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Citation: Cruz-Rosa, S.; Pérez-Reyes,
O. Neurotoxicity and Oxidative
Stress Development in Adult Atya
lanipes Shrimp Exposed to Titanium
Dioxide Nanoparticles. Toxics 2023,
11, 694. https://doi.org/10.3390/
toxics11080694
Academic Editor: Elena Maria Scalisi
Received: 9 June 2023
Revised: 3 August 2023
Accepted: 9 August 2023
Published: 11 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
toxics
Article
Neurotoxicity and Oxidative Stress Development in Adult Atya
lanipes Shrimp Exposed to Titanium Dioxide Nanoparticles
Stefani Cruz-Rosa * and Omar Pérez-Reyes *
Department of Environmental Sciences, College of Natural Sciences, University of Puerto Rico,
Rio Piedras Campus, San Juan 00925, Puerto Rico
*Correspondence: stefani.cruz@upr.edu (S.C.-R.); omar.perez15@upr.edu (O.P.-R.)
Abstract:
Titanium dioxide is a type of nanoparticle that is composed of one titanium atom and
two oxygen atoms
. One of its physicochemical activities is photolysis, which produces different
reactive oxygen species (ROS). Atya lanipes shrimp affect detrital processing and illustrate the potential
importance of diversity and nutrient availability to the rest of the food web. It is essential in
removing sediments, which have an important role in preventing eutrophication. This study aimed
to determine the toxic effect of changes in behavior and levels of oxidative stress due to exposure to
titanium dioxide nanoparticles in Atya lanipes and to determine the effective concentration (EC50) for
behavioral variables. The concentrations of TiO
2
NPs tested were 0.0, 0.50, 1.0, 2.0, and 3.0 mg/L with
the positive controls given 100
µ
g/L of titanium and 3.0 mg/L of TiO
2
NPs
±
100
µ
g/L of titanium.
After 24 h of exposure, significant hypoactivity was documented. The EC50 was determined to
be a concentration of 0.14 mg/L. After the exposure to 10 mg/L of TiO
2
NPs, oxidative stress in
gastrointestinal and nervous tissues was documented. The toxic effects of this emerging aquatic
pollutant in acute exposure conditions were characterized by sublethal effects such as behavior
changes and oxidative stress.
Keywords: acute exposure; catalase; effective concentration; macroinvertebrates; nanotoxicity
1. Introduction
Titanium dioxide is a type of nanoparticle that is composed of one titanium atom and
two oxygen atoms. One of its physicochemical activities is photolysis, which produces
different reactive oxygen species (ROS) [
1
,
2
]. In this way, it is a chemically inert and photo-
catalytic nanoparticle that reflects all the colors of the light spectrum (since it reflects white
light). Also, titanium dioxide is a white and fine powder [
3
]; it can exist in different forms,
such as rutile, anatase, and brookite [
4
–
6
]. The anatase form is the most photocatalytic
arrangement of this nanoparticle [7,8].
Due to their photocatalytic activities, titanium dioxide nanoparticles (TiO
2
NPs) are of
great interest because they are used in paints and coatings as self-cleaning, antimicrobial,
and antifouling agents, a food additive, and as a UV absorber in cosmetics [
9
,
10
]. From
2006 to 2010, the commercial production of titanium dioxide was 5000 metric tons per
year; from 2011 to 2014, it was more than 10,000 metric tons, and it is estimated to reach
2.5 million metric tons by 2025 [11].
The rapid production and use of TiO
2
NPs result in a direct and indirect release into aquatic
environments through bathing, industrial effluent, and engineering
applications [12,13]
. In this
way, one of the ecosystems affected by the presence of TiO
2
NPs is the freshwater ecosystem.
The environmental concentrations of these NPs in freshwater ecosystems are variable in the
scientific literature. However, the health of the river can be affected by TiO
2
NPs in ways
that are not yet quantified. Nevertheless, some studies have demonstrated the presence
of TiO
2
NPs in high concentrations in natural surface waters and rivers [
14
]. However,
most studies focus on determining TiO
2
NP levels in surface waters, and few studies have
Toxics 2023,11, 694. https://doi.org/10.3390/toxics11080694 https://www.mdpi.com/journal/toxics
Toxics 2023,11, 694 2 of 14
studied these in sediments. Despite the lack of standardized quantification of TiO
2
NPs in
aquatic ecosystems, European studies have determined that the concentrations of TiO
2
NPs
in freshwater range from 0.015 to 24.5 micrograms/liter [
15
]. On the other hand, studies
have documented that soil concentrations may exceed 100 micrograms/liter [
14
]. However,
concentrations of titanium (including TiO
2
NPs) were measured in the United States and
Canada in a study involving 15 rivers. The concentrations of titanium in these rivers ranged
from 0.5 to15 micrograms/liter and, in some soils and sediments, about 10–100 g/kg with
an average of less than 5 g/kg [16].
In the Caribbean and Puerto Rican freshwater ecosystems, we have an important
shrimp species named Atya lanipes Holthuis 1963. Atya lanipes are a scraper/filter feeder
shrimp that live part of their life as planktonic organisms until they reach the youth
stage. This amphidromous life cycle of the Caribbean freshwater shrimp represents an
important relationship between the headwaters and estuaries [
17
]. Crowl (2001) [
18
],
among others, demonstrated that Atya lanipes affect detrital processing and illustrate the
potential importance of diversity and nutrient availability to the rest of the food web. Also,
Atya lanipes are essential in removing sediments from streams, which have an important role
in preventing eutrophication in this aquatic environment. For this reason, any contaminant
that can both settle or accumulate/dissolve in the water could harm this species and alter
its role in the ecosystem. Therefore, TiO
2
NPs, like other hydrophobic nanomaterials, tend
to sediment as their fate in aquatic ecosystems. In this way, Atya lanipes will be susceptible
to the presence of these engineering nanoparticles in two different aquatic ecosystems:
estuaries and rivers. This makes Atya lanipes an excellent nano-toxicological model.
Studies with TiO
2
NPs have found that these NPs, acting as adsorptive agents, interact
with metals and are stored in sediments [
19
]. In addition, it has been shown that that
adsorptive ability of TiO
2
NPs can exacerbate the bioavailability and neurotoxicity of
organic pollutants and pesticides [
19
]. There have also been several studies that focused on
using the photocatalysis (formation of oxidative agents) of TiO
2
NPs for remediation and
treatment of wastewater but many of these studies did not consider the possible toxicity to
aquatic organisms [20,21].
Researchers have recognized the reaction between nanomaterials and biological sys-
tems in many ecotoxicology studies [
22
]. However, most of these studies have been
conducted in bacteria, cell lines, and rodent animals [
23
]. Nevertheless, these studies reveal
the development of oxidative stress as the principal biological effect. Oxidative stress is
produced by free radicals, which contain one unpaired electron and are highly reactive
and capable of damaging molecules and transforming them into reactive molecules. These
produce a redox chain reaction that damages cells and tissues in biological systems. This
includes cell toxicity by oxidation of lipids, proteins, carbohydrates, and nucleotides lead-
ing to the formation of intracellular aggregates, mitochondrial dysfunction, excitotoxicity,
and apoptosis. All biological systems have an antioxidant defense to avoid oxidative stress.
