Received: 15 April 2019 Revised: 13 May 2019 Accepted: 14 May 2019
DOI: 10.1002/jat.3833
R E S E A R CH AR T I C L E
High‐throughput COPAS assay for screening of developmentaland reproductive toxicity of nanoparticles using the nematodeCaenorhabditis elegans
MinA Kim | Jaeseong Jeong | Heejin Kim | Jinhee Choi
School of Environmental Engineering,
University of Seoul, Dongdaemun‐gu, Seoul,South Korea
Correspondence
Jinhee Choi, School of Environmental
Engineering, University of Seoul, 163
Seoulsiripdae‐ro, Dongdaemun‐gu, Seoul02504, South Korea.
Email: [email protected]
Funding information
Mid‐Career Researcher Program, Grant/Award
Number: NRF‐2017R1A2B3002242;Nano·Material Technology Development Pro-
gram, Grant/Award Number: NRF‐2017M3A7B6052457
MinA Kim and Jaeseong Jeong contributed equally to this
J Appl Toxicol. 2019;1–10.
Abstract
With the rapid advancement and numerous applications of engineered nanomaterials
(ENMs) in science and technology, their effects on animal health, environment and
safety should be considered carefully. However, quick assessment of their effects
on developmental and reproductive health and an understanding of how they cause
such adverse toxic effects remain challenging, because of the fast‐growing number
of ENMs and the limitations of the different toxicity assays currently in use as well
as lack of suitable animal model systems. In this study, we performed a high‐
throughput complex object parametric analyzer and sorter (COPAS) assay for
assessing the developmental and reproductive toxicity of ENMs using Caenorhabditis
elegans and provide descriptions of the data and their subsequent analysis. The
results showed significant reproductive and developmental toxicity potential of dif-
ferent ENMs. We assessed the usefulness of this method in terms of error‐free data,
user‐friendliness and results being consistent with those of visual, molecular and cel-
lular studies. Moreover, the COPAS Biosort system could be used on a larger scale to
screen thousands of chemicals, drugs, pharmaceuticals and ENMs. This study also
indicates that the COPAS‐based high‐throughput screening system is highly reliable
for the assessment of toxicity and health risks of NMs.
KEYWORDS
C. elegans, developmental toxicity, engineered nanomaterials, high‐throughput screening,
reproductive toxicity
1 | INTRODUCTION
The booming nanotechnology industry has raised concerns among the
public about the environmental health and safety of engineered
nanomaterials (ENMs). Worldwide research on ENMs and their com-
mercial success have led to a rapid increase in the number of applica-
tions; however, limited understanding of aspects related to the
environmental health and safety of ENMs remains a major obstacle
for the future progress of nanotechnology (Qu, Brame, Li, & Alvarez,
paper.
wileyonlinelibrary.com/jour
2013). Currently, a key challenge is the very limited and often conflict-
ing data available in the published literature and the fact that different
ENMs are physicochemically heterogeneous makes it difficult to gen-
eralize the health risks associated with exposure to ENMs (Brohi et al.,
2017). Therefore, there is an urgent need to have a clear understand-
ing of the toxic effects of ENMs and to elucidate the mechanisms
involved in their toxicity. In this regard, high‐throughput screening
(HTS) techniques aimed at accurately predicting and assessing the
toxicity of ENMs are definitely and urgently needed.
With the growing numbers of ENMs, there is a huge demand from
the scientific community as well as from legislative institutions for
© 2019 John Wiley & Sons, Ltd.nal/jat 1
2 KIM ET AL.
methods to test the safety of ENMs accurately and rapidly. HTS is
considered a leading paradigm for tackling this challenge (Damoiseaux
et al., 2011). It allows the examination of a large number of samples,
concentrations, materials and experimental variations in a time‐ and
cost‐effective manner (Collins et al., 2017). Several HTS methods have
been used for testing of ENM toxicity (Barrick, Châtel, Bruneau, &
Mouneyrac, 2017; Jung et al., 2015; Nel et al., 2013). One of the bet-
ter solutions for HTS of large numbers of ENMs is the complex object
parametric analyzer and sorter (COPAS) Biosort system (Union
Biometrica) (Pulak, 2006). The COPAS Biosort facilitates the rapid,
accurate and efficient analysis, dispensing and/or sorting of a large
number of nematodes by measuring their axial length (time of flight,
TOF) as well as their size and internal structure (extinction, EXT).
