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: jinhchoi@uos.ac.kr

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

REFERENCES

Allard, P., Kleinstreuer, N. C., Knudsen, T. B., & Colaiácovo, M. P. (2013). A

C. elegans screening platform for the rapid assessment of chemical dis-

ruption of germline function. Environmental Health Perspectives, 121(6),

717–724. https://doi.org/10.1289/ehp.1206301

Ban, Z., Zhou, Q., Sun, A., Mu, L., & Hu, X. (2018). Screening priority factors

determining and predicting the reproductive toxicity of various

KIM ET AL. 9

nanoparticles. Environmental Science and Technology, 52(17),

9666–9676. https://doi.org/10.1021/acs.est.8b02757

Barrick, A., Châtel, A., Bruneau, M., & Mouneyrac, C. (2017). The role of

high‐throughput screening in ecotoxicology and engineered

nanomaterials. Environmental Toxicology and Chemistry, 36(7),

1704–1714. https://doi.org/10.1002/etc.3811

Boyd, W. A., McBride, S. J., Rice, J. R., Snyder, D. W., & Freedman, J. H.

(2010). A high‐throughput method for assessing chemical toxicity

using a Caenorhabditis elegans reproduction assay. Toxicology and

Applied Pharmacology, 245(2), 153–159. https://doi.org/10.1016/j.

taap.2010.02.014

Boyd,W. A., Smith, M. V., Co, C. A., Pirone, J. R., Rice, J. R., Shockley, K. R., &

Freedman, J. H. (2016). Developmental effects of theToxCast™ phase I

and phase II chemicals in caenorhabditis elegans and corresponding

responses in zebrafish, rats, and rabbits. Environmental Health Perspec-

tives, 124(5), 586–593. https://doi.org/10.1289/ehp.1409645

Boyd, W. A., Smith, M. V., Kissling, G. E., & Freedman, J. H. (2010).

Medium‐ and high‐throughput screening of neurotoxicants using C.

elegans. Neurotoxicology and Teratology, 32(1), 68–73. https://doi.org/10.1016/j.ntt.2008.12.004

Boyd, W. A., Smith, M. V., Kissling, G. E., Rice, J. R., Snyder, D. W., Portier,

C. J., & Freedman, J. H. (2009). Application of a mathematical model to

describe the effects of chlorpyrifos on Caenorhabditis elegans develop-

ment. PLoS ONE, 4(9), 1–10. https://doi.org/10.1371/journal.

pone.0007024

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics, 77(1),

71–94. Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/

4366476

Brohi, R. D., Wang, L., Talpur, H. S., Wu, D., Khan, F. A., Bhattarai, D., …Huo, L. J. (2017). Toxicity of nanoparticles on the reproductive system

in animal models: A review. Frontiers in Pharmacology, 8(SEP), 1–22.https://doi.org/10.3389/fphar.2017.00606

Brooking, J., Davis, S. S., & Illum, L. (2001). Transport of nanoparticles

across the rat nasal mucosa. Journal of Drug Targeting, 9(4), 267–279.Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/11697030,

https://doi.org/10.3109/10611860108997935

Chatterjee, N., Jeong, J., Yoon, D., Kim, S., & Choi, J. (2018). Global meta-

bolomics approach in in vitro and in vivo models reveals hepatic

glutathione depletion induced by amorphous silica nanoparticles.

Chemico‐Biological Interactions, 293(July), 100–106. https://doi.org/

10.1016/j.cbi.2018.07.013

Cho, C. F., Behnam Azad, B., Luyt, L. G., & Lewis, J. D. (2013). High‐throughput screening of one‐bead‐one‐compound peptide libraries

using intact cells. ACS Combinatorial Science, 15(8), 393–400. https://doi.org/10.1021/co4000584

Choi, J., Tsyusko, O. V., Unrine, J. M., Chatterjee, N., Ahn, J. M., Yang, X., …Meyer, J. N. (2014). A micro‐sized model for the in vivo study of nano-

particle toxicity: What has Caenorhabditis elegans taught us?

Environmental Chemistry, 11(3), 227–246. https://doi.org/10.1071/

EN13187

Collins, A. R., Annangi, B., Rubio, L., Marcos, R., Dorn, M., Merker, C., …Dusinska, M. (2017). High throughput toxicity screening and intracellu-

lar detection of nanomaterials. Wiley Interdisciplinary Reviews:

Nanomedicine and Nanobiotechnology, 9, e1413. https://doi.org/

10.1002/wnan.1413

Damoiseaux, R., George, S., Li, M., Pokhrel, S., Ji, Z., France, B., … Nel, A.

