Journal Pre-proof

Acute and chronic effects of environmental realistic concentrations of

simvastatin in Danio rerio: evidences of oxidative alterations and
endocrine disruptive activity<!–<ForCover>Rebelo D, Correia AT, Nunes
B, Acute and chronic effects of environmental realistic concentrations of
simvastatin in Danio rerio: evidences of oxidative alterations and
endocrine disruptive activity, Environmental Toxicology and
Pharmacology, doi: 10.1016/j.etap.2020.103522</ForCover>–>

D. Rebelo (Methodology) (Validation) (Formal analysis)

(Investigation) (Data curation) (Writing - original draft), A.T. Correia
(Methodology) (Validation) (Formal analysis) (Investigation) (Data
curation) (Writing - review and editing), B. Nunes
(Conceptualization) (Methodology) (Validation) (Resources) (Writing
- review and editing) (Supervision) (Project administration) (Funding
acquisition)

PII: S1382-6689(20)30198-8
DOI: https://doi.org/10.1016/j.etap.2020.103522
Reference: ENVTOX 103522

To appear in: Environmental Toxicology and Pharmacology

Received Date: 16 December 2019

Revised Date: 1 July 2020
Accepted Date: 20 October 2020

Please cite this article as: { doi: https://doi.org/

This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.

© 2020 Published by Elsevier.

Acute and chronic effects of environmental realistic concentrations of simvastatin
in Danio rerio: evidences of oxidative alterations and endocrine disruptive activity

Rebelo, D.1,2, Correia, A.T.3,4, Nunes, B.1,2,* nunes.b@ua.pt

1
Departamento de Biologia, Universidade de Aveiro, Campus de Santiago, 3810-193
Aveiro, Portugal

2
Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus
de Santiago, 3810-193 Aveiro, Portugal

of
3
Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR),
Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos S/N, 4550-

ro
208 Matosinhos, Portu=gal

4 -p
Faculdade de Ciências da Saúde, Universidade Fernando Pessoa (UFP), Rua Carlos da
Maia 296, 4200-150, Porto, Portugal
re
lP

*Corresponding author

Bruno Nunes
na

Departamento de Biologia da Universidade de Aveiro, Campus Universitário de Santiago,

3810-193 Aveiro, Portugal
ur

Highlights
 Simvastatin exposure in zebrafish affected behaviour through hiperactivity.
 Simvastatin’s antioxidant properties caused a decrease in biochemical biomarkers.
Jo

 This antihyperlipidemic drug did not act as a reproductive disruptor.

Abstract

1
Due to their wide use, pharmaceuticals can be discarded, metabolized and excreted into
the environment, potentially affecting aquatic organisms. Lipid-regulating drugs are
among the most prescribed medications around the world, to control human cholesterol
levels, in more than 20 million patients. Despite this massive use of lipid-regulating drugs,
particularly simvastatin, the role of these drugs is not fully characterized and understood
in terms of its potential toxicological effects at the environmental level. This work
intended to characterize the toxicity of an acute (120 hours post-fertilization) and chronic
(60 days) exposure to the antihyperlipidemic drug simvastatin (in concentrations of 92.45,
184.9, 369.8, 739.6 and 1479.2 ng L-1), in the freshwater species of zebrafish (Danio
rerio). The concentrations hereby mentioned were implemented in both exposures, and
were based on levels found in wastewater treatment plant influents (11.7±3.2 µg L-1),

of
effluents (2.65±0.8 µg L-1) and Apies River (1.585±0.3 µg L-1), located in Pretoria, South
Africa and, particularly in the maximum levels found in effluents from wastewater

ro
treatment plants in Portugal (369.8 ng L-1). The acute effects were analysed focusing on
behavioural endpoints (erratic and purposeful swimming), total distance travelled and
-p
swimming time), biomarkers of oxidative stress (the activity of the enzymes superoxide
dismutase, catalase, glutathione peroxidase), biotransformation (the activity of
re
glutathione S-transferases) and lipid peroxidation (thiobarbituric acid reactive
substances). Animals chronically exposed were also histologically analysed for sex
lP

determination and gonadal developmental stages identification. In terms of acute

exposure, significant alterations were reported in terms of behavioural alterations
(hyperactivity), followed by a general reduction in all tested biomarkers. Also, the
na

analysis of chronically exposed fish evidenced no alterations in sex ratio and maturation
stages. In addition, the analysis of chronically exposed fish evidenced no alterations in
terms of sexual characteristics, suggesting that the chronic exposure of Danio rerio to
ur

simvastatin does not alter the sex ratio and maturation stages of individuals. This
assumption suggests that simvastatin did not act as an endocrine disruptor. Moreover, the
Jo

metabolism, neuronal interactions and the antioxidant properties of SIM seem to have
modulated the hereby-mentioned results of toxicity. Results from this assay allow
inferring that simvastatin can have an ecologically relevant impact in living organisms.