The antioxidants act by decreasing the concentration of oxidants, preventing the initiation
of the chain reaction by “sweeping” (covering or stopping a very highly reactive chemical)
the first free radicals to form, binding to metal ions to prevent the formation of reactive
species, transforming peroxides into less reactive products, and stopping the spread and
increase in free radicals. One example of a very important antioxidant enzyme is catalase
which acts as a metabolizer, transforming peroxides into H
2
O and O
2
. In this way, this
enzyme minimizes oxidative stress damage [24].
The biological effects from oxidative stress induced by TiO
2
NPs are known as dose-
dependent toxicity indices. Some studies focused on green algae (Desmodesmus subspicatus)
have determined that EC50 = 44 mg/L [
25
]. There are no dose–response toxicological
analyses on Atya lanipes shrimp for behavioral variables (another important sublethal
effect). These indices are imperative to explore in this species due to its susceptibility
to contamination in water bodies, particularly for nanomaterials, as we have previously
discussed. It is necessary to determine the toxicological indices for TiO
2
NPs in terms of
behavioral variables to analyze the environmental risk from this type of nanoparticles in
Toxics 2023,11, 694 3 of 14
the freshwater ecosystems inhabited by this species [
23
]. Also, this shrimp needs a healthy
nervous system to complete its complex life cycle (which includes migrations), acquire
food, avoid predation, and fulfill its other ecological roles.
This study aimed to determine the toxic effects of exposure to titanium dioxide
nanoparticles reflected in changes in behavior and development of oxidative stress during
the adult life cycle of Atya lanipes shrimp and to determine the dose–response index (EC50),
for behavioral variables after exposure to different TiO
2
NP suspension concentrations. We
found that acute exposure to TiO
2
NPs produced a toxic effect on the nervous system of
Atya lanipes shrimp, resulting in hypoactivity as has been determined in the larval stage of
this species [
26
]; we also observed the effects of TiO
2
NPs at concentrations at the mg/L
scale due to the capacity of anatase form of this nanoparticle to produce oxidative stress. A
Probit analysis for behavioral variables showed an EC50 of TiO
2
NPs at a low concentration.
Also, the development of oxidative stress was evident and remained constant during the
acute exposure, reflected in an increase in catalase enzyme activity.
2. Materials and Methods
2.1. Characterization of Titanium Dioxide Nanoparticles Suspensions (TiO2NPs)
Titanium dioxide nanopowder, with a particle size until 25 nm (Sigma Aldrich Chemi-
cal Company St. Louis, MO, USA, Titanium IV Oxide, Anatase nanopowder), was used to
conduct the experiments. The titanium dioxide powder was spread on a weighing paper
and gently picked up by the sticky carbon surface above the aluminum stubs. An S4700 II
cFEG SEM (Hitachi High Technologies-America, Schaumburg, IL, US) with a silicon drift
EDX detector (Oxford Instruments, X-MaxN, Abingdon, UK) was used to measure the
surface morphology, elemental composition, and distribution of elements. All the SEM data
reported were obtained at an acceleration voltage of 10 kV, and the images were collected
with a Secondary Electron detector. The elemental mapping and energy spectrums were
acquired with Aztec tools (Oxford Instruments, UK). The elemental analysis through the
energy dispersive spectrum indicated the presence of Ti, O, C, and S elements (61, 36.1, 2.6,
and 0.3 wt%, respectively) (Figure 1).
Toxics 2023, 11, x FOR PEER REVIEW 3 of 15
discussed. It is necessary to determine the toxicological indices for TiO
2
NPs in terms of
behavioral variables to analyze the environmental risk from this type of nanoparticles in
the freshwater ecosystems inhabited by this species [23]. Also, this shrimp needs a healthy
nervous system to complete its complex life cycle (which includes migrations), acquire
food, avoid predation, and fulfill its other ecological roles.
This study aimed to determine the toxic effects of exposure to titanium dioxide na-
noparticles reflected in changes in behavior and development of oxidative stress during
the adult life cycle of Atya l a nipes shrimp and to determine the dose–response index
(EC50), for behavioral variables after exposure to different TiO
2
NP suspension concen-
trations. We found that acute exposure to TiO
2
NPs produced a toxic effect on the nervous
system of Atya l anipes shrimp, resulting in hypoactivity as has been determined in the lar-
val stage of this species [26]; we also observed the effects of TiO
2
NPs at concentrations at
the mg/L scale due to the capacity of anatase form of this nanoparticle to produce oxida-
tive stress. A Probit analysis for behavioral variables showed an EC50 of TiO
2
NPs at a low
concentration. Also, the development of oxidative stress was evident and remained con-
stant during the acute exposure, reflected in an increase in catalase enzyme activity.
2. Materials and Methods
2.1. Characterization of Titanium Dioxide Nanoparticles Suspensions (TiO
2
NPs)
Titanium dioxide nanopowder, with a particle size until 25 nm (Sigma Aldrich Chem-
ical Company St. Louis, MO, USA, Titanium IV Oxide, Anatase nanopowder), was used
to conduct the experiments. The titanium dioxide powder was spread on a weighing pa-
per and gently picked up by the sticky carbon surface above the aluminum stubs. An
S4700 II cFEG SEM (Hitachi High Technologies-America, Schaumburg, IL, US) with a sil-
icon drift EDX detector (Oxford Instruments, X-MaxN, Abingdon, UK) was used to meas-
ure the surface morphology, elemental composition, and distribution of elements. All the
SEM data reported were obtained at an acceleration voltage of 10 kV, and the images were
collected with a Secondary Electron detector. The elemental mapping and energy spec-
trums were acquired with Aztec tools (Oxford Instruments, UK). The elemental analysis
through the energy dispersive spectrum indicated the presence of Ti, O, C, and S elements
(61, 36.1, 2.6, and 0.3 wt%, respectively) (Figure 1).
Figure 1. Elemental analysis through energy dispersive spectrum. The composition of the nanopow-
der of titanium dioxide in the analyzed sample demonstrates 61.0 wt% titanium, 36.1 wt% oxygen,
2.6 wt% carbon and 0.3 wt% sulfur.
2.2. Atya lanipes Specimens Collection
Adult Atya lani pes specimens were collected and identified [27], using baited minnow
traps at the Sonadora Stream at the El Verde Field Station, Rio Grande, Puerto Rico. The
pools at the Sonadora Stream have a depth that range between 0.5 and 1.5 m, but we se-
lected pools with a depth of 0.5 m. The sampling period was from January 2022 to June
2022 when the climate and specimen availability were favorable. Baited traps (dry cat food
Figure 1.
Elemental analysis through energy dispersive spectrum. The composition of the nanopow-
der of titanium dioxide in the analyzed sample demonstrates 61.0 wt% titanium, 36.1 wt% oxygen,
2.6 wt% carbon and 0.3 wt% sulfur.
2.2. Atya lanipes Specimens Collection
Adult Atya lanipes specimens were collected and identified [
27
], using baited minnow
traps at the Sonadora Stream at the El Verde Field Station, Rio Grande, Puerto Rico. The
pools at the Sonadora Stream have a depth that range between 0.5 and 1.5 m, but we
selected pools with a depth of 0.5 m. The sampling period was from January 2022 to June
2022 when the climate and specimen availability were favorable. Baited traps (dry cat food
was used as the bait) were set in different pools along the stream and removed 24 h later.
