Humic Acid Alleviates the Toxic Effect of AgNPs on Tigriopus

Japonicus

Jiaying Zhao

and Xinwei Wang

College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China

Keywords:

HA, AgNPs, Tigriopus, Feeding Behavior, Toxic Effect.

Abstract: Silver nanoparticles (AgNPs) with excellent physical and chemical properties are one of the most widely used

nanoparticles (NPs). However, with the increase of the production of AgNPs, there will be certain risks to

aquatic organisms when they are discharged into the water environment. Here, we systematically studied the

ecotoxic effect of AgNPs on Tigriopus under the influence of humic acid (HA) in natural organic matter

(NOM). From the perspective of individual level, the effects of AgNPs on feeding behavior of Tigriopus

under the action of HA were explored. As a link between primary and higher nutrient levels in the ocean, it is

of great significance to study the feeding behavior of copepods. Compared with acute and chronic toxicity

indexes, behavioral indexes are more sensitive to evaluate the toxic effects of pollutants. With the increase of

AgNPs concentration, the feeding rate and water filtration rate of Tigriopus decreased, indicating that the

presence of nanoparticles (NPs) could significantly inhibit the feeding behavior of the tested organisms. The

presence of humic acid (HA) alleviated the inhibition of AgNPs on feeding rate and water filtration rate of

Tigriopus. In addition, this indicated that there was a certain interaction between NPs and HA, which would

alleviate the toxic effects of nanoparticles on copepods, and ultimately affected the individual level and

population dynamics of copepods.

1 INTRODUCTION

Nanoparticles are defined as ultra-fine particles with

a particle size of 1-100 nm. Due to their chemical

composition, shape, size, density, aggregation,

surface properties and unique physical and chemical

properties (such as magnetic, optical and

electrochemical properties) (Shevlin, 2018). In the

process of synthesis, production and use,

nanomaterials will inevitably be discharged into

water through a variety of ways. Their unique

physical and chemical properties can be transmitted

and transferred through the food chain of aquatic

organisms, enrich in the body of organisms with

higher trophic levels, and have toxic effects on

biological cells and individuals (Vance, 2015).

Existing nanomaterials have confirmed that silver

nanoparticles (AgNPs) are toxic to aquatic organisms,

playing a toxic role by causing membrane damage,

production of reactive oxygen species, protein

degeneration, mitochondrial dysfunction, DNA

damage, and inhibition of cell proliferation Silver

nanoparticles: Toxicity in model organisms as an

overview of its hazard for human health and the

environment (Tortella, 2020). Their effects on aquatic

organisms should be fully studied (Williams, 2019).

Their position in marine food chains is very

prominent, especially with regard to the transfer of

energy (Jeong, 2020). From the study's endpoints,

Tigriopus is not only suitable for short-term acute

exposure but also for multigenerational subchronic or

chronic toxicity studies. From the perspective of

research methods, there are individual level of

apparent research, biochemical level of micro

research and individual level of research indicators,

such as mortality, growth and development, sex ratio,

spawning capacity, reproduction and feeding.

Natural organic matter (NOM), which is a

complex mixture of a wide range of molecular

weights, is presents in the environment at

concentrations ranging from 1 to 10 mg·L

-1

(Li,

2018). The results showed that Suwannee River

humic acid (SRHA) could decompose the surface

layer of AgNPs at the concentration of 10 mg·L

-1

, and

the release amount of Ag

decreased with the increase

of SRHA concentration (Gunsolus, 2015). At the

same time, there may be a reversible process in the

light environment where NOM (e.g.HA) reduces Ag

Zhao, J. and Wang, X.

Humic Acid Alleviates the Toxic Effect of Agnps on Tigriopus Japonicus.

DOI: 10.5220/0012012800003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 46-49

ISBN: 978-989-758-637-8

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

to AgNPs, depending on the concentrations of Ag

and HA (Zhou, 2016).