Keywords: zebrafish; antihyperlipidemic; reproductive disruptors; behaviour;

biomarkers; histology.

Introduction
2
 The use of pharmaceuticals to prevent and treat several diseases has led to a
worldwide increase in their consumption and environmental presence (Jelic et al., 2011;
Kaczala & Blum, 2015; Ebele et al., 2017). Some pharmaceuticals are discarded directly
into the wild or sent to wastewater treatment plants (WTTPs) from which are eliminated
after treatment, being ultimately discarded into receiving waters (Boxall, 2004). The
results of previous investigations have shown that classic WWTPs cannot completely
remove many pharmaceuticals from residual waters (Paíga et al., 2019). Physicochemical
properties of drugs, such as biodegradability, lipophilicity, solubility, photosensitivity,
and volatility, as well as operation and climate conditions during the process of treatment,
can affect the removal rate of pharmaceuticals (Boxall, 2004; Gracia-Lor et al., 2012).

of
Thus, the occurrence has been detected worldwide, in sewage treatment plants, seawater,
surface water and groundwater (Nikolaou et al., 2007).

ro
When in the environment, these substances may potentially affect aquatic
organisms and cause short and long-term effects, including toxicological interactions with
-p
varied pathways and receptors (El-Saad & Elgerbed, 2010). By attaining this purpose,
drugs may damage the proper functioning of the liver and the central nervous system
re
(CNS), affecting behaviour (El-Saad & Elgerbed, 2010; Monat-Descamps & Deschamps,
2012; Omar & Mahmoud, 2017). One type of behavioural parameter that may be
measured is the swimming patterns of fish, as the individuals can develop altered
lP

locomotory responses, and the frequency of swimming movements and duration of

activity can change due to the exposure to xenobiotics (Sharma, 2019). Movement
na

analysis is often conducted by automated biomonitoring systems because of their

sensitivity, involving the quantification of swimming patterns and videography
(Dagneaux et al., 2009).
ur

The components and extent of biological response to contaminants can also be

measured by biochemical biomarker assessment (Jemec et al., 2010). One of the most
Jo

recurrent responses to chemical compounds involves their metabolism through the

activation of phase I of enzymatic complexes, namely cytochrome monooxygenases P450
enzymes (CYP450) (Holth et al., 2008; Sharma et al., 2012). Commonly, the poor
coupling of the CYP450 catalytic cycle results in the continuous production of reactive
oxygen species (ROS), that are relevant in signalling pathways but may be responsible
for oxidative stress, where the production of ROS surpasses the efficacy of antioxidant
defences (Betteridge, 2000; Ray et al., 2012; Banerjee et al., 2016). Some examples of
3
biomarkers of oxidative stress are superoxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GPx). SOD constitutes protection against the radical superoxide
(O2-) toxic effects, as it catalyzes its dismutation into hydrogen peroxide (H2O2) and
molecular oxygen (O2). As H2O2 is continuously produced in the organism, two enzymes
ensure its removal, namely CAT and GPx (Lumb, 2017). CAT is part of the antioxidant
defence system that occurs in peroxisomes (Modesto & Martinez, 2010), subcellular
structures where many enzymes that are responsible for the production of H2O2 are
located (Djordjević, 2004). GPx also acts against H2O2, by the conversion of reduced
glutathione (GSH) and H2O2 into water and oxidized glutathione (GSSG) (Blondet et al.,
2018).