The collected shrimp were transported to the laboratory in a cooler under constant aeration.
The shrimp were transferred individually to glass tanks (15 cm
×
15 cm
×
15 cm) with 1 L
Toxics 2023,11, 694 4 of 14
of dechlorinated water and constant aeration. The animals were acclimated for 72–120 h
(three to five days) in the laboratory environment before the bioassay.
2.3. The Microcosm
Fifty microcosms (25 for the control and 25 for each treated group) were designed. They
consisted of a square aquarium containing 1 L of dechlorinated water; 150 g of synthetic
sediment that was previously sterilized with activated carbon and heated in an oven at a
temperature of 50–60
◦
C; an air stone with an air pump; and LED lamps with a 10 h/14 h
light/dark photoperiod (controlled by an automatic timer). The water temperature was
kept between 19 and 21
◦
C (Figure 2). These variables reflect the environmental conditions
in which Atya lanipes live in the wild.
Toxics 2023, 11, x FOR PEER REVIEW 4 of 15
was used as the bait) were set in different pools along the stream and removed 24 h later.
The collected shrimp were transported to the laboratory in a cooler under constant aera-
tion. The shrimp were transferred individually to glass tanks (15 cm × 15 cm × 15 cm) with
1 L of dechlorinated water and constant aeration. The animals were acclimated for 72–120
h (three to five days) in the laboratory environment before the bioassay.
2.3. The Microcosm
Fifty microcosms (25 for the control and 25 for each treated group) were designed.
They consisted of a square aquarium containing 1 L of dechlorinated water; 150 g of syn-
thetic sediment that was previously sterilized with activated carbon and heated in an oven
at a temperature of 50–60 °C; an air stone with an air pump; and LED lamps with a 10 h/14
h light/dark photoperiod (controlled by an automatic timer). The water temperature was
kept between 19 and 21 °C (Figure 2). These variables reflect the environmental conditions
in which Atya l a n ipes live in the wild.
Figure 2. Microcosm: aquarium with 1 L of water, 150 g of artificial sediment, air stone connected to
the air pump, light, and one Atya l a nip e s adult individual.
2.4. Acute Toxicity Tests
Before the bioassay, we mixed the titanium dioxide nanopowder with one L of
dechlorinated and oxygenated (for 24 h) water using a magnetic stirrer at maximum speed
for 30 min. Subsequently, TiO
2
NPs were weighed to obtain final concentrations of 0.0,
0.50, 1.0, 2.0, and 3.0 mg/L. The nanoparticles were added into the fish tanks and left to
fall by gravity; the aquarium water was mixed with a glass stirrer to obtain homogeneity
in the microcosm. For each treatment, we set up 25 replicates each for the treated group
and controls. The positive controls were exposed to titanium (100 mg/L) and TiO
2
NPs and
titanium (3.0 mg/L; 100 mg/L). The microcosms were set in an exposure bench covered
with a piece of blue fabric to prevent the entry of external light and to control any possible
contamination. Physicochemical parameters (temperature, salinity, dissolved oxygen, dis-
solved solids, and pH) of the water of each microcosm were taken before and after the
exposure period using a Hanna HI98129 and Sper Scientific 850045 dissolved oxygen me-
ter pen. After preparing the microcosms with their respective treatments, the Atya l a nipes
adults were randomly assigned to each treatment. Measurements of the post-orbital and
cephalothorax of the shrimp were collected. Then, shrimp were individually introduced
into the microcosms and left exposed to the treatments for an acute exposure of 24 h. No
separation by sex was made because this species does not present clear sexual dimorphism
traits. Instead, we randomly selected specimens ensuring a similar range of sizes for each
treated and control group. Gravid shrimp were excluded from the bioassay. Shrimp were
not fed during the bioassay.
Figure 2.
Microcosm: aquarium with 1 L of water, 150 g of artificial sediment, air stone connected to
the air pump, light, and one Atya lanipes adult individual.
2.4. Acute Toxicity Tests
Before the bioassay, we mixed the titanium dioxide nanopowder with one L of dechlo-
rinated and oxygenated (for 24 h) water using a magnetic stirrer at maximum speed for
30 min. Subsequently, TiO
2
NPs were weighed to obtain final concentrations of 0.0, 0.50,
1.0, 2.0, and 3.0 mg/L. The nanoparticles were added into the fish tanks and left to fall
by gravity; the aquarium water was mixed with a glass stirrer to obtain homogeneity in
the microcosm. For each treatment, we set up 25 replicates each for the treated group and
controls. The positive controls were exposed to titanium (100 mg/L) and TiO
2
NPs and
titanium
(3.0 mg/L
; 100 mg/L). The microcosms were set in an exposure bench covered
with a piece of blue fabric to prevent the entry of external light and to control any possi-
ble contamination. Physicochemical parameters (temperature, salinity, dissolved oxygen,
dissolved solids, and pH) of the water of each microcosm were taken before and after the
exposure period using a Hanna HI98129 and Sper Scientific 850045 dissolved oxygen meter
pen. After preparing the microcosms with their respective treatments, the Atya lanipes
adults were randomly assigned to each treatment. Measurements of the post-orbital and
cephalothorax of the shrimp were collected. Then, shrimp were individually introduced
into the microcosms and left exposed to the treatments for an acute exposure of 24 h. No
separation by sex was made because this species does not present clear sexual dimorphism
traits. Instead, we randomly selected specimens ensuring a similar range of sizes for each
treated and control group. Gravid shrimp were excluded from the bioassay. Shrimp were
not fed during the bioassay.
2.5. Behavioral Analysis
A movement analysis was carried out after acute exposure to TiO
2
NPs for 24 h. Atya
lanipes shrimp are more active at night; therefore, in order to analyze these patterns in
the laboratory setting, we created a recording scenario that simulates nighttime for these
Toxics 2023,11, 694 5 of 14
organisms. The shrimp recordings were performed in a red box with red light because they
do not detect this light wavelength. Also, in control organisms, it was observed that, in the
box, they were active at the corners and never went toward the center of the recording box.
Moving to the center of the box has the possibility of being preyed on.
Each specimen of Atya lanipes in the experiment was removed from the exposure tank
and acclimatized for 3 min in a red plastic box with 2 L of dechlorinated water that was
previously oxygenated for 24 h (it was changed with new water for each shrimp analyzed
to avoid contamination and to minimize errors in the analysis). The shrimp were set in the
red box with new water because the artificial sediment alters the video, creating artifacts
that result in errors in the video; in addition, the movement of the TiO
2
NPs in the water to
the red box will result in the displacement of the nanoparticles that were set in the sediment.
This acclimatization was performed in the recording room under total darkness and with
no sound. After the acclimation period, the movement of the shrimp in the red tank was
recorded for 5 min using a camera (Go Pro Hero 6
®
, GoPro, Inc., San Mateo, CA, USA).
These visual patterns are analyzed from the recording and, through Loligo Systems
©
5th
version software (Loligo Systems, Viborg, Denmark), we quantified movement variables
such as distance traveled and active time, among others (Figure 3).
Toxics 2023, 11, x FOR PEER REVIEW 5 of 15
2.5. Behavioral Analysis
A movement analysis was carried out after acute exposure to TiO
2
NPs for 24 h. Aty a
lanipes shrimp are more active at night; therefore, in order to analyze these paerns in the
laboratory seing, we created a recording scenario that simulates nighime for these or-
ganisms. The shrimp recordings were performed in a red box with red light because they
do not detect this light wavelength. Also, in control organisms, it was observed that, in the
box, they were active at the corners and never went toward the center of the recording
box. Moving to the center of the box has the possibility of being preyed on.