(2011). No time to lose—High throughput screening to assess

nanomaterial safety. Nanoscale, 3(4), 1345–1360. https://doi.org/

10.1039/c0nr00618a

Daniele, J. R., Esping, D. J., Garcia, G., Parsons, L. S., Arriaga, E. A., & Dillin,

A. (2017). High‐throughput characterization of region‐specific

mitochondrial function and morphology. Scientific Reports, 7(1), 1–16.https://doi.org/10.1038/s41598‐017‐05152‐z

Ema, M., Kobayashi, N., Naya, M., Hanai, S., & Nakanishi, J. (2010).

Reproductive and developmental toxicity studies of manufactured

nanomaterials. Reproductive Toxicology, 30(3), 343–352. https://doi.

org/10.1016/j.reprotox.2010.06.002

Epstein, H. F., Shakes, D. C., & American Society for Cell Biology (1995).

Caenorhabditis elegans: Modern biological analysis of an organism.

Cambridge, Massachusetts: Academic Press.

Fernandez, A. G., Bargmann, B. O. R., Mis, E. K., Edgley, M. L., Birnbaum, K.

D., & Piano, F. (2012). High‐throughput fluorescence‐based isolation of

live C. elegans larvae. Nature Protocols, 7(8), 1502–1510. https://doi.org/10.1038/nprot.2012.084

Gao, S., Chen, W., Zeng, Y., Jing, H., Zhang, N., Flavel, M., … Li, G. (2018).

Classification and prediction of toxicity of chemicals using an

automated phenotypic profiling of Caenorhabditis elegans. BMC

Pharmacology and Toxicology, 19(1), 1–11. https://doi.org/10.1186/

s40360‐018‐0208‐3

Hunt, P. R. (2017). The C. elegans model in toxicity testing. Journal of

Applied Toxicology, 37(1), 50–59. https://doi.org/10.1002/jat.3357

Hunt, P. R., Keltner, Z., Gao, X., Oldenburg, S. J., Bushana, P., Olejnik, N., &

Sprando, R. L. (2014). Bioactivity of nanosilver in Caenorhabditis

elegans: Effects of size, coat, and shape. Toxicology Reports, 1,

923–944. https://doi.org/10.1016/j.toxrep.2014.10.020

Hunt, P. R., Olejnik, N., Bailey, K. D., Vaught, C. A., & Sprando, R. L. (2018).

C. elegans development and activity test detects mammalian develop-

mental neurotoxins. Food and Chemical Toxicology, 121(September),

583–592. https://doi.org/10.1016/j.fct.2018.09.061

Hunt, P. R., Olejnik, N., & Sprando, R. L. (2012). Toxicity ranking of heavy

metals with screening method using adult Caenorhabditis elegans and

propidium iodide replicates toxicity ranking in rat. Food and

Chemical Toxicology, 50(9), 3280–3290. https://doi.org/10.1016/j.

fct.2012.06.051

Jeong, J., Song, T., Chatterjee, N., Choi, I., Cha, Y. K., & Choi, J. (2018).

Developing adverse outcome pathways on silver nanoparticle‐induced reproductive toxicity via oxidative stress in the nematode

Caenorhabditis elegans using a Bayesian network model.

Nanotoxicology, 12(10), 1182–1197. https://doi.org/10.1080/

17435390.2018.1529835

Judson, R. S., Houck, K. A., Kavlock, R. J., Knudsen, T. B., Martin, M. T.,

Mortensen, H. M., … Dix, D. J. (2010). In vitro screening of environ-

mental chemicals for targeted testing prioritization: The toxcast

project. Environmental Health Perspectives, 118(4), 485–492. https://doi.org/10.1289/ehp.0901392

Jung, S. K., Qu, X., Aleman‐Meza, B., Wang, T., Riepe, C., Liu, Z., … Zhong,

W. (2015). Multi‐endpoint, high‐throughput study of nanomaterial tox-

icity in Caenorhabditis elegans. Environmental Science and Technology,

49(4), 2477–2485. https://doi.org/10.1021/es5056462

Kim, J. H., Lee, S. H., Cha, Y. J., Hong, S. J., Chung, S. K., Park, T. H., & Choi,

S. S. (2017). C. elegans‐on‐a‐chip for in situ and in vivo Ag nanoparti-

cles' uptake and toxicity assay. Scientific Reports, 7(October 2016),

1–11. https://doi.org/10.1038/srep40225

Knudsen, T. B., Martin, M. T., Kavlock, R. J., Judson, R. S., Dix, D. J., &

Singh, A. V. (2009). Profiling the activity of environmental chemicals

in prenatal developmental toxicity studies using the US EPA's

ToxRefDB. Reproductive Toxicology, 28(2), 209–219. https://doi.org/10.1016/j.reprotox.2009.03.016

Martin, M. T., Judson, R. S., Reif, D. M., Kavlock, R. J., & Dix, D. J. (2009).