In recent years, reproductive and developmental toxicity has increas-
ingly been recognized as an important component of the overall toxicity
of ENMs. In fact, several reports show that nanoparticles (NPs) can pass
through biological membranes. This has been a cause of great concern
with regard to the possible reproductive and developmental toxicity of
ENMs (Brooking, Davis, & Illum, 2001; Ema, Kobayashi, Naya, Hanai, &
Nakanishi, 2010; Williams, 2018). In this regard, Caenorhabditis elegans,
a popular model organism for reproductive and developmental biology
research is now being recognized as an attractive invertebrate model
for high‐throughput toxicological studies (Hunt, 2017). C. elegans
growth assay is currently being used to screen theToxCast Phase I and
II libraries of the US Environmental Protection Agency (EPA), which
comprise 1011 chemicals (Judson et al., 2010; Knudsen et al., 2009). In
addition, the COPAS‐based C. elegans HTS assay has been used in the
screening of neurotoxicants (Boyd, Smith, Kissling, & Freedman, 2010)
and for assessing the reproductive toxicity of drugs and environmental
chemicals (Maurer, Ryde, Yang, & Meyer, 2015), effects of environmen-
tal toxicants on germline cells (Shin, Cuenca, Karthikraj, Kannan, &
Colaiácovo, 2019), larval growth and development (Boyd, et al., 2010),
and chemical disruption of germline function (Allard, Kleinstreuer, Knud-
sen, & Colaiácovo, 2013). It has also been used for studying the mito-
chondrial function and morphology (Daniele et al., 2017) and
developmental toxicity in axenic liquid cultures of C. elegans (Sprando,
Olejnik, Cinar, & Ferguson, 2009), zebrafish, rats and rabbits (Boyd
et al., 2016). C. elegans is also widely used in nanotoxicity assessment
because it has many advantages that are useful for assessing
nanotoxicity, such as transparent body and short reproductive life cycle
(Choi et al., 2014; Wang, 2018;Wu, Xu, Liang, & Tang, 2019). However,
reports on the reproductive and developmental toxicity potential of
ENMs have been scarce and inconclusive. Therefore, in the present
study, we performed COPAS Biosort HTS for the reproductive and
developmental toxicity of selected ENMs in C. elegans.
2 | MATERIALS AND METHODS
2.1 | Maintenance of C. elegans
C. elegans strains were cultured at 20°C on nematode growth medium
plates seeded with Escherichia coli strain OP50, as previously
described (Brenner, 1974). The N2 Bristol strain was used as the
wild‐type strain, obtained from the Caenorhabditis Genetics Center
(CGC). We synchronized a number of gravid adult C. elegans with
10% hypochlorite solution to isolate the eggs (Stiernagle, 2006), which
were used in all the experiments.
2.2 | Selection and preparation of chemicals
To predict the toxic level in the screening assay using C. elegans by
comparing with mammalian in vivo data associated with reproduction
present in the Toxicological Reference Database (ToxRefDB, http://
www.epa.gov/ncct/toxrefdb), we selected 16 chemicals for which
in vivo multigenerational reproductive toxicity data are available.
Descriptions of the selected chemicals and their mammalian in vivo
endpoint data are presented in Table 1. The chemicals were divided
based on their lowest‐observed‐adverse‐effect level in a multigenera-
tional study (MGLEL) (Martin, Judson, Reif, Kavlock, & Dix, 2009).
Eight chemicals with one or more endpoints in MG‐LEL were defined
as positive reproductive toxicants, and eight chemicals that had no
MG‐LEL endpoints ≤500 mg/kg/day were considered as negative
controls. All the chemicals were purchased from Sigma‐Aldrich
(Korea), and dissolved in dimethyl sulfoxide. The final concentrations
of dimethyl sulfoxide and chemicals were 0.1% and 100 μM, respec-
tively. Testing solution of chemicals was prepared in K‐media
(0.032 M KCl and 0.051 M NaCl) containing OP50 as food for C.
elegans.