Each specimen of Atya lan ipe s in the experiment was removed from the exposure tank
and acclimatized for 3 min in a red plastic box with 2 L of dechlorinated water that was
previously oxygenated for 24 h (it was changed with new water for each shrimp analyzed
to avoid contamination and to minimize errors in the analysis). The shrimp were set in the
red box with new water because the artificial sediment alters the video, creating artifacts
that result in errors in the video; in addition, the movement of the TiO
2
NPs in the water
to the red box will result in the displacement of the nanoparticles that were set in the
sediment. This acclimatization was performed in the recording room under total darkness
and with no sound. After the acclimation period, the movement of the shrimp in the red
tank was recorded for 5 min using a camera (Go Pro Hero 6
®
, GoPro, Inc., San Mateo, CA,
USA). These visual paerns are analyzed from the recording and, through Loligo Sys-
tems
©
5th version software (Loligo Systems, Viborg, Denmark), we quantified movement
variables such as distance traveled and active time, among others (Figure 3).
Figure 3. Movement analysis performed on Atya l anip es exposed to different concentrations of TiO
2
NPs for 24 h. (a) The microcosm prepared for the bioassay with the Atya lani pes shrimp exposed to
the nanoparticles; (b) the acclimatization process of the shrimp in the recording area inside a red
plastic box; (c) the analysis of the movement in the videos using Loligo Systems
®
5th version soft-
ware.
2.6. Oxidative Stress Analysis
To analyze oxidative stress from 0 to 11 days after an exposure in Atya lani pes, we
dissected five specimens that had been previously exposed to each TiO
2
NPs concentration
and the control treatments. These shrimps were dissected every two days. The shrimp
exposed to the nanoparticles were not fed during the 11 days. The Atya l anipe s specimens
were preserved by gradually puing the shrimp at a cold temperature to decrease their
biological activity without causing strong stress that would alter our results. This was
performed in approximately five minutes; then, we removed the gastrointestinal tract
(gut), the ventral nervous system (nerve cord), and the gills (Figure 4).
We used the Catalase (CAT) Activity Assay kit (Catalog No: MBS2540413; colorimet-
ric method; sensitivity 0.27 U/mL) to determine the catalase enzymatic activity in each
tissue sample for each period. Then, we calculated the enzymatic activity in U/mgprot
with the formula CAT activity = ΔA × 32.5/1 × V × f/Cpr where 32.5 is the reciprocal of the
slope, 1 is the reaction time, ΔA is the ODcontrol—ODsample, V is the volume of the sam-
ple (mL), f is the l factor of the sample before the test, and Cpr is the concentration of
protein in the sample (g/L).
Figure 3. Movement analysis performed on Atya lanipes exposed to different concentrations of TiO2
NPs for 24 h. (
a
) The microcosm prepared for the bioassay with the Atya lanipes shrimp exposed to the
nanoparticles; (
b
) the acclimatization process of the shrimp in the recording area inside a red plastic
box; (c) the analysis of the movement in the videos using Loligo Systems®5th version software.
2.6. Oxidative Stress Analysis
To analyze oxidative stress from 0 to 11 days after an exposure in Atya lanipes, we
dissected five specimens that had been previously exposed to each TiO
2
NPs concentration
and the control treatments. These shrimps were dissected every two days. The shrimp
exposed to the nanoparticles were not fed during the 11 days. The Atya lanipes specimens
were preserved by gradually putting the shrimp at a cold temperature to decrease their
biological activity without causing strong stress that would alter our results. This was
performed in approximately five minutes; then, we removed the gastrointestinal tract (gut),
the ventral nervous system (nerve cord), and the gills (Figure 4).
Toxics 2023, 11, x FOR PEER REVIEW 6 of 15
Figure 4. Diagram of Atya l ani pes shrimp and the anatomy of the dissected tissues for the oxidative
stress assessment. (A) Gut, (B) gills, and (C) nerve cord.
2.7. Statistical Analyses
Parametric statistical approach was performed because of the normality of the data.
Descriptive statistics were used to summarize the values of the physicochemical parame-
ters. To compare the physicochemical parameters among treatments and control, one-way
ANOVA was conducted. The shrimp movement was compared among treatments by a
one-way ANOVA and Tukey tests. A Probit analysis was used to compare the toxicity
levels in the movements. Lastly, we used the formula suggested by the catalase kit to an-
alyze the development of oxidative stress. All the descriptive and statistical analyses were
performed using Minitab 17 Statistical Software [28].
3. Results
3.1. Specimens of Atya lanipes and Physicochemical Parameters
The sample of 25 organisms for the control and exposure groups was chosen to nor-
malize the average shrimp size to standardize the bioassay and obtain reliable data. Ceph-
alothorax lengths (CL) of the control and TiO
2
NP-exposed groups ranged from 14.6 to
16.9 mm on average, and the post-orbital lengths (POL) were between 11.3 and 13.9 mm
on average.
To maintain the internal environmental conditions of the microcosms stable during
the bioassay, we measured the physicochemical parameters of the water before and after
the 24 h exposure to TiO
2
NPs. The temperature of the microcosm was the same as labor-
atory temperature which varied between 19 and 21 °C for the entire exposure period (Ta-
ble 1).
The one-way ANOVA for the physicochemical properties showed no significant dif-
ferences between any pre- and post-exposure time variable. No changes in pH, conduc-
tivity, or salinity measurements before and after exposure were observed. However, dis-
solved the oxygen measurements showed a decrease in groups exposed to TiO
2
NPs; this
result was not observed in the controls. An increase in dissolved oxygen concentrations
was observed after 24 h of exposure. This was expected due to the presence of the constant
oxygen pump in each microcosm during the entire exposure period.
Figure 4.
Diagram of Atya lanipes shrimp and the anatomy of the dissected tissues for the oxidative
stress assessment. (A) Gut, (B) gills, and (C) nerve cord.
Toxics 2023,11, 694 6 of 14
We used the Catalase (CAT) Activity Assay kit (Catalog No: MBS2540413; colorimetric
method; sensitivity 0.27 U/mL) to determine the catalase enzymatic activity in each tissue
sample for each period. Then, we calculated the enzymatic activity in U/mgprot with the
formula CAT activity =
∆
A
×
32.5/1
×
V
×
f/Cpr where 32.5 is the reciprocal of the slope,
1 is the reaction time,
∆
A is the ODcontrol—ODsample, V is the volume of the sample (mL),
f is the l factor of the sample before the test, and Cpr is the concentration of protein in the
sample (g/L).
2.7. Statistical Analyses
Parametric statistical approach was performed because of the normality of the data.
Descriptive statistics were used to summarize the values of the physicochemical parameters.
To compare the physicochemical parameters among treatments and control, one-way
ANOVA was conducted. The shrimp movement was compared among treatments by a
one-way ANOVA and Tukey tests. A Probit analysis was used to compare the toxicity
levels in the movements. Lastly, we used the formula suggested by the catalase kit to
analyze the development of oxidative stress. All the descriptive and statistical analyses
were performed using Minitab 17 Statistical Software [28].
