\documentclass[11pt,a4]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage[round]{natbib}
\let\cite\citep




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}



\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,english]{babel}










\begin{document}

\title{in vivo toxicity impact of the organo-metallic photovoltaic perovskites
tested on Caenorhabditis elegans}



\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup








\sloppy


\section*{Abstract}

{\label{293933}}

The organo-metallic photovoltaic perovskites are making a breakthrough
in light to electricity conversion efficiency to that extent that their
commercialization is imminent.~ However, there is still a hidden
parameter which concerns the health hazards related to their production,
transportation and break-down of the installed photovoltaic devices in
our environment. This needs to be addressed by in vivo toxicity studies
and life cycle assessment. We hereby show for the two most studied
photovoltaic perovskites, CN3NH3PbI3 and CN3NH3SnI3 their impact on the
life span, development and fertility of Caenorhabditis elegans (\emph{C.
elegans}), a model organism in toxicology. We highlight the novelty of
our method which is based on microfluidic device allowing a rapid and
accurate monitoring of the assay in many replicates, for various
concentrations at the same time, and analyzing the faith of \emph{C.
elegans} by video-recorded images. Given the increased frequency at
which application-oriented novel compounds emerge every year, this quick
and high-throughput method is badly needed for toxicity studies of new
compounds before their industrial production

\par\null\par\null\par\null

\section*{Introduction}

{\label{555775}}

The main challenges of photovoltaic solar cells today stem in the
complexity of manufacturing, the low energy conversion efficiency, the
price and the low long-term benefit yield. Moreover, the latter is more
pronounced when it comes to maintenance and recycling. In the past five
years, the scientific community pointed halide perovskites as promising
compounds for photovoltaic applications
\cite{Lee_2012}\cite{Luo_2014} due to the high photon to electron
conversion efficiency, low price, and the simplicity of device
architectures. The CH3NH3PbI3, (abbreviated as MAPbI3), was the most
commonly used and developed material. Nevertheless, its lead content
raised much more concerns\cite{Benmessaoud_2016} regarding health hazards
during handling and processing, as well as its potential toxic release
into the environment. Therefore, active research in materials science,
chemistry and physics is currently developing novel materials, which
could be non-toxic but still having high conversion efficiency. The
CH3NH3SnI3, (hereafter MASnI3), was proposed as such by the scientific
community. However, tin is also a heavy metal which could be toxic in
specific forms \cite{Blunden_2003,Browne_1974,22931871}. There is an emergency in verifying
their potential toxic effects since they are promised to be on the
market in 2018 (cite dyesol nesli and oxford).

We have shown recently by in vitro studies how these perovskites can
impact human cells(tox. Res. 2016 and paper snami in vitro-in
preparation), and the group of Conings claimed to report the toxic
effects of a sister compound, the non-photovoltaic and~ commercially
available PbI2~ and SnI2 on zebrafish(cite alibaba). Here, we report on
the severe toxic effects for both MAPbI3 and MASnI3 by in vivo study on
\emph{Caenorhabditis elegans} (\emph{C. elegans}) using a microfluidic
platform.

\par\null

\emph{C. elegans} is a free living, non parasitic soil nematode that can
be cheaply and easily cultivated in the laboratory, where it is grown on
agar plates with liquid cultures of \emph{E. coli} as the food source.
The 1 mm long organism is transparent, which facilitates organs
observation and dissection, and can be housed in large numbers (10'000
worms per Petri dish). Its life span is about 3 weeks and its genome has
been fully sequenced and annotated.(Sulston \& Brenner, 1974) The adult
body-plan is anatomically simple with 959 somatic cells and produces up
to 300 eggs per adult. It reproduces within a life cycle from egg to
young adult of about 3 days under optimal conditions. Another key
advantage of \emph{C. elegans} model is that feeding by RNAi facilitates
gene silencing\cite{11178279}.

