Chronic Toxicity of Silver Nanoparticles to Tigriopus Japonicus

Xinwei Wang

and Jiaying Zhao

Key Laboratory of Marine Environment and Ecology, Ministry of Education (Ocean University of China),

Qingdao, P. R. China

Keywords: Silver Nanoparticles, Chronic Toxicity.

Abstract: Silver nanoparticles (Ag NPs) have been widely used because of their excellent bactericidal properties, and

with them comes their massive discharge, which may pose potential risks to marine ecology and the

environment, but relatively few studies have been conducted on the chronic toxicity of Ag NPs to marine

organisms. Here, the marine copepod Tigriopus japonicus was used to investigate the effects of Ag NPs on

the survival, development, and reproduction under two generations. The results showed that Ag NPs

significantly increased the mortality of T. japonicus at 0.2 mg/L. In the F

generation, 0.3 mg/L Ag NPs

significantly prolonged the developmental time of T. japonicus, while in the F

generation, it was significantly

prolonged at only 0.1 mg/L. And Ag NPs significantly inhibited the hatching number of T. japonicus at F

generation while it was alleviated at F

generation.

1 INTRODUCTION

Silver nanoparticles (Ag NPs) are widely used in a

variety of consumer products, including textiles, care

products, and food packaging, due to their

physicochemical properties, especially their excellent

bactericidal effect. It was estimated that the global

production of Ag NPs was 500 tons/year. and more

than 60 tons of Ag NPs were released into the water

environment each year (Handy, 2012). Several

studies have shown that Ag NPs can have acute toxic

effects on a variety of organisms, including oxidative

stress, genotoxicity, and behavioral effects, but their

possible chronic toxicity to organisms was less well

studied.

As a key link in the marine food web, zooplankton

plays an important role in the process of material

cycling and energy transfer and influences the

transport of pollutants (Batel, 2016). Tigriopus

japonicus belongs to Arthropoda, Crustacea,

Harpacticoida, is a common species in the estuaries

of the western Pacific Ocean, with short generation

time, strong reproduction, and easy cultivation,

widely used in the detection of microplastics, heavy

metals, organic matter, and other pollutants toxicity

(Juan, 2020). It has been classified as a standard

organism for toxicity testing by OECD.

In this paper, we investigated the effects of Ag

NPs on the growth, development, and reproduction of

T. japonicus under two generations. The chronic

toxicity data of Ag NPs were supplemented to

provide information and methods for assessing the

effects of Ag NPs on marine invertebrates and to

provide a reference for understanding the

reproductive toxicity of Ag NPs.

2 MATERIAL AND METHODS

2.1 Ag NPs and Organisms

Silver nanoparticles (PVP - Ag NPs, ﹤100nm) were

obtained from Sigma-Aldrich (Germany). Tigriopus

japonicus and natural seawater were taken from the

sea near Qingdao, China, and have been continuously

cultured in the laboratory for many generations. The

culture conditions were: 32‰ of filtered and

sterilized natural seawater as culture medium, 2100 lx

of light, 12 h: 12 h of light to dark ratio, and 24℃.

During the culture period, the algae were fed with a

1:1 mixture of Phaeodactylum tricornutum and

Isochrysis galbana every 3 days at a feeding density

of 1×10

cells/mL.

2.2 Experimental Methods

Before the formal experiment, the acute toxicity of

Ag NPs to T. japonicus was tested, and the 96 h - LC

Wang, X. and Zhao, J.

Chronic Toxicity of Silver Nanoparticles to Tigriopus Japonicus.

DOI: 10.5220/0012011900003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 12-16

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

was 0.892 mg/L (0.778 - 1.079 mg/L). Before starting

the experiment, T. japonicus females with eggs were

transferred to 6-well culture plates, one per well, and

fed with bait. The nauplius with hatching time <24h

were randomly selected for the experiment. Four

concentration groups (0.1, 0.2, 0.3, 0.4 mg/L) and

control groups were set up, with three parallels in

each group. The nauplius (F

) were transferred to 12-

well plates with 10 per well, and the culture

conditions were as above. After 12 h of exposure,

baits were fed and the experimental solution was

changed after 24 h. The development of T. japonicus

(nauplius - copepodite - adult) and the number of dead

individuals were observed and recorded, and the dead

individuals were removed. For the nauplius stage, 5

mL of experimental solution was added to each

group, and 10 mL was added to the copepodite and

adult stage. After the females of T. japonicus held

eggs, the females were transferred to 12-well culture,

one in each well, and the same concentration of Ag

NPs was added. Similarly, fed after 12 h of exposure

and changed the solution after 24 h. Females were

observed for reproduction, and the number of eggs

carried, incubations, and hatching were recorded for

10 d. At the peak of reproduction in each group of

females, nauplius hatchlings with an incubation time

<24 h were randomly selected for the second

generation (F

) experiment. The experimental

methods were the same as those for F

2.3 Statistical Analysis

The experimental data were analyzed by one-way

ANOVA and LSD multiple comparison analysis

using SPSS 16.0 to compare the significance of

differences between concentrations, with P ﹤0.05

indicating a significant difference.

3 RESULTS

3.1 Effects of Ag NPs on the Growth

and Development of T. Japonicus

The mortality rate of T. japonicus F

and F

generations after 21 days of exposure to Ag NPs was

shown in Fig. 1. The mortality rate in the control

group was 3.33%, and 13.4%, 33.33%, 70%, and

86.7% for each concentration of the F

generation,

while 23.3% and 36.7% for the F

generation,

respectively. The results showed that the mortality

rate of T. japonicus increased gradually with the

increase of Ag NPs concentration. Compared with F

the mortality rate increased in all groups in F

. It can

be seen that Ag NPs had a greater effect on the

survival of T. japonicus at higher concentrations.