2.3 | Nanoparticles and physicochemicalcharacterization
Seven types of commercially available NPs were used. These
included the following: two types of amorphous silica NPs (aSiNPs),
namely aSiNPs‐189 (18.3 nm), which were obtained from the
NANoREG project (http://www.nanoreg.eu), and aSiNPs‐116, which
were purchased from Sigma‐Aldrich; two types each of TiO2NPs
(6 nm and 24.7 nm), CeO2NPs (33 nm) and AgNPs (16.7 nm), dis-
persed in deionized water containing 7% ammonium nitrate as a sta-
bilizing agent and 8% emulsifiers, which were provided by
NANoREG; AgNPs (<150 nm), which were purchased from Sigma‐
Aldrich. The stock solutions were prepared in distilled water at a
concentration of 2560 mg/L by sonication and were immediately
used as test materials. K‐medium was used as the exposure media
for all NPs, except AgNPs (<150 nm), for which EPA moderately hard
water (NaHCO3 96 mg/L, CaSO4·2H2O 60 mg/L, MgSO4 60 mg/L
and KCl 4 mg/L) was used. NPs in the exposure media were charac-
terized by photon dynamic light scattering (DLS, DLS‐7000; Otsuka
Electronics) and transmission electron microscopy (TEM; LIBRA 120;
Carl Zeiss) as described previously (Chatterjee, Jeong, Yoon, Kim, &
Choi, 2018). The exposure concentrations ranged from 0.0625 to
100 mg/L.
TABLE 1 List of negative and positive control chemicals with multigenerational reproductive toxicity data from the ToxRefDB
CASnumber Chemical Usage
Reproductive toxicity endpoint (NOAEL, mg/kg/day) (rat data from ToxRefDB‐US EPA)
Fertility ImplantationsLittersize Ovary
Reproductiveoutcome
Reproductiveperformance
Viabilitypostnatalday 4
Negative control (8)
101200‐48‐0 Tribenuron‐methyl Pesticide 0 0 0 0 0 0 0
34014‐18‐1 Tebuthiuron Herbicide 0 0 0 0 0 0 0
87820‐88‐0 Tralkoxydim Herbicide 0 0 0 0 0 0 0
51‐03‐6 Piperonyl butoxide Pesticide 0 0 0 0 0 0 0
181274‐15‐7 Propoxycarbazone‐sodium Herbicide 0 0 0 0 0 0 0
21087‐64‐9 Metribuzin Herbicide 0 0 0 0 0 0 0
23103‐98‐2 Pirimicarb Insecticide 0 0 0 0 0 0 0
22781‐23‐3 Bendiocarb Insecticide 0 0 0 0 0 0 0
Positive control (8)
116714‐46‐6 Novaluron Insecticide 0 0 895 0 895 0 0
741‐58‐2 Bensulide Herbicide 68.2 0 0 0 0 68.2 86.5
12427‐38‐2 Maneb Fungicide 0 0 0 25.1 0 106 0
333‐41‐5 Diazinon Insecticide 35.2 0 35.2 0 35.15 35.15 35.2
2310‐17‐0 Phosalone Insecticide 0 0 29.4 0 29.4 0 29.4
115‐32‐2 Dicofol Pesticide 0 0 0 2.4 23.2 0 11.2
68694‐11‐1 Triflumizole Fungicide 0 0 8.5 0 8.5 1.5 8.5
60168‐88‐9 Fenarimol Fungicide 2.5 0 0 0 1.2 2.5 0
NOAEL, no‐observed‐adverse‐effect level; ToxRefDB, Toxicological Reference Database.
KIM ET AL. 3
2.4 | High‐throughput screening with the COPASBiosort analysis
Development of C. elegans from L1 larvae to adult stage takes approx-
imately 72 hours at 20°C. This time period was chosen for growth
assays to allow for maximum development, while avoiding the produc-
tion of offspring at later times. To determine the potential toxicity of
the chemicals on larval development, 20 synchronized L1 nematodes
were distributed by COPAS Biosort to each well of a 96‐well plate.
After 24, 48 and 96 hours of incubations, the sizes of worms in each
well were recorded using COPAS Biosort by aspirating the exposure
medium from the sample wells and measuring TOF, EXT and fre-
quency for individual C. elegans. The sizes of nematodes were linked
to specific C. elegans larval stages (Boyd et al., 2009). In the full repro-
duction assay, single young adult individuals were placed in a 96‐well
plate and incubated at 20°C for 72 hours. After the exposure, the
number of offspring from one young‐adult were counted using
COPAS Biosort. All samples were prepared with three replicates in a
96‐well plate.