3. Results
3.1. Specimens of Atya lanipes and Physicochemical Parameters
The sample of 25 organisms for the control and exposure groups was chosen to
normalize the average shrimp size to standardize the bioassay and obtain reliable data.
Cephalothorax lengths (CL) of the control and TiO
2
NP-exposed groups ranged from 14.6
to 16.9 mm on average, and the post-orbital lengths (POL) were between 11.3 and 13.9 mm
on average.
To maintain the internal environmental conditions of the microcosms stable during the
bioassay, we measured the physicochemical parameters of the water before and after the
24 h
exposure to TiO
2
NPs. The temperature of the microcosm was the same as laboratory
temperature which varied between 19 and 21 ◦C for the entire exposure period (Table 1).
Table 1.
Average (
±
standard error, SE) of the microcosm’s chemical parameters during the assay (pre-
and post-exposure to TiO2NPs for 24 h) for each control and TiO2NP suspension concentrations.
Group Time pH + S.E. Conductivity + S.E.
(ms)
Salinity + S.E.
(ppm)
O2, + S.E.
(mg/L)
0.0 mg/L; TiO2NPs Pre 8.2 ±0.04 328.2 ±19.0 0.17 ±0.01 8.7 ±0.2
Post 8.2 ±0.04 341.5 ±15.0 0.17 ±0.01 8.8 ±0.1
0.50 mg/L; TiO2NPs Pre 7.8 ±0.003 468.7 ±1.0 0.23 ±0.001 9.2 ±0.02
Post 7.7 ±0.02 436.0 ±9.1 0.22 ±0.004 8.0 ±0.1
1.0 mg/L; TiO2NPs Pre 8.3 ±0.00 353.2 ±0.8 0.23 ±0.0001 9.3 ±0.1
Post 7.7 ±0.02 362.8 ±2.3 0.22 ±0.0001 8.4 ±0.1
2.0 mg/L; TiO2NPs Pre 7.8 ±0.01 469.6 ±3.6 0.23 ±0.002 9.5 ±0.1
Post 7.7 ±0.02 475.2 ±4.2 0.24 ±0.002 7.9 ±0.1
3.0 mg/L; TiO2NPs Pre 8.2 ±0.04 332.2 ±17.3 0.17 ±0.01 8.6 ±0.1
Post 8.1 ±0.04 337.4 ±16.2 0.17 ±0.01 8.9 ±0.1
Titanium (100 mg/L) Pre 8.2 ±0.04 332.4 ±16.0 0.17 ±0.01 8.6 ±0.1
Post 8.1 ±0.04 367.2 ±21.0 0.18 ±0.01 8.9 ±0.1
TiO2NPs (3 mg/L) ±
Titanium (100 mg/L)
Pre 8.2 ±0.04 340.4 ±21.0 0.17 ±0.01 8.6 ±0.1
Post 8.1 ±0.04 371.8 ±22.4 0.19 ±0.01 8.9 ±0.1
The one-way ANOVA for the physicochemical properties showed no significant differ-
ences between any pre- and post-exposure time variable. No changes in pH, conductivity,
or salinity measurements before and after exposure were observed. However, dissolved the
Toxics 2023,11, 694 7 of 14
oxygen measurements showed a decrease in groups exposed to TiO
2
NPs; this result was
not observed in the controls. An increase in dissolved oxygen concentrations was observed
after 24 h of exposure. This was expected due to the presence of the constant oxygen pump
in each microcosm during the entire exposure period.
3.2. Movement Assessment
The analysis of movement after the exposure to TiO
2
NPs for 24 h showed significant
changes leading to hypoactivity. The heat maps for the adult shrimp in the control group
(0.0 mg/L of TiO
2
NPs) showed a preference for the corner of the box over any other
location. Their movements were limited to the corners of the box. In contrast, the exposure
group showed erratic preferences and less exploration movements, especially in the TiO
2
NP-exposed groups (Figure 5).
Toxics 2023, 11, x FOR PEER REVIEW 7 of 15
Table 1. Average (±standard error, SE) of the microcosm’s chemical parameters during the assay
(pre- and post-exposure to TiO2 NPs for 24 h) for each control and TiO2 NP suspension concentra-
tions.
Group Time pH + S.E. Conductivity + S.E.
(ms)
Salinity + S.E.
(ppm)
O2, + S.E.
(mg/L)
0.0 mg/L; TiO2 NPs Pre 8.2 ± 0.04 328.2 ± 19.0 0.17 ± 0.01 8.7 ± 0.2
Post 8.2 ± 0.04 341.5 ± 15.0 0.17 ± 0.01 8.8 ± 0.1
0.50 mg/L; TiO2 NPs Pre 7.8 ± 0.003 468.7 ± 1.0 0.23 ± 0.001 9.2 ± 0.02
Post 7.7 ± 0.02 436.0 ± 9.1 0.22 ± 0.004 8.0 ± 0.1
1.0 mg/L; TiO2 NPs Pre 8.3 ± 0.00 353.2 ± 0.8 0.23 ± 0.0001 9.3 ± 0.1
Post 7.7 ± 0.02 362.8 ± 2.3 0.22 ± 0.0001 8.4 ± 0.1
2.0 mg/L; TiO2 NPs Pre 7.8 ± 0.01 469.6 ± 3.6 0.23 ± 0.002 9.5 ± 0.1
Post 7.7 ± 0.02 475.2 ± 4.2 0.24 ± 0.002 7.9 ± 0.1
3.0 mg/L; TiO2 NPs Pre 8.2 ± 0.04 332.2 ± 17.3 0.17 ± 0.01 8.6 ± 0.1
Post 8.1 ± 0.04 337.4 ± 16.2 0.17 ± 0.01 8.9 ± 0.1
Titanium (100 mg/L) Pre 8.2 ± 0.04 332.4 ± 16.0 0.17 ± 0.01 8.6 ± 0.1
Post 8.1 ± 0.04 367.2 ± 21.0 0.18 ± 0.01 8.9 ± 0.1
TiO2 NPs (3 mg/L) ± Ti-
tanium (100 mg/L)
Pre 8.2 ± 0.04 340.4 ± 21.0 0.17 ± 0.01 8.6 ± 0.1
Post 8.1 ± 0.04 371.8 ± 22.4 0.19 ± 0.01 8.9 ± 0.1
3.2. Movement Assessment
The analysis of movement after the exposure to TiO2 NPs for 24 h showed significant
changes leading to hypoactivity. The heat maps for the adult shrimp in the control group
(0.0 mg/L of TiO2 NPs) showed a preference for the corner of the box over any other loca-
tion. Their movements were limited to the corners of the box. In contrast, the exposure
group showed erratic preferences and less exploration movements, especially in the TiO2
NP-exposed groups (Figure 5).
Figure 5.
Heat maps for the adult Atya lanipes shrimp behavior in all treatment and control groups.
Red areas = more time spent in that area by the shrimp. (
a
) Control, (
b
) titanium (100 micrograms/mL),
(c) titanium (100 micrograms/mL) ±TiO2NPs (3 mg/L), (d) TiO2NPs (3 mg/L).