Figure 1 21 - day mortality of T. japonicus F

(a) and F

(b) generations exposed to Ag NPs. The data of 0.3 mg/L and 0.4

mg/L in (b) were missing because T. japonicus in F

did not incubate enough nauplius to continue the experiment. Values are

shown as mean ± S.D., different letters: P ﹤0.05.

Effect of Ag NPs on the time from nauplius to

copepodite (N-C) and from nauplius to adults (N-A)

in the F

and F

generations of T. japonicus are shown

in Fig. 2. In the F

generation, the duration of N-C and

N-A in the control was 4.67 and 12.33 days,

respectively, and 5.33, 5.67, 6.33, and 7.67 days for

each concentration of N-C; and 12.33, 13.33, 14.67,

and 16.33 days for N-A, respectively. In the F

generation, the duration of N-C and N-A in the

control group was 4.67 and 12.67 days, respectively.

Chronic Toxicity of Silver Nanoparticles to Tigriopus Japonicus

The duration of N-C and N-A for each concentration

group was 7.33 days, 8.67 days, and 15 days, 18.33

days, respectively. The results showed that the N-C,

N-A of both T. japonicus F

and F

increased with the

increase of Ag NPs concentration, and the growth

time of F

was longer than F

. There was no

significant difference in N-C and N-A of F

generation compared to the control at concentrations

of 0.1 mg/L and 0.2 mg/L.

Figure 2 Effect of Ag NPs on the time from nauplius to copepodite (N-C) and from nauplius to adults (N-A) in the F

(a) and

(b) generations of T. japonicus. The data of 0.3 mg/L and 0.4 mg/L in (b) were missing because T. japonicus in F

did not

incubate enough nauplius to continue the experiment. Values are shown as mean ± S.D., different letters: P ﹤0.05.

Figure 3 Effect of Ag NPs on the number of three hatchings in T. japonicus females F

(a) and F

(b) generations. The data

of 0.3 mg/L and 0.4 mg/L were missing because the mortality rate was too high for the hatchings to be known. Values are

shown as mean ± S.D., different letters: P ﹤0.05.

3.2 Effects of Ag NPs on the

Reproduction of T. japonicus

The effect of Ag NPs on the number of three

hatchings of T. japonicus females for 10 days was

shown in Fig. 3. In F

generation, the total number of

hatchings in 10 days of the control group was 25.89,

and 22.00 and 14.46 at 0.1 mg/L and 0.2 mg/L,

respectively; in the F

generation, the total number of

hatchings in 10 days of the control group was 29.40,

and 24.18 and 20.28 at 0.1 mg/L and 0.2 mg/L,

respectively. The results showed that the hatching

number of T. japonicus gradually decreased with the

increase of concentration and the number of hatchings

increased with the number of incubations at the same

concentration in both F

and F

generations.

ICBB 2022 - International Conference on Biotechnology and Biomedicine

4 DISCUSSION

In the ocean, the survival, growth, and reproduction

of T. japonicus were affected by a variety of factors.

For example, seawater acidification and various

pollutants (heavy metals, organic matter,

microplastics, etc.). It has been shown that 4-

methylbenzylidene camphor (4-MBC) (Hong, 2021),

Ni (Mohammed, 2010), the microplastics

polyethylene (PE), and polyamide-nylon 6 (PA 6)

(Yu, 2020) all reduced T. japonicus survival in a

dose-dependent manner, and that nauplius was more

sensitive to pollutants than copepodite and adults,

similar to Ag NPs. In addition to its effect on survival,

the impact on growth and reproduction was also a

major concern. Hong et al. (2021) noted that 4-MBC

reduced the developmental time of T. japonicus at the

N-C stage and the number of female hatchings

decreased with increasing concentration. For the

incubation number, 4-MBC barely affected the F

generation, but at high concentrations (5 and 10

μg/L), the F

and F

generations were significantly

inhibited, and inhibition was relieved at the F

generation (Chen, 2018). This differs from Ag NPs,

which in this study significantly prolonged the

developmental time of T. japonicus although they

inhibited the hatching number of females. Of course,

some pollutants did not adversely affect the survival

of T. japonicus but inhibit reproduction. For example,

dibutyl phthalate (DBP) did not have a lethal effect

on T. japonicus at the concentrations tested, but

prolonged incubation time and inhibited hatching

numbers (Li, 2020). Most contaminants reduced the

hatching number while prolonging the development

of T. japonicus. It has been shown that when T.

japonicus were exposed to oil-contaminated

sediments, the growth rate of nauplius decreased,

developmental time increased significantly, and the

number of egg-bearing females decreased and

hatching was significantly reduced (Won, 2018).

Seawater contaminated with various metals

(containing Cr, Zn, Ni, As, etc.) can significantly

inhibit the survival and reproduction rate of T.

japonicus. And ZnO nanoparticles completely

inhibited the reproduction of T. japonicus at 0.5 mg/L

(Jeong, 2019). Similar to the metal contaminants

mentioned above, Ag NPs had adverse effects on the

growth, development and reproduction of T.

japonicus.

5 CONCLUSIONS

Ag NPs inhibited the survival and development of T.

japonicus at all stages, and the inhibition increased

with increasing concentration and generations. In

contrast, the inhibition of hatching numbers

decreased with increasing generations. In future

studies, the chronic toxicity of Ag NPs should

continue to be investigated and attention the effects

on T. japonicus reproduction under multiple

generations.

ACKNOWLEDGMENTS

This work was supported by the National Natural

Science Foundation of China (Grant No. 41276104),

and Public Science and Technology Research Funds

Projects of Ocean (Grant No. 201505034-2).

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