2.5 | Data and statistical analysis
To exclude measurements of objects other than nematodes (NPs
aggregates), values were deleted if they deviated by one standard
deviation at that EXT value from the TOF vs. EXT plots for an entire
data set in all concentrations. The significance of differences
among/between treatments was determined using one‐way analysis
of variance followed by post‐hoc tests (Tukey, P < .05) in SPSS
12.0KO (SPSS Inc.). We used MATLAB (R2017a) to carry out data
plotting with P < .05 considered statistically significant.
3 | RESULTS
3.1 | Optimization of COPAS analysis
The COPAS Biosort EXT measurement reflects the amount of light
absorbed as an object passes through the laser and has been success-
fully used as an indicator of size of C. elegans (Pulak, 2006). Before
toxicity testing, we optimized the assessment of the developmental
stage of C. elegans using COPAS‐SELECT. Age‐synchronized L1 larvae
were monitored at 0, 24, 48 and 72 hours, by measuring EXT, TOF and
frequency. To simplify the identification of the growth cycles of the
nematode, a grid with log(EXT) and a histogram of all observations
was plotted (Figure 1). Over the duration of the growth assay, the
nematodes grew at an exponential rate. The growth in the control
group varied from day‐to‐day and at different stages of nematodes
over the 72‐hour observation period.
FIGURE 1 Growth of Caenorhabditis elegans from the L1 to adultstage. Untreated C. elegans were incubated at 20°C and sampled at0, 24, 48 and 72 h. Histogram shows optical density (log(EXT)) versusfrequency distributions. At 72 h, adult nematodes (high EXT) and theiroffspring (low EXT) were observed. EXT, extinction
FIGURE 2 Observed frequency distributions of log(EXT) of Caenorhabddevelopment and initial reproduction analysis, age‐synchronized L1 larvaewere measured every 24 h. A, Negative control group chemicals. B, Positiv
4 KIM ET AL.
3.2 | Validation of COPAS‐based reproductive anddevelopmental toxicity test in C. elegans
COPAS‐based development and reproduction screening tests for C.
eleganswere validated using eight chemicals, which are verified for their
reproductive and developmental toxicity potential in mammalian
models, as positive controls, and eight chemicals with no such indica-
tions as negative controls (Figure 2). The chemicals in the positive con-
trol group included dicofol, bensulide, phosalone, fenarimol, diazinon,
maneb, triflumizole and navaluron, and those in the negative control
group included tebuthiuron, metribuzin, bendiocarb, tribenuron‐
methyl, piperonyl butoxide, tralkoxydim, propoxycarbazone sodium
and pirimicarb. In the results of developmental tests for the negative
control group, the log(EXT) value at which the peak appeared increased
with time although it varied for different chemicals (Figure 2A). It was
also found that the 72‐hour data had a high frequency peak at low
log(EXT) value and therefore the negative control had little effect on
the early reproductive potential. On the other hand, in the results for
the positive control group, it was difficult to identify the peak or the
increase over time clearly (Figure 2B). In addition, unlike in the negative
control group, no peak corresponding to L1 was observed at 72 hours,
itis elegans exposed to negative and positive control chemicals. Forwere exposed to each chemical for 96 h and EXT and frequencye control group chemicals. EXT, extinction
KIM ET AL. 5
indicating that the chemicals in the positive control group affected not
only the development but also the early reproductive potential. To con-
firm the reproductive potential by measuring the number of offspring,
age‐synchronized young adults were exposed to each chemical for
72 hours (Figure 3). No reproductive toxicity was observed for any of
the chemicals in the negative control group, and the number of offspring
tended to be lower in the positive control group than in the negative
control group. Severe reproductive toxicity was observed for dicofol,
phosalone, diazinon and triflumizole. We validated the developmental
and reproductive toxicity assay using COPAS Biosort, which directly
counts the number of nematode offspring, and the number of larvae
produced during this time period is an indication of the effect of the tox-
icant on nematode fecundity/reproductive potentials. Overall, our
results indicate that the COPAS Biosort system is a good screening tool
for the assessment of reproductive and developmental toxicity in mam-
mals using C. elegans as a model system.