In this study, Tigriopus was selected as the

research object, and the research method combined

with laboratory culture, determination and analysis

was planned to conduct the following explorations. In

terms of individual level, this paper explored the

effects of different concentrations of AgNPs on

feeding behavior of Tigriopus, and the effects of

AgNPs on feeding behavior of Tigriopus in the

presence of humic acid (HA). This study provides

data support and scientific basis for evaluating the

potential effects of silver nanoparticles (AgNPs) on

marine copepods under the influence of humic acid

(HA) in natural organic matter (NOM).

2 MATERIALS AND METHODS

2.1 Preparation of Experimental

Materials

(1) Preparation of AgNPs reserve solution

AgNPs (<100 nm) powder purchased from

Sigma-Aldrich. When preparing the reserve solution,

0.015g AgNPs powder was placed in a 100 mL

beaker, and the volume was fixed in a 250 mL

volumetric flask. The powder was prepared into 60

mg/L reserve solution and stored in a refrigerator at

4℃. Before each experiment, the reserve solution

was diluted into solutions of different concentrations,

and ultrasound was required for 30 min in advance.

(2) Preparation of HA and AgNPs mixed solution

In the preparation of mixed solutions of HA and

AgNPs, quantitative AgNPs and HA should be added

into the test tube with sterilized seawater and

oscillated continuously for 24 h in an oscillating

chamber (37℃, rotation speed 180 r/min). The

process of sterilizing seawater was as follows: the

purchased natural seawater was aerated with an air

pump, filtered by 0.45 μm cellulose nitrate membrane

before the experiment, and then sterilized with an

autoclave. The sterilization condition was 121℃ for

20 min.

(3) Culture of Tigriopus

The Tigriopus used in this experiment was

provided by Ecotoxicology Laboratory of Ocean

University of China. Isochrysis galbana and

Phaeodactylum tricornutum were used as mixed bait

and fed once a day. The density of microalgae was 1

× 10

cell·mL

-1

. Incubate in a constant temperature

incubator with light to dark ratio of 12 h: 12 h, light

intensity of 2100 lux and light temperature of 24℃.

The seawater was changed once a week. The seawater

was extracted and filtered by 0.45 μm cellulose nitrate

lipid membrane. The seawater was sterilized in an

autoclave at 121℃ for 20 min and cooled to room

temperature for later use. The pH of the sterilized

seawater is 8.2 and the salinity is 31‰.

(4) Culture of microalgae

Isochrysis galbana and Phaeodactylum

tricornutum used in this experiment were provided by

Ecotoxicology Laboratory of Ocean University of

China. Shake well once every morning and evening

to prevent microalgae from settling or sticking to the

wall.

2.2 Experimental Method

In order to study whether the presence of humic acid

(HA) would affect the feeding behavior of AgNPs on

Tigriopus, a feeding experiment was designed. There

were 5 adult in each culture plate. The volume of

solution in each well of culture plate was 5 mL. The

experiment was carried out under 24 light protection

conditions. The vibration was carried out every half

hour in the vibration chamber. After exposure for 4 h,

the number of algal cells was counted under the

microscope with a blood cell counting plate. The algal

cells used in the experiment were Isochrysis galbana.

3 RESULTS AND DISCUSSION

3.1 Feeding, Filtering Rates of AgNPs

to Tigriopus in the Presence of HA

As shown in Fig.1, compared with the control group,

even in the experimental group with the smallest

concentration of AgNPs, the feeding rate and water

filtration rate were reduced by 41.14% and 50.64%,

respectively. The experimental group with the largest

concentration of AgNPs had even lower feeding rate

and water filtration rate 67.28%, 74.55%. It can be

seen that the presence of AgNPs inhibited the feeding

and filtering behavior of the Tigriopus. However,

after adding different concentrations of HA, the

feeding rate and water filtration rate changed. For the

low concentration AgNPs experimental group, the

feeding rate of the experimental group with 5 mg·L

-1

HA increased by 18.26% compared with the AgNPs

experimental group, and the water filtration rate also

showed an upward trend, increasing by 23.08%.