This hypoactivity characteristic in the movement assessment was statistically evalu-
ated using one-way ANOVA with a Tukey test. During the 24 h of exposure, we observed
significant differences between the exposed and control groups in the total distance moved
and active time (min) (p< 0.05) (Figure 6). The average of the total distance (n= 25) trav-
eled by the adult shrimp in the negative control group was
15,372.6 mm ±/−2581.8 mm
with a minimum of 592.1 mm and a maximum of 46,759.8 mm during the 24 h of expo-
sure. Also, for the positive controls exposed to titanium, they moved a total distance
of
21,039.8 mm ±10,070.0 mm
with a range of 403.7 mm to 254,938.2 mm on average.
In the second positive control (titanium
±
TiO
2
NPs), the average total distance was
16,525.2 mm ±4188.4 mm
with a range of 272.5 mm to 95,032.1 mm. For the different
treatments of TiO
2
NPs, (0.5, 1.0, 2.0, and 3.0 mg/L), we observed a total distance average
of 6072.7
±
1150.8, 9644.3
±
1585.3, 5429.7
±
626.0, and 6571.0
±
1388.9 mm, respec-
tively. Moreover, the total distance moved for the exposed shrimp ranged from 7.1 mm
to
35,659.7 mm
. The one-way ANOVA for the comparison of the total distance moved of
Toxics 2023,11, 694 8 of 14
the adult Atya lanipes shrimp in the control groups and the exposure groups showed a
significant difference (F (6167) = 2.6; p< 0.05) among groups.
Toxics 2023, 11, x FOR PEER REVIEW 8 of 15
Figure 5. Heat maps for the adult Atya l anip es shrimp behavior in all treatment and control groups.
Red areas = more time spent in that area by the shrimp. (a) Control, (b) titanium (100 mi-
crograms/mL), (c) titanium (100 micrograms/mL) ± TiO
2
NPs (3 mg/L), (d) TiO
2
NPs (3 mg/L).
This hypoactivity characteristic in the movement assessment was statistically evalu-
ated using one-way ANOVA with a Tukey test. During the 24 h of exposure, we observed
significant differences between the exposed and control groups in the total distance
moved and active time (min) (p < 0.05) (Figure 6). The average of the total distance (n = 25)
traveled by the adult shrimp in the negative control group was 15,372.6 mm ±/−2581.8 mm
with a minimum of 592.1 mm and a maximum of 46,759.8 mm during the 24 h of exposure.
Also, for the positive controls exposed to titanium, they moved a total distance of 21,039.8
mm ± 10,070.0 mm with a range of 403.7 mm to 254,938.2 mm on average. In the second
positive control (titanium ± TiO
2
NPs), the average total distance was 16,525.2 mm ± 4188.4
mm with a range of 272.5 mm to 95,032.1 mm. For the different treatments of TiO
2
NPs,
(0.5, 1.0, 2.0, and 3.0 mg/L), we observed a total distance average of 6072.7 ± 1150.8, 9644.3
±1585.3, 5429.7 ± 626.0, and 6571.0 ± 1388.9 mm, respectively. Moreover, the total distance
moved for the exposed shrimp ranged from 7.1 mm to 35,659.7 mm. The one-way ANOVA
for the comparison of the total distance moved of the adult Atya lani p es shrimp in the con-
trol groups and the exposure groups showed a significant difference (F (6167) = 2.6; p <
0.05) among groups.
The active time (min) of the adult shrimp (n = 25) was 3.18 min ± 0.17 min with a
minimum of 0.8 min and a maximum of 4.32 min for the negative control during the 24 h
of exposure. Consequently, the active time for the positive controls was 2.74 min ± 0.22
min and 2.98 min ± 0.19 min with minimums of 0.6 and 1.51 and maximums of 4.77 and
4.84 min, respectively. The exposure groups’ (TiO
2
NP suspension concentrations of 0.5,
1.0, 2.0, and 3.0 mg/L) average active time were 2.37 ± 0.18, 2.96 ± 0.18, 2.62 ± 0.17, and 2.54
± 0.22 min. We observed minimums of 0.47, 0.85, 0.06, and 0.35 min with maximums of
3.90, 4.37, 4.02, and 4.26 min for the TiO
2
NP suspension concentrations of 0.5, 1.0, 2.0, and
3.0 mg/L, respectively. The one-way ANOVA for the comparison of all TiO
2
NP suspen-
sion concentrations and the positive and negative controls for the active time variable after
24 h of exposure showed a significant difference (F (6167) = 2.24; p < 0.05) between groups.
The Tukey test analysis showed values of p < 0.05 for the treatment of 0.5 mg/L TiO
2
NPs.
Figure 6.
Assessment of movement of adult Atya lanipes shrimp (n= 25 for each treatment). (
a
) Total
distance moved (mm) and (
b
) active time (min) of Atya lanipes shrimp after 24 h acute exposure
to different TiO
2
NP suspension concentrations, titanium (100 mg/L), and TiO
2
NPs + titanium
(3 mg/L; 100 mg/L). * = outliers.
The active time (min) of the adult shrimp (n= 25) was 3.18 min
±
0.17 min with a
minimum of 0.8 min and a maximum of 4.32 min for the negative control during the 24 h of
exposure. Consequently, the active time for the positive controls was
2.74 min ±0.22 min
and 2.98 min
±
0.19 min with minimums of 0.6 and 1.51 and maximums of 4.77 and
4.84 min,
respectively. The exposure groups’ (TiO
2
NP suspension concentrations of 0.5,
1.0, 2.0, and 3.0 mg/L) average active time were 2.37
±
0.18, 2.96
±
0.18, 2.62
±
0.17, and
2.54 ±0.22 min
. We observed minimums of 0.47, 0.85, 0.06, and 0.35 min with maximums
of 3.90, 4.37, 4.02, and 4.26 min for the TiO
2
NP suspension concentrations of 0.5, 1.0,
2.0, and 3.0 mg/L, respectively. The one-way ANOVA for the comparison of all TiO
2
NP suspension concentrations and the positive and negative controls for the active time
variable after 24 h of exposure showed a significant difference (F (6167) = 2.24; p< 0.05)
between groups. The Tukey test analysis showed values of p< 0.05 for the treatment of
0.5 mg/L TiO2NPs.
For the movement variable of active time, a Probit analysis was conducted. The
lognormal probability plot at a 95% CI (
−
6.320–2.424) showed a coefficient of 0.20 for the
different concentrations, standard error = 0.19, Z= 1.03, and a pvalue = 0.30. Also, the
regression data showed a chi-square = 0.025, Df = 2, and Pearson = 0.99. The EC values for
50, 60, 70, and 90% of events are presented in Table 2.
3.3. Oxidative Stress Assessment
The development of oxidative stress, defined as increased catalase enzyme activity,
in shrimp exposed to TiO
2
NPs was compared against that of the unexposed shrimp. The
branchial, nervous, and gastrointestinal tissues were obtained from five shrimp for each
analysis time and for each nanoparticle suspension concentration and control group, after
24 to 264 h of exposure. The average environmental conditions of the microcosms of the
shrimp used to obtain the fresh tissue samples are summarized in Table 3. During the
Toxics 2023,11, 694 9 of 14
0–264 h
exposure to TiO
2
NPs, the temperature varied between 18.5 and 19.9. The pH
ranged from 5.6 to 7.7. A pH below 6.0 was seen during 216 to 264 h of exposure. The
conductivity varied from 0.3 to 0.5
µ
s. Dissolved oxygen during the exposure ranged from
7.8 to 9.2 mg/L and the salinity from 0.1 to 0.2 ppm.