These unique properties make the worm an important model system for
biological research in many fields including not only genomics, cell
biology, neuroscience and aging but also toxicity\cite{Hunt}.
\emph{C. elegans} model in biology is sorting the worms by age, and
regarding their fast development rate, the experimenter needs to be
highly trained to sort a consequent number of worms quickly by hand for
a large statistic, and isolate groups for all the test conditions
without biasing the experiment. Furthermore, in our study, the
continuous worm feeding as well as the high-resolution imaging implicate
manual removal of the worms from the incubator to the bench at each time
point, which triggers temperature changes undesirable stress to the
animal. To overcome this issue, we opted for a method using a
microfluidics platform combined with optical microscopy to allow
sorting, continuous feeding and imaging of the worms. Although many
other researchers developed microfluidic tools to simplify \emph{C.
elegans} studies\selectlanguage{ngerman}\cite{Charão_Souto_Brucker_Barth_Jornada_Ávila_Eifler-lima_Guterres_Pohlmann_Garcia_2015,Hanna_Cooksey_Dong_Nelson_Mao_Elliott_Petersen_2016,Jung_Qu_Aleman-meza_Wang_Riepe_Liu_Li_Zhong_2015,Kim_Lee_Cha_Hong_Chung_2017,Kim_Kwak_An_2013,Ma_Kabengi_Bertsch_Unrine_Glenn_Williams_2011,Mohan_Chen_Hsieh_Wu_2010,Rohde_Zeng_Gonzalez-rubio_Angel_Yanik_2007}, their approaches did not allow for a
high-throughput screening. (Matteo/Laurent), please elaborate a bit the
advantages, differences in respect to the method of Rhode et al etc.

Our system was developed at EPFL (Laboratory of Integrative Systems
Physiology and Laboratory of Microsystems)(cite Cornaglia et al., 2015,
2016) HERE LAURENT AND MATTEO WILL EMPHASIZE.

We cultivated a wild type strain and used high-resolution optical
microscopy to probe the changes in real-time. Contrary to the
expectations, we found a stronger negative influence of the tin than of
the lead compound on life span, development and fertility of \emph{C.
elegans}. Since in the community it is considered that toxicity studies
of \emph{C. elegans} results in predictive power for higher eukaryotes
so these results are important for human exposure, as well.~ They
underline the potential health hazards related to large-scale production
of MAPbI3 and/or MASnI3, and invite the photovoltaic community to take
safety measures and to amplify research on encapsulated systems to
prevent the release of the materials into the environment.

\par\null

\section*{Results}

{\label{926058}}

The following toxicity study was performed on the microfluidic platform
on \emph{C. elegans} by continuous injection of S-medium containing
\emph{E. coli} with or without perovskites during one week.

The sketch of the platform is shown in figure 2, with typical snapshots
of the chambers with the evolution of \emph{C. elegans}. During the one
week exposure of the worms an image was taken every 20 min and analyzed.
We have deduced the survival rate, the evolution of size and the
development of worms. The data were obtained from 3 independent repeats.
The major results are shown and discussed below.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Screenshot-2017-04-20-15.13.32/Screenshot-2017-04-20-15.13.32}
\caption{{a) Sketch of the microfluidic platform. It allows several replica for a
given concentration (6 are marked), and several concentration of the
perovskite introduced into the flow channel. (NT stands for the
reference water flow, non-treated with perovskite; Pb 1 and 2 for two
concentrations of MAPbI3 and , Sn 1, 2 for two concentrations of MASnI3
in the fluid. In principle, one can use as many parallel channels as
reasonable; b. Typical snapshots of chambers after 7 days of continuous
flow of pure water (NT) and 100 mg/ml concentration of MAPbI3 and
MASnI3. The changes in the population after exposure to the two
perovskites is striking. (Matteo/Laurent), please update it.
{\label{156567}}%
}}
\end{center}
\end{figure}

\par\null

\subsection*{Effects on the worm's
survival}

{\label{742757}}

The survival rate of treated worms was normalized to controls and
plotted as a function of time (Figure 2) for samples containing 50, 100
and 200 ug/ml of MAPbI3 (top panels) or MASnI3 (bottom panels).
Non-treated worms (blue line) exhibited a survival rate of 100\% then
started to decay after 6 days because the compartments were filled with
worms and they suffered a space limitation. When worms were exposed to
MAPbI3, the population began to die after 60 hours from the beginning of
the experiment. The dead worms are recognized by immobile, stiff shapes.
This trend was more pronounced with higher doses (green and red lines),
especially at the highest dose where no living worm could be observed
after 5 days of exposure. MASnI3 started interrupting the worms'
survival as early as 20 hours, also in a time and dose-dependent
fashion. At 200 ug/ml, the population suffered extensively the exposure
and perished after 60 hours.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/LifeSpanVer/LifeSpanVer}
\caption{\selectlanguage{ngerman}{Life span/survival rates of \emph{C. elegans} exposed to MAPbI3 or
MASnI3 perovskites. (a) L3-stage larvae were inoculated into
microfluidic device in different chambers where regular bacterial food
containing or not MAPbI3 or MASnI3 was sequentially injected. Survival
rate was evaluated every 20 min by live imaging. All the histograms show
an average of at least 3 independent repeats (each condition in
triplicate). Bars are means ± S.D. One-way ANOVA test followed by a
Tukey-Kramer post-hoc test were performed (non-exposed vs MAPbI3 or
MASnI3 exposed flies or worms), *p\textless{}0.05, **p\textless{}0.005~
and ***\textless{}0.0005.%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}