3.3 | Physicochemical characterization of engineerednanomaterials
The DLS and TEM analyses were performed for physicochemical char-
acterization of ENMs in the C. elegans exposure media. The hydrody-
namic diameters and electrophoretic mobility of ENMs are
summarized in Table 2. The TEM images show that the sizes of the
NMs in deionized water were close to their labeled monomeric sizes.
In the tested media, the measured hydrodynamic diameter ranged from
126 to 379 nm, with the zeta potential ranging from −47.5 to 33 mV.
3.4 | Developmental and reproductive toxicityassays of engineered nanomaterials using COPASanalysis
We tested both developmental and early reproductive toxicity assays
as HTS tools to estimate the potential toxicities of the ENMs using
FIGURE 3 COPAS measurement of thecomplete reproduction process ofCaenorhabditis elegans exposed to positive andnegative control chemicals. For completereproduction analysis, age‐synchronizedyoung adults were exposed to each chemicalfor 72 h and frequency was measured(**P < .01)
COPAS. The 72‐hour developmental dynamics was first screened
using AgNPs, SiO2NP, TiO2NPs and CeO2NPs in C. elegans
(Figure 4). Results show that significant early reproductive toxicity
was observed in the AgNP‐treated group, whereas no reproductive
toxicity was observed in the CeO2NP‐, TiO2NP‐ and SiO2NP‐treated
groups. However, in the TiO2NP‐treated group, an abnormally high
peak was observed between ln (EXT) 2 and 3 at 72 hours, which
seemed to be due to the presence of aggregated NPs that were aspi-
rated along with the nematodes, and resulted in high frequency values.
In this study, the optimized COPAS screening method for soluble com-
pounds were applied for NPs. However, unlike for the chemicals in the
positive and negative control groups, NPs are not soluble and were in
dispersed condition in the culture media. In COPAS analysis, dispersed
NPs in the media create interference with reproduction (frequency)
data and this is considered as one of the limitations of COPAS for
HTS of NPs.
3.5 | Assessment of dose‐ and size‐specific responseon the development of C. elegans by COPAS analysis
Using the COPAS analysis, we examined the developmental toxicity of
four NMs at different concentrations. Developmental toxicity of NPs
was observed at 48 hours for a broad range of exposure concentra-
tions from 0.0625 to 100 mg/L (Figure 5). No developmental toxicity
was observed for any of the four NMs up to a concentration of
0.5 mg/L. However, concentration‐dependent toxicity was observed
for AgNPs at concentrations higher than 1 mg/L. Similarly, SiO2NP‐
and TiO2NP‐treated groups showed significant developmental toxicity
at concentrations higher than 10 mg/L, whereas, 25 mg/L CeO2NPs
caused toxicity in C. elegans.
Additionally, we studied whether the 48‐hour TOF values for the
developmental toxicity index could be applied to test the size‐
dependent toxicity of NPs by using different‐sized NPs (16.7 and
<150 nm for AgNPs; 16.2 and 200 nm for SiO2NPs; 6 and 24.7 nm
TABLE
2List
ofen
gine
eringna
nomaterialsan
dtheirph
ysicoch
emical
prope
rtiesin
thetest
med
ia
AgN
Ps
AgN
Ps
aSiN
P‐189
aSiN
P‐116
TiO
2NPs
TiO
2NPs
CeO
2NPs
Polymorph
Metallic
Metallic
Amorpho
usAmorpho
usAna
tase
Rutile
–
Diameter
(nm)
16.7
<150
18.3
20–5
06
24.7
33
Transmissionelectron
microscopicim
age
Zetapo
tential(m
V)
–11±3
N/A
−47.5
−10.02
N/A
pH
<4+30mV;
pH
>10‐40mV
33±2
Hyd
rody
namic
diam
eter
(nm)
166.63±2.87
333.10±11.19
220.78±4.69
202.6
379.30±7.67
126.83±0.85
170.10±3.37
N/A
,notap
plicab
le;NPs,na
nopa
rticles.