Under the influence of HA, the change trend of

feeding rate and water filtration rate in the high

concentration AgNPs group was consistent with that

in the low concentration group.

Humic Acid Alleviates the Toxic Effect of Agnps on Tigriopus Japonicus

Behavioural effects of organisms are of great

significance in the study of the toxic effects of

pollutants such as nanoparticles. Compared with

mortality, behavioral indicators are more sensitive to

the evaluation of the toxic effects of pollutants.

Therefore, the influence of AgNPs on the feeding

behavior of Tigriopus was investigated in the

presence of different concentrations of HA. As can be

seen from Fig.1, the feeding rate of Tigriopus

decreased with the increase of AgNPs concentration,

which may be because Tigriopus transferred the

energy used for feeding behavior to resist the external

environmental stress, resulting in a decrease in

feeding rate. Studies have shown that the ability of

Tigriopus to feed may be related to metabolism, as

hard-to digest food will stay in the intestines for

longer, resulting in a sense of satiety, resulting in a

decreased ability to feed (Yu, 2020). The exposure of

AgNPs in Tigriopus produced oxidative stress effect.

To resist oxidative stress effect, the organism must

consume energy, and the energy consumption

accumulated to a certain extent can further affect the

normal physiological activities of the organism and

lead to toxicity effects at the individual level (such as

changes in feeding behavior). According to the study

of Mattsson et al. (Mattsson, 2016), 24 h after daphnia

was exposed to silver nanowires (Ag NWs), Ag NWs

would accumulate in the intestine of daphnia and

transfer to the body cavity through the intestinal

epithelium. Due to the damage of digestive organs,

nanoparticles would destroy the nutrition and

digestive function of daphnia. Keller et al. (Keller,

2012) pointed out that after the organism ingents

nanoparticles, nanoparticles adsorb on the surface of

the cell membrane through coordination and

electrostatic action, interfering with signal

transmission on the cell membrane, leading to

abnormal cell membrane function and even changing

the permeability of the cell membrane, resulting in

cell damage. The feeding rate of Tigriopus in the

presence of HA was higher than that in the presence

of AgNPs alone. This may be the Zeta potential

change caused by the adsorption of HA on the surface

of AgNPs. Meanwhile, the surface charge of NPs was

increased to form a higher double electric layer

repulsed energy, which lead to the uniform dispersion

of aggregates in the medium and inhibits the uptake

of NPs by Tigriopus (Mohd, 2014).

Figure 1: Effects of AgNPs on feeding rate (a) and filtering rate (b) of Tigriopus in the presence of different concentrations

of HA.

4 CONCLUSIONS

With the increase of AgNPs concentration, the

feeding rate and filtrating rate of Tigriopus decreased,

indicating that the presence of nanoparticles (NPs)

could significantly inhibit the feeding behavior of the

tested organisms. This may be that nanoparticles

(NPs) were ingested by Tigriopus and entered the

intestinal mucosa, which caused mechanical damage

to the intestinal mucosa. The nanoparticles (NPs)

accumulated in the intestinal mucosa were difficult to

digest and affected the feeding behavior of Tigriopus.

The presence of humic acid (HA) alleviated the

inhibition effect of Ag NPs on the feeding rate and

water filtration rate of Tigriopus. This may be

because the adsorption of HA on the surface of

AgNPs causes a change in Zeta potential, resulting in

agglomerates being evenly dispersed in the medium,

reducing the intake of AgNPs by Tigriopus. In

addition, due to the adsorption of HA on the surface

of AgNPs, the total amount of Ag

released was

ICBB 2022 - International Conference on Biotechnology and Biomedicine

reduced, which weakened the degree of

internalization of AgNPs by organisms and alleviated

the toxic effect of AgNPs on Tigriopus.

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