Table 2.
Effective concentration of TiO
2
NPs (mg/L) for active time variable of Atya lanipes shrimp
after an acute 24 h exposure.
Point
Concentration (mg/L)
24 h
EC50 0.143
EC60 0.520
EC70 2.073
EC80 10.467
EC90 98.868
Table 3.
Average
±
S.E. of the microcosms’ chemical parameters during the bioassay for each
exposure time (10 mg/L of TiO2NPs after 24 to 264 h).
Time
(Hours)
Temperature + S.E.
(◦C) pH + S.E. Conductivity + S.E.
(µs)
DO + S.E.
(mg/L)
Salinity + S.E.
(ppm)
Control group 19.4 ±0.01 7.0 ±0.1 0.4 ±0.01 8.9 ±0.04 0.2 ±0.01
24 19.9 ±0.02 7.1 ±0.2 0.3 ±0.004 8.3 ±0.1 0.2 ±0.01
72 18.5 ±0.1 6.1 ±0.1 0.4 ±0.01 9.0 ±0.1 0.2 ±0.01
120 18.8 ±0.02 6.8 ±0.1 0.4 ±0.0 8.7 ±0.2 0.2 ±0.001
168 18.6 ±0.02 7.1 ±0.1 0.4 ±0.0 7.8 ±0.01 0.2 ±0.0
216 19.2 ±0.1 5.8 ±0.1 0.4 ±0.01 8.4 ±0.1 0.2 ±0.004
264 18.7 ±0.1 6.0 ±0.04 0.4 ±0.001 8.9 ±0.04 0.2 ±0.01
The specimen sizes were determined by measuring their POL and CL lengths (Table 4).
The POS lengths of the samples ranged from 10 to 17 mm and the CEF from 11 to
22 mm
.
The average POS lengths of the Atya lanipes specimens were 12.7
±
1.3, 13.3
±
1.3,
12.1 ±1.0,
11.1
±
0.2, 12.2
±
1.0, 12.0
±
1.0, and 10.8
±
/
−
0.2 mm for the control, 24 h, 72 h, 120 h,
168 h, 216 h, and 264 h exposure specimens, respectively, and the average CEF lengths
were 14.3
±
2.0, 16.6
±
1.4, 14.6
±
1.4, 13.6
±
0.2, 14.2
±
1.1, 14.9
±
1.2, and 14.0
±
0.4 mm,
respectively. The POS and CEF measurements for the control and treated groups were not
significant different.
Table 4.
Average
±
S.E. of the size of the dissected specimens at each exposure time presented as
post-orbital and cephalothorax lengths.
Time of Exposure
(Hours)
POS + S.E.
(mm)
CEF + S.E.
(mm)
Control 12.7 ±/−1.3 14.3 ±/−2.0
24 13.3 ±/−1.3 16.6 ±/−1.4
72 12.1 ±/−1.0 14.6 ±/−1.4
120 11.1 ±/−0.2 13.6 ±/−0.2
168 12.2 ±/−1.0 14.2 ±/−1.1
216 12.0 ±/−1.0 14.9 ±/−1.2
264 10.8 ±/−0.2 14.0 ±/−0.4
Toxics 2023,11, 694 10 of 14
Catalase activity in gastrointestinal tissues between 24 and 264 h of exposure to
10 mg/L
of TiO
2
NPs was accessed. The average catalytic activity of catalase in the control
group was 10.97
±
/
−
0.2 U/mg prot. The catalase activity at each exposure time (24, 72,
120, 168, 216, and 264 h) was 0.79, 15.93, 27.94, 55.90, 19.5, and 17.77 U/mg prot, respectively.
After 24 h of exposure, the catalase activity was approximately 0, which indicates a state of
extreme shock and oxidative stress development in a short period. However, after 264 h of
exposure, there was higher catalase enzymatic activity compared to the control group.
The analysis of catalase activity in the gill tissues showed that, after 24 h of exposure,
the enzyme activity was significantly higher in the exposure group (54.38 U/mgprot) than
the control group (18.09
±
/
−
0.2 U/mg prot). This result differed from the values we
obtained in the analysis of catalase activity in the gastrointestinal tissues. In this case, the
enzymatic activity was higher after 24 h but after 72 h (22.48 U/mg prot), 120 h (
28.87 U/mg
prot), 216 h (22.60 U/mg prot), and 264 h (20.39 U/mg prot), the enzyme activity was
similar to that of the control group, except for the exposure time of 168 h (104.73 U/mg
prot) when the enzyme activity was higher than that after 24 h of exposure.
In the nervous tissues from the control group, the average enzyme activity was
258.04 ±/−0.3 U/mg
prot. However, the catalase activity for each exposure time was
69.44, 62.23, 31.70, 135.0, 24.01, and 147.02 U/mg prot for the exposures times of 24, 72, 120,
168, 216, and 264 h, respectively (Figure 7).
Toxics 2023, 11, x FOR PEER REVIEW 11 of 15
(a)
(b)
(c)
Figure 7. Atya lan ipes catalase activity in tissues after 24 to 264 h of exposure to 10 mg/L of TiO
2
NPs.
The red triangle is the catalase activity in the control group. (a) Gastrointestinal tissues, (b) nervous
tissues, and (c) gill tissues.
4. Discussion
This study assessed the neurotoxicity of TiO
2
NPs in Atya lan ip es shrimp after a 24 h
acute exposure and documented the development of oxidative stress after 24 to 264 h of
exposure. Currently, more studies must be carried out related to the biocompatibility and
toxicity of engineering nanomaterials [29,30]. This is because their use continues to in-
crease in many sectors globally including medicine, the food industry, and technology
[31]. The biological and ecosystem interactions with nanomaterials are important in de-
fining and preventing future impacts on ecosystems such as freshwater ones. Also, these
data could help in the production, management, and future environmental regulation of
engineered nanoparticles. Therefore, their possible interactions with living organisms and
their toxicity must be considered before commercial use.
Figure 7.
Atya lanipes catalase activity in tissues after 24 to 264 h of exposure to 10 mg/L of TiO
2
NPs.
The red triangle is the catalase activity in the control group. (
a
) Gastrointestinal tissues, (
b
) nervous
tissues, and (c) gill tissues.
Toxics 2023,11, 694 11 of 14
4. Discussion
This study assessed the neurotoxicity of TiO
2
NPs in Atya lanipes shrimp after a 24 h
acute exposure and documented the development of oxidative stress after 24 to 264 h of
exposure. Currently, more studies must be carried out related to the biocompatibility and
toxicity of engineering nanomaterials [
29
,
30
]. This is because their use continues to increase
in many sectors globally including medicine, the food industry, and technology [
31
]. The
biological and ecosystem interactions with nanomaterials are important in defining and
preventing future impacts on ecosystems such as freshwater ones. Also, these data could
help in the production, management, and future environmental regulation of engineered
nanoparticles. Therefore, their possible interactions with living organisms and their toxicity
must be considered before commercial use.
Most of the nano-toxicological studies have been carried out in organisms that are
not exposed to nanomaterials in the aquatic environment where they naturally live [
32
].