6 KIM ET AL.
for TiO2NPs) (Figure 6). Distinct size‐dependent toxicity was observed
in nematodes treated with AgNPs and SiO2NPs at all concentrations
higher than 10 mg/L. However for TiO2NPs, size‐dependent toxicity
was observed only at the highest concentration tested (i.e.,
100 mg/L), which might be because the size difference of the two
TiO2NPs was not as evident as it was for AgNPs or SiO2NPs. These
results collectively suggest that the COPAS‐based HTS assay could
be applicable for testing the developmental toxicity of NPs, and the
exposure concentration and size‐dependent toxicity can be success-
fully assessed using this HTS method.
4 | DISCUSSION
NMs are being used extensively in a variety of applications at such a
rate that cannot be matched by safety/toxicity assessments. There is
an urgent need for consistent and accurate HTS systems for rapid tox-
icity assessment of emerging NMs (Ban, Zhou, Sun, Mu, & Hu, 2018).
The present study showed that the HTS strategy using C. elegans can
be successfully applied for toxicity assessment of ENMs, as has been
reported in previous studies on NMs and various environmental toxi-
cants (Hunt et al., 2014; Jung et al., 2015; Mashock et al., 2016; Shin
et al., 2019). The developmental and reproductive toxicity assay
described in this study used the COPAS Biosort assay for counting
the number and measuring the size of C. elegans at L1 larval and adult
stages. This period was chosen to coincide with the developmental
stage when the number of germ cells reached its maximum, but before
the embryonic membrane became impermeable (Epstein et al., 1995).
Before testing the toxicity of ENMs, preliminary experiments focused
on optimizing the method using the toxicity potential of different
known reproductive toxicants. An exposure time of 48‐72 hours was
chosen to maintain a relatively low number of offspring. This exposure
time avoided overcrowding in the wells and allowed nematode counts
to remain within the sampling limits of the Biosort (Boyd, et al., 2010).
Exposures to ENMs and toxicants were started at the L1 stage to max-
imize the chance of observing potential chemical effects on reproduc-
tion and development, while avoiding potential effects on the growth
of offspring (Boyd, et al., 2010). Our results showed that C. elegans is
an efficient animal model for HTS of the reproductive and develop-
mental toxicities using COPAS analysis.
Among several HTS assays, the COPAS Biosorters represent
recent advances in HTS analyses of C. elegans (Pulak, 2006). Before
the invention of COPAS, library screening and toxicity assessment of
compounds in model organisms used manual processes and were,
thus, very slow, tedious and simply impractical as they could only be
performed for one organism at a time under a microscope. The COPAS
system can analyze about 1 million C. elegans individuals in 8 hours
(Pulak, 2006). The screening time with the COPAS analysis consists
of 48‐72 hours of exposure and 45 minutes of reading for each 96‐
well plate. Because exposures can be performed simultaneously and
each plate adds only an additional 45 minutes of reading time, a library
of 1000 compounds can be screened in triplicate in only 4 days. We
used COPAS for assessing the developmental and reproductive
FIGURE 4 COPAS measurement of the development and initial reproduction of Caenorhabditis elegans exposed to engineered nanomaterials.For development and initial reproduction analysis, age‐synchronized L1 larvae were exposed to each chemical for 96 h and EXT and frequencywere measured every 24 h. EXT, extinction; NPs, nanoparticles
FIGURE 5 COPAS measurement of the development of Caenorhabditis elegans exposed to engineered nanomaterials at nine concentrations. Fordevelopmental analysis, age‐synchronized L1 larvae were exposed to each nanoparticle at different doses for 48 h (*P < .05; **P < .01). NPs,nanoparticles; TOF, time of flight
KIM ET AL. 7
toxicity of AgNPs, SiO2NPs, TiO2NPs and CeO2NPs, and our results
prove that it is an efficient and reliable technology for fast screening
of ENMs or other toxicants that alter the reproduction and growth
of C. elegans. We focused on known reproductive toxicants to address
the gap in our current ability to assess the potential toxicity of NPs.
However, the assay described here is also applicable to other
FIGURE 6 A, AgNPs. B, SiO2NPs. C, TiO2NPs. COPAS measurement of the development of Caenorhabditis elegans exposed to AgNPs, SiO2NPsand TiO2NPs. For development analysis, age‐synchronized L1 larvae were exposed to each nanoparticle at different doses for 48 h. NPs,nanoparticles; TOF, time of flight
8 KIM ET AL.
chemicals and ENMs for the safety assessment of NPs and small mol-
ecule assays by analyzing the reproductive toxicity in animals.
