papers to the DF.
DF is currently arranged in sections, which may be chosen by a prospective author(s) to contributeto: Main text, Opinion paper, Discussion corner, Useful methods, Student experiments, Student
spotlight, Science meets art, and Cover photo(s). 1,000 words are suggested as the upper limit foreach contribution, but can be extended on request to the Steering Committee if the reason for thewaiver request is warranted.

Presubmissions
In addition to invitees by a Duckweed Steering Committee member, if you are considering submitting
a contribution to DF but are unsure about the fit of your idea, please feel free to contact one of themembers in the Duckweed Steering Committee in order to obtain feedback as to theappropriateness of the subject for DF. Please include a few sentences describing the overall topic
that you are interested to present on, and why you think it is of interest to the general duckweed
community. If you have the abstract or draft text prepared, please include it. The Duckweed Steering
Committee will discuss the material in one of its meetings and the decision to formally invite
submission will be given shortly afterwards.

Copyright and co-author consent
All listed authors must concur in the submission and the final version must be seen and approved by
all authors of the contribution. As a public forum, we do not carry out any Copyright application. If
you need to copyright your material, please do so beforehand.

Formatting requirements:
A commonly used word processing program, such as Word, is highly recommended.

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DUCKWEED FORUM

Formatting requirements: 8.5-by-11-inch (or 22 cm-by-28 cm) paper size (standard US letter).

Single-spaced text throughout.

One-inch (or 2.5 cm) left and right, as well as top and bottom margins.

11-point Times New Roman font.



Number all pages, including those with figures on the bottom and center of each page.

Title:


Should be intelligible to DF readers who are not specialists in the field and should convey
your essential points clearly.

Should be short (no more than 150 characters including spaces) and informative.

Should avoid acronyms or abbreviations aside from the most common biochemical
abbreviations (e.g., ATP). Other acronyms or abbreviations should either:obe introduced in their full form (e.g., Visualization of Polarized Membrane Type 1
Matrix Metalloproteinase (MT1-MMP) Activity in Live Cells by Fluorescence
Resonance Energy Transfer (FRET) Imaging); orobe clarified by use as a modifier of the appropriate noun (e.g., FOX1 transcription
factor, ACC dopamine receptor).

Authors:All authors are responsible for the content of the manuscript.

Provide the complete names of all authors.



Identify which author will receive correspondence regarding the contribution.

Provide the corresponding author’s name, telephone number, and current e-mail address.

Image resolution and submission:
It is extremely important that figures be prepared with the proper resolution for publication in order
to avoid inaccurate presentation of the data. The minimum acceptable resolution for all figures is
300 dpi. Excessive file compression can distort images, so files should be carefully checked after
compression. Note that figures that contain both line art (such as graphs) and RGB/grayscale areas(such as photographs) are best prepared as EPS (vector) files with embedded TIFF images for the
RGB/grayscale portions. The resolution of those embedded TIFF images should be at least 300 dpi.
Original images should be submitted as a separate file to the text file. It would be helpful to insert
the intended into the Word file as well, if desired, to indicate the location for it. The legend to theimage/figure should be added at the end of the text file and labeled as "Legend to Figures".

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DUCKWEED FORUM

Links for Further Reading
http://www.ruduckweed.org/ Rutgers Duckweed Stock Cooperative, New Brunswick, New Jersey
State University. Prof. Dr. Eric Lam
http://www.InternationalLemnaAssociation.org/ Working to develop commercial applications for
duckweed globally, Exec. Director, Tamra Fakhoorian
http://www.mobot.org/jwcross/duckweed/duckweed.htm Comprehensive site on all things
duckweed-related, By Dr. John Cross.
http://plants.ifas.ufl.edu/ University of Florida’s Center for Aquatic & Invasive Plants.

Community Resources - Updated Table for
Duckweed Collections in the Community
For information related to the location, collection size and contact email for duckweed collections in
our community, please access the website of the RDSC (Rutgers Duckweed Stock Cooperative)
under the heading "List of Worldwide Duckweed Collections". This Table will be updated as new
entries for duckweed collections are being supplied to members of the International Steering
Committee for Duckweed Research and Applications (ISCDRA). We also plan to publish the updated
table in the first issue of each Duckweed Forum newsletter volume starting in 2021.

Note to the Reader
Know of someone who would like to receive their own copy of this newsletter? Would you like tooffer ideas for future articles or have comments about this newsletter? Need to be added or
removed from our contact list?
Please let us know via email to the Chair of ISCDRA, Prof. Eric Lam: ericL89@hotmail.com

vol 8 (4), page 126 of 126