\subsection*{}

{\label{850001}}

\subsection*{Effects on the worms' growth
size}

{\label{920248}}

The body area of living worms was measured using a logarithm and the
values are reported in Figure 3. Theblue-lined curves show the
increasing body area of non-treated nematodes. Dueto their natural
sinusoidal movement allowed by muscular contraction and thelack of space
inside the microfluidic chambers the body size shrinks after 70 hours.
After treatment with MAPbI3 at 50(orange line) the worms' body size
reaches a plateau at the same time point ascontrol however it has a
smaller area. This observation is even more strikingwith the curve
profile of worms exposed to 100 ug/ml (green line) and to200 ug/ml (red
line) of MAPbI3. On the other hand exposure of nematodes to MASnI3
resulted in a much stunning body size freezing (Figure 3 bottompanels).
In addition the area values reached a plateau earlier thannon-treated
samples and this effect was dose-dependent. From these observations we
assumed that perovskites preventthe larvae to reach the young adult
stage. Therefore we ought to verify if thedecrease in the worms' size is
due to an affected development by tracking theyoung adults and
quantifying the time-lapse when they developed.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/TestDevVer/TestDevVer}
\caption{\selectlanguage{ngerman}{Effects of MAPbI3 and MASnI3 on the development of: (a) \emph{C.
elegans}, L3-stage larvae were inoculated into microfluidic device in
different chambers where regular bacterial food containing or not~
MAPbI3 or MASnI3 was sequentially injected, the development of the
larvae was assessed until old adult stage. All the histograms show an
average of at least 3 independent repeats (each condition in
triplicate). Bars are means ± S.D. One-way ANOVA test followed by a
Tukey-Kramer post-hoc test were performed (non-exposed vs MAPbI3 or
MASnI3 exposed flies or worms), *p\textless{}0.05, **p\textless{}0.005~
and ***\textless{}0.0005.
{\label{272240}}%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}

\par\null

\subsection*{Effects on the developpement of
worms}

{\label{384116}}

The first young adult observed in control samples was between 20 and 22
hours from the L3 larva stage (Figure 4). Injection of MAPbI3 delayed
the developmental process by 3 hours at the lowest concentration and by
5.5 hours at the highest one. Treatment with MASnI3 induced a worse
delay in development already at 50 ug/ml, where the majority of larvae
became adults after 30 hours. For higher concentrations, most of the
nematodes either perished or remained small, which explains the absence
of young adult ``phenotype''.\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=1.00\columnwidth]{figures/YASVer/YASVer}
\caption{{Young adult stage reached by C elegans after the 3rd larval stage, when
exposed or not to MAPbI3 (a) or MASnI3 (b) at increasing concentrations:
50 (orange), 100 (green), and 200 ug/ml (red). All plots show an average
of standard deviation (S.D.) using One-way ANOVA test followed by a
Tukey-Kramer post-hoc test (non-treated vs. treated conditions),
p-value\textless{}0.002.
{\label{397899}}%
}}
\end{center}
\end{figure}

\subsection*{Effects on fertility}

{\label{677462}}

The next live-imaging analysis concerned the extraction of the time
point where the first egg was observed. This was acquired for the
non-treated nematodes before 23 hours after the 3rd larval stage (Figure
5, a, b. Blue boxes). As for the worms exposed to MAPbI3, the first egg
was observed after 23 hours at the lowest dose, and after 26 hours at
the highest one (Figure 5a, orange, green and red boxes). Clearly, there
is a shift in the worms' fertility over exposure to perovskites,
especially to the tin-based one (figure 5b), where we can observe a more
marked delay for a concentration as low as 50 ug/ml.