In recent years, the incorporation of COPAS assays has been
attempted in toxicological screening studies (Pulak, 2006). The results
of these studies showed that COPAS is a suitable and reliable system
for assaying the changes in mitochondrial morphology and activity
(Daniele et al., 2017; Hunt, Olejnik, Bailey, Vaught, & Sprando, 2018),
heavy metal toxicity (Hunt, Olejnik, & Sprando, 2012), toxicity assess-
ment of neurotoxicants (Boyd, et al., 2010), and environmental toxi-
cants (Boyd, McBride, Rice, Snyder, & Freedman, (2010), and enables
the screening of drug libraries (Cho, Behnam Azad, Luyt, & Lewis,
2013), metal NMs (Jeong et al., 2018; Kim et al., 2017) and phenotype
profiling of C. elegans in different chemical environments (Gao et al.,
2018). However, COPAS has several limitations. One of the limitations
of using COPAS for measurements in C. elegans is the possibility of
extraneous materials, such as bacteria, discarded cuticles or chemical
precipitates being aspirated along with the nematodes (Smith et al.,
2009). Another limitation of the COPAS assay is its susceptibility to
the effects of ambient temperature and humidity, and the introduction
of artifacts from improper gating or handling (Collins et al., 2017; Hunt
et al., 2012). The instrumentation for the COPAS assay is also very
expensive, and is not widely available in many laboratories, which limits
its use for toxicity testing with C. elegans (Fernandez et al., 2012). In
addition, the COPAS system only provides a limited range of data on
features such as length and width of worm, and fluorescence intensity.
Besides these minor limitations, COPAS provides a low‐cost, high‐
speed and strong predictive value, and fulfills the requirement for the
first‐pass assessment of toxicity. Furthermore, it offers insights into
the mechanism of reproductive and developmental toxicity in animals.
5 | CONCLUSIONS AND FUTUREAPPLICATIONS
We showed that the COPAS HTS assay is extremely powerful and
suitable to determine the developmental and reproductive toxicity of
ENMs using C. elegans. We have also provided an unbiased discussion
of the major technical advantages and limitations of COPAS HTS for
evaluating the reproductive and developmental potential of ENMs.
With the increasing diversity of engineered NMs being considered
for large‐scale use, this COPAS HTS assay would facilitate rapid
screening of the toxicity of NMs, allowing risk assessment of NPs in
a timely manner. In addition, with the recent technical advances in C.
elegans handling, culture and phenotyping, it is now increasingly possi-
ble to conduct mass screens in whole, intact organisms for develop-
mental toxicity risk assessment assays. Therefore, the COPAS HTS
assay using C. elegans can be applied at a larger scale for screening
thousands of chemicals, drugs, pharmaceuticals and ENMs. However,
our results clearly demonstrate several limitations of using COPAS
HTS that would need to be technically improved in the future for bet-
ter HTS assays. There is a need for modification of the COPAS assay
with better computational tools, which will provide an opportunity
for the creation of faster and error‐free toxicity screening on a wider
scale using C. elegans. It is our hope that, in future, COPAS technology
will be widely used for ecotoxicity and health risk assessment of
emerging pollutants, such as microplastics and nanoplastics.
CONFLICTS OF INTERESTS
The authors have no conflicts of interest to report.
ACKNOWLEDGMENTS
This work was supported by the Mid‐Career Researcher Program
(NRF‐2017R1A2B3002242) and Nano·Material Technology Develop-
ment Program (NRF‐2017M3A7B6052457) through the National
Research Foundation of Korea (NRF), funded by the Ministry of
Science and ICT.
ORCID
Jinhee Choi https://orcid.org/0000-0003-3393-7505
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How to cite this article: Kim MA, Jeong J, Kim H, Choi J.
High‐throughput COPAS assay for screening of developmental
and reproductive toxicity of nanoparticles using the nematode
Caenorhabditis elegans. J Appl Toxicol. 2019;1–10. https://doi.
org/10.1002/jat.3833
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