Profiling chemicals based on chronic toxicity results from the US EPA

ToxRef Database. Environmental Health Perspectives, 117(3), 392–399.https://doi.org/10.1289/ehp.0800074

10 KIM ET AL.

Mashock, M. J., Zanon, T., Kappell, A. D., Petrella, L. N., Andersen, E. C., &

Hristova, K. R. (2016). Copper oxide nanoparticles impact several toxicolog-

ical endpoints and cause neurodegeneration in Caenorhabditis elegans.

PLoS ONE, 11(12), 1–19. https://doi.org/10.1371/journal.pone.0167613

Maurer, L. L., Ryde, I. T., Yang, X., & Meyer, J. N. (2015). Caenorhabditis

elegans as a model for toxic effects of nanoparticles: lethality,

growth, and reproduction. Current Protocols in Toxicology, 66(1),

20.10.1–20.10.25. https://doi.org/10.1002/0471140856.tx2010s66

Nel, A., Xia, T., Meng, H., Wang, X., Lin, S., Ji, Z., & Zhang, H. (2013).

Nanomaterial toxicity testing in the 21st century: Use of a predictive tox-

icological approach and high‐throughput screening. Accounts of Chemical

Research, 46(3), 607–621. https://doi.org/10.1021/ar300022h

Pulak, R. (2006). Techniques for analysis, sorting, and dispensing of C.

elegans on the COPAS™ Flow‐Sorting System. In C. elegans (Vol. 351)

(pp. 275–286). New Jersey: Humana Press. https://doi.org/10.1385/

1‐59745‐151‐7:275

Qu, X., Brame, J., Li, Q., & Alvarez, P. J. J. (2013). Nanotechnology for a safe

and sustainable water supply: Enabling integrated water treatment and

reuse. Accounts of Chemical Research, 46(3), 834–843. https://doi.org/10.1021/ar300029v

Shin, N., Cuenca, L., Karthikraj, R., Kannan, K., & Colaiácovo, M. P. (2019).

Assessing effects of germline exposure to environmental toxicants by

high‐throughput screening in C. elegans. PLoS Genetics, 15(2),

e1007975. https://doi.org/10.1371/journal.pgen.1007975

Smith, M. V., Boyd, W. A., Kissling, G. E., Rice, J. R., Snyder, D. W., Portier,

C. J., & Freedman, J. H. (2009). A discrete time model for the analysis of

medium‐throughput C. elegans growth data. PLoS ONE, 4(9), e7018.

https://doi.org/10.1371/journal.pone.0007018

Sprando, R. L., Olejnik, N., Cinar, H. N., & Ferguson, M. (2009). A method to

rank order water soluble compounds according to their toxicity using

Caenorhabditis elegans, a Complex Object Parametric Analyzer and

Sorter, and axenic liquid media. Food and Chemical Toxicology, 47(4),

722–728. https://doi.org/10.1016/j.fct.2009.01.007

Stiernagle, T. (2006). Maintenance of C. elegans. WormBook, 1–11.https://doi.org/10.1895/wormbook.1.101.1

Wang, D. (2018). Nanotoxicology in Caenorhabditis elegans.

Nanotoxicology in Caenorhabditis elegans. Springer Nature Singapore

Pte Ltd. https://doi.org/10.1007/978‐981‐13‐0233‐6

Williams, H. M. (2018). The application of magnetic nanoparticles in the

treatment and monitoring of cancer and infectious diseases. Bioscience

Horizons: The International Journal of Student Research, 10, 1–10.https://doi.org/10.1093/biohorizons/hzx009

Wu, T., Xu, H., Liang, X., & Tang, M. (2019). Caenorhabditis elegans as

a complete model organism for biosafety assessments of

nanoparticles. Chemosphere, 221, 708–726. https://doi.org/10.1016/j.chemosphere.2019.01.021

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