The first generation of eggs from the non-treated worms progressed to
the larval stage for the first time before 36 hours (Figure 5c, d. Blue
boxes) and continued to proliferate until the end of the experiment (140
hours), where space in the chambers represented a limitation to continue
the study. In parallel, eggs generated by treated nematodes developed to
larvae much later for MAPbI3 (between 36 and 42 hours). Additionally,
the delay was even more pronounced in the case of exposure to MASnI3
(around 38 hours at 50ug/ml). Finally, the absence of progeny at 100 and
200 ug/ml of perovskites confirms the delay in development.

\par\null\selectlanguage{english}
\begin{figure}[h!]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/1stEggVer/1stEggVer}
\caption{\selectlanguage{ngerman}{Fertility investigation of \emph{C. elegans} over MAPbI3 (left panels)
or MASnI3 (right panels) exposure
{\label{485956}}
at increasing concentrations: 50 (orange), 100 (green), and 200 ¹g /ml
(red). The time point where the first egg was
observed in each of the six chambers in all channels (or conditions) was
monitored after the 3rd larval stage and
extracted using live-imaging then plotted in the top panels (a, b); and
time point of the first observed larva in all
chambers of each condition is plotted in the bottom panels (c, d). All
plots show an average of standard deviation
(S.D.) using One-way ANOVA test followed by a Tukey-Kramer post-hoc test
(non-treated vs. treated conditions),
p-value (\textless{}0.003) is indicated on each panel.
{\label{485956}}%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}

\section*{Discussion}

{\label{351404}}

Despite the recent reports highlighting the potential toxic effects of
photovoltaic devices (\textbf{cite Patel, 2015; Hailegnaw, 2015; Spross,
2014; and Benmessaoud, 2015}), industrial production of
perovskites-based photovoltaic devices is on going with a project of
commercialization in 2018. We showed in this paper the effects of
perovskites on the \emph{Caenorhabditis elegans}. Although nematodes are
invertebrate small animals, they represent the gold standard in
toxicology with their intact and metabolically active digestive,
reproductive, endocrine, sensory and neuromuscular systems.
Unfortunately, mammalian models will continue to be used to predict safe
human exposure levels because no combination of current alternative
assays can replicate the complexity of interacting metabolism,
homeostasis and signaling mechanisms that are present in mammals
(\textbf{Tice et al.,2013}). However, given the high percentage of
commercially available chemicals for which there is little or no
toxicity data available (\textbf{Dix et al., 2007; Judson et al.,
2009}), toxicity screenings that can at least flag for further studies
those chemicals with the most potential for harm are urgently needed. As
an intermediate between in vitro and mammalian testing, combining
various \emph{C. elegans} assays on a single experiment using our
microfluidic device contributes significantly to the field of
toxicology.

The in vivo experimentation presented in this paper strengthens the in
vitro outcomes presented previously (\textbf{CITE benmessaoud etal
2016}), and validates the toxic profile of perovskites, which will
potentially constitute the next generation of solar panels that will
cover our buildings. The eco-toxic effects observed in our study
demonstrate the hazards on small model organisms ( nematode), and are
translated in a decrease of viability, a retard in growth and
development, as well as in an altered fertility. These effects may be
due by an induced mitochondrial stress, as predicted by the RNA-seq data
(\textbf{TO BE PUBLISHED}). We believe a homolog of the transcription
factor p53 in \emph{C. elegans} (CEP-1 )\textbf{(Hoffman et al., 2014;
Schumacher et al., 2001}) may be at the origin of the mitochondrial
stress, which, together with DNA damage, could be responsible of the
adverse effects of perovskites observed on the whole organisms.
Mitochondrial stress and DNA damage may target the hormone ecdysone,
which directs progression through larval stages. (\textbf{Ahn et al.,
2014; Posgai et al., 2011}) Additionally, strong correlation of
mitochondrial or DNA damage and cell cycle or apoptosis have been made
to evidence developmental impacts and even a shortened life
span.(\textbf{Stergiou et al., 2004}) These publications may hold an
explanation of our results, and encourage our research to assess
mitochondrial correlation of mitochondrial or DNA damage and cell cycle
or apoptosis have been made to evidence developmental impacts and even a
shortened life span.199 These publications may hold an explanation of
our results, and encourage our research to assess mitochondrial
imbalance as well as metal traces in \emph{C. elegans}. Considering the
genome conservation between humans and \emph{C. elegans}, (\textbf{Bier
et al., 2005}) and the analysis of the genome expression profile
obtained from human cells using RNA-seq (\textbf{TO BE PUBLISHED}), the
data presented in this paper could predict the potential hazards of the
photovoltaic perovskites on humans. As an outlook of this in vivo study,
our protocols must obviously be improved for a more accurate and
predictable outcome. There is room for debate regarding dose, time lapse
and pathway of exposure to perovskites. These tests should be performed
on other small organisms such as Drosophila melanogaster or Danio rerio,
and can be even pursued to verify whether generations n2 and n3 still
suffer the exposure of their grand-parents, and determine when the
observed toxic effects stop inter-generation transmission. For \emph{C.
elegans}, because of a lack of space after one week \emph{(Figure?)},
worms should be transferred into petri dishes and evaluation of toxicity
should be continued without adding extra perovskites.