However, in the communities and populations of freshwater shrimp, there has been little
attention on this matter. It has been shown in studies related to other aquatic contaminants
such as pesticides, that these macroinvertebrates are a good toxicological model [
33
]. For
Atya lanipes and TiO
2
NPs, their lethal and sublethal effects in larval stages [
26
] have been
evaluated and documented. A significant hypoactivity in the movement of the larvae
after acute exposure was observed. In this study, in the adult stage of the Atya lanipes
shrimp, we observed that, after a 24 h acute exposure to low concentrations of TiO
2
NPs,
the shrimp exhibited hypoactive movements. Significantly, the heat maps documenting
the “normal” movements of exploration of the Atya lanipes shrimp to be in the corners of
the box rather than the center. This behavior is expected in this shrimp species since it
is a standard mechanism to remain still or in the center to evade predation. Thus, when
not exposed to any treatment, healthy adult shrimp showed constant movement in this
pattern for more than half of the recording time. However, shrimp exposed to titanium
treatment or titanium and titanium dioxide nanoparticles began to show a decrease in this
pattern of exploration. In particular, the shrimp exposed to 3 mg/L TiO
2
NPs did not show
movement within the recording box. This is consistent with other studies that evaluated
animal behavior after exposure to this nanomaterial, as neotropical tadpoles and zebrafish
also presented this characteristic hypoactivity [34,35].
Scientific investigations have shown the relevance of the sublethal effects of toxicants.
In the past, we referred to toxicity primarily as the ability of contaminants to cause the
death of the organisms under study. Today, we know that sublethal effects can produce
a great disparity in the organism’s functioning that can be extrapolated to effects on the
ecosystem it is part of. Therefore, it is necessary to know the probability that a specific
contaminant concentration will produce a defined effect, in this case, a sublethal effect.
When carrying out the Probit analysis for the variable of active time of the shrimp, we
obtained an average index of 0.14 mg/L. These data demonstrate the susceptibility of this
species to an acute exposure to TiO
2
NPs of only 24 h. The need to evaluate toxicological
indices for sublethal effects for this species at different exposure times is evident.
In general, acute toxicity studies (
≤
96 h) in bacteria (V. fischeri), green algae (Pseu-
dokirchneriella sub-capitata and Chydorus sphaericus), some crustaceans (D. magna and T.
platyurus), and fish embryos (D. rerio) have shown little or no toxicity when exposed to TiO
2
NPs [
36
–
38
]. Recent research shows that exposure to many emerging contaminants can
induce biochemical responses such as oxidative stress (as an example of sublethal effects)
after an acute exposure without any lethality for that same exposure time [
39
]. However, a
recent study evaluated the nanotoxicity of TiO
2
toward the freshwater shrimp Atya lanipes
in their zoea larval stages and the results showed that exposure to these nanoparticles
induced mortality after 48 h of exposure, with edema, less pigmentation development, and
hypoactivity in the larvae [26].
The characteristic biological effect of TiO
2
NPs is the development of oxidative stress.
Oxidative stress occurs when reactive oxygen species (ROS) are produced uncontrol-
lably [
24
]. ROS are interrelated with many cellular mechanisms. They are highly reactive
Toxics 2023,11, 694 12 of 14
species which allows them to react with many biomolecules, affecting their function and/or
damaging them and potentially leading to cellular apoptosis [
40
,
41
]. Fortunately, the cell
has molecules and enzymes that have antioxidant roles. One of the most important is
the enzyme catalase. The activity of this enzyme below or above the expected or normal
values may indicate an increase in ROS and therefore the presence of oxidative stress [42].
This excess ROS in response to a xenobiotic contaminant can overwhelm the antioxidant
mechanisms present in the organism [
43
]. This can cause oxidative damage and loss of
compensatory mechanisms and a suppression of antioxidant enzymatic activities [
43
,
44
].
In this study, the catalase enzymatic activity of the groups exposed to TiO
2
NPs were signif-
icantly different from that of the control group. It was shown that a 24 h acute exposure
to TiO
2
NPs resulted in the development of oxidative stress in gill, gastrointestinal, and
nervous tissues.
The neurotoxicity after a 24 h acute exposure to TiO
2
NPs and the development of
oxidative stress in the freshwater shrimp species Atya lanipes was documented in this study.
The toxic effects of acute exposure to this emerging aquatic pollutant was characterized
by sublethal effects such as behavioral changes and induction of oxidative stress. Also,
we suggest that freshwater shrimp are an excellent nano-toxicological model because
of their life cycle and susceptibility to the presence of nanomaterials in their freshwater
ecosystems and their ecological role in the biofiltration of natural organic particles in these
ecosystems [45–47]
. Future research should evaluate other sublethal effects such as bioac-
cumulation of the TiO2NPs in Atya lanipes shrimp to understand their biological routes.
Some limitations of this study are in the assessment of the movement of the shrimp
when exposed to TiO
2
NPs. It was not possible to record the shrimp in the same exposure
tank due to the presence of artificial sediment that was part of the microcosm and because
the system that was used to analyze the behavior of the shrimp did not recognize the
sediment as different from the shrimp due to their similar dark colors. It was not possible
to determine “locomotion” so the “movement” of the shrimps was analyzed. Although it
was recorded in a red box with red lights, which cannot be detected by these shrimps, and
we promoted their normal active behavior at night, the removal of the shrimp from the
exposure microcosm to fresh, clean, and oxygenated water prevented us from determining
their locomotion. However, a short acclimatization period was allowed to avoid losing
the effect of the TiO
2
NPs in the exposure medium. All other variables were successfully
controlled to obtain a movement assessment after exposure to TiO2NPs.
This research contributes to our understanding of the nanotoxicity of TiO
2
NPs and
provides a starting point to determine the importance of regulating this type of nanomaterial
and controlling concentrations of this contaminant in freshwater ecosystems. These results
are very relevant in the scientific community because they present data for an area that has
not received much attention in the field of nano-ecotoxicology and can promote research
towards understanding the biocompatibility of these nanomaterials. At the same time,
it shows us the need to prevent biological, ecological, and environmental impacts in the
short, medium, and long term, for the purpose of conserving aquatic environments and
biodiversity both in Puerto Rico and around the world.
Author Contributions: Conceptualization, S.C.-R. and O.P.-R.; methodology, S.C.-R.; software, S.C.-
R.; validation, O.P.-R.; formal analysis S.C.-R. and O.P.-R.; investigation, S.C.-R. and O.P.-R.; resources,
O.P.-R.; data curation, S.C.-R.; writing—original draft preparation, S.C.-R.; writing—review and
editing, O.P.-R.; visualization, S.C.-R. and O.P.-R.; supervision, O.P.-R.; project administration, S.C.-R.
and O.P.-R.; funding acquisition, S.C.-R. and O.P.-R. All authors have read and agreed to the published
version of the manuscript.
Funding:
This study was supported by the Puerto Rico Center for Environmental Neuroscience
(PRCEN), grant # HRD-11736019 (PRCEN2), and the Shrimp and Fish Ecology Laboratory at the
University of Puerto Rico—Río Piedras Campus.
Institutional Review Board Statement:
Ethical review and approval were waived for this study due
to the University of Puerto Rico, Rio Piedras does not requires the approval for invertebrates models.
Toxics 2023,11, 694 13 of 14
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
We thank Edgar Lozada and Fernando Villar for their help in preparing the
materials used for the methodology.
Conflicts of Interest: The authors declare no conflict of interest.
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