\section*{Conclusion}

{\label{286567}}

In summary, the marked changes observed in \emph{C. elegans}' viability,
growth, development and fertility; reinforce our previous results
obtained in vitro(Iness R. Benmessaoud et al., 2016), and clearly
demonstrate the toxic impact of the photovoltaic perovskites on the
environment. Furthermore, the microfluidic device used in our study
simplified the screening of worms and allowed for analyzing many
parameters including development, lifespan and fertility in a single
assay. The device is very useful in the field of toxicology when using
\emph{C. elegans} as a model, and could be employed in other disciplines
such as neurodegeneration, immunology and pharmacology.

\section*{Acknowledgments}

{\label{810619}}

The work was performed in the frame of the ERC Advanced Grant
``Picoprop''.

\section*{Materials and methods}

{\label{855239}}

\subsection*{Culturing and treatment of the worm
strain}

{\label{787724}}

\emph{Caenorhabditis elegans} wild type Bristol N2 strain was cultured
at 20 °C on nematode growth media (NGM) agar plates seeded with the
Escherichia coli strain OP50. For microfluidic experiments, the E. coli
strain HT115 was suspended in S-medium at a concentration of 1.4\selectlanguage{english} x\selectlanguage{english} 109
cells/ml. Worms were exposed to perovskite compounds from L3 stage the
day of the experiment. The injected media and perovskite solutions were
freshly prepared the day of the experiment as follows. MAPbI3 and MASnI3
were suspended in S-medium of 50 mg/ml. After filtration at 0.22 um,
this stock solution was diluted in bacteria-containing S-medium to 50,
100 and 200 ug/ml.

\subsection*{Microfluidics experiments}

{\label{772622}}

The microfluidics device fabricated and tested by Cornaglia et
al.(Cornaglia et al., 2016) contains 8 separated channels and allows for
testing 8 biological conditions during one experiment. Each channel is
subdivided into 6 connected chambers offering the possibility for
biological replicates. The whole setup was placed within an inverted
microscope (Axio Observer, Zeiss) equipped with two illumination
systems: (i) a precisExcite High-Power LED illumination system
(Visitron, Puchheim, Germany) for brightfield imaging. The microscope
had a motorized xy-stage and the automated imaging process was
controlled using VisiView Premier Image acquisition software (Visitron,
Puchheim, Germany). Images were acquired every 20 minutes during one
week through a Hamamatsu Orca-ER CCD camera (Hamamatsu, Solothurn,
Switzerland).

\subsection*{Data analysis and
statistics}

{\label{738075}}

The recorded images allowed for survival studies, growth measurements,
and fertility assessment. Image processing was performed using Fiji
software(Schindelin et al., 2012) (version 2.0.0-rc-15/1.49k). For
Survival, we plotted the time point where we observed a dead worm in
each condition. Worm areas were measured by processing time-lapse
brightfield pictures as follows;

Each frame was first converted to a binary image by applying a threshold
to the full stack of time-lapse images and transforming it into a set of
binary masks. Each stack of masks was then analysed using the ``particle
analysis'' Fiji plugin, which allows directly extracting area values for
each picture in the stack. For fertility assessment, we tracked the
sequence where the first egg was laid, and then we probed the time point
where the first larva emerged. The experiment was repeated 3 times
independently using new batches of media, perovskites and by inverting
the position of the conditions in the device channels. We analysed each
dataset alone by averaging the sextuplicate of each condition and
measuring the standard deviation (S.D.) using One-way ANOVA test
followed by a Tukey-Kramer post-hoc test (non-treated vs. treated
conditions).

\selectlanguage{english}
\FloatBarrier
\nocite{*}

\bibliographystyle{plainnat}
\bibliography{bibliography/converted_to_latex.bib%
}

\end{document}