of
One example of phase II enzymes is the group of metabolic isoenzymes
glutathione S-transferases (GSTs), which act by conjugating GSH with electrophilic

ro
centers, resulting in significant water solubility of the newly formed complex, and the
ultimate detoxification of xenobiotics. In that way, GSTs prevent the interaction of
-p
xenobiotic substrates with nucleic acids and cellular proteins (Dzoyem & Eloff, 2014).
GSTs can also transport proteins and some of their isoenzymes can reduce organic
re
hydroperoxides and are responsible for the isomerization of unsaturated substances,
protecting the organism against oxidative stress, and therefore, oxidative damage
(Rahman, 2007; Smith et al., 2013; Dzoyem & Eloff, 2014).
lP

Lipid peroxidation can result because of the depletion or inefficacy of the previous
oxidative damage preventive measures (Sharma et al., 2012). Free radicals interact with
na

lipids present in membranes, particularly unsaturated fatty acids (Halliwell, 2009). As

secondary products, various aldehydes are formed, such as malondialdehyde (MDA)-like
substances, whose levels are used as one of the most comprehensive indicators of the total
ur

amount of final products of lipid peroxidation (Lykkesfeldt, 2007; Ayala et al., 2014).
The quantification of such substances may be performed by measuring the levels of
Jo

TBARS (Dzoyem & Eloff, 2014).

Some xenobiotics, known as Endocrine-Disrupting Chemicals (EDCs), act by

favouring endocrine disruption (Voss et al., 2005; Bogers, 2008). Some examples of this
type of EDCs of anthropogenic origin are pharmaceuticals (Metzler, 2006). One of the
most sensitive and important effects caused by exposure to EDCs are deleterious
histological modifications of gonadal tissues, which may result in changes in sex ratio

4
and maturation stages of affected individuals (Bogers, 2008). Danio rerio was suggested
as a model organism for the assessment of EDCs effects in terms of gonad development
(Örn, 2006).

Lipid-regulating drugs are one of the most prescribed medications around the
world, to control human cholesterol levels (Davidson, 2009). There are several lipid-
regulating drugs, such as fibrates, inhibitors of absorption of cholesterol, nicotinic acid,
and fish oil derivatives, but statins are the predominant group (Rang et al., 2007). Statins
exert their therapeutic activity by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase (Davidson, 2009; Rang et al., 2007). HMG-CoA reductase
catalyzes the conversion of HMG-CoA to mevalonic acid, a cholesterol precursor

of
(Chavan-Gautam et al., 2018). Thus, the most evident effect of statins is the reduction of
plasma (low-density lipoproteins) LDL, some reduction in plasma triglycerides, and an

ro
increase in HDL (high-density lipoproteins) (Rang et al., 2007). One of the most sold
statin worldwide is simvastatin (SIM). SIM can also increase mitochondrial and
-p
peroxisomal β-oxidation of fatty acids (Park et al., 2016). The degradation of fatty acids
has the primary goal of the formation of acetyl-CoA, to serve as a carbon and energy
re
source, essential for the metabolism of the individual. Thus, it is possible to expect an
increase in locomotion of individuals exposed to this substance (Bhagavan & Ha, 2015).
The antioxidant properties of SIM should also decrease erratic behaviour, through the
lP

reduction of stress and anxiety-like behaviour (Bouayed, 2011; Hassan et al., 2014; De
Carvalho et al., 2019). β-oxidation and the Krebs cycle are closely related to the electron
na

transport chain. In turn, the final electron acceptor of the electron transport chain consists
in O2, which leads to the production of H2O, and ultimately to the formation of ROS, such
as O2-, HO- and H2O2 (Speijer et al., 2014). The production of ROS can, therefore, lead
ur

to oxidative stress, and ultimately to oxidative damage, thus altering the activity of such
enzymes. However, the antioxidant properties of SIM could counteract this effect, by
Jo

decreasing the activity of these enzymes (Hassan et al., 2014). HMG-CoA reductase
inhibitors, such as statins, are thought to affect sex hormone biosynthesis, inhibiting the
synthesis of cholesterol, a precursor of estradiol and androstenedione, which should affect
sex ratio and maturation stages of individuals exposed to SIM (Ser et al., 2010).

Considering its wide and continuous use by humans, the occurrence of SIM in
wastewater all over the world has been reported in various studies, in the range of 0.04-
718 ng L-1, in multiple countries, such as Greece, with concentrations reaching 718 ng L-
5
1
, in wastewater influents (Papageorgiou et al., 2016); in the UK, reaching 115 and 5 ng
L-1, in wastewater influents and effluents, respectively (Kasprzyk-Hordern et al., 2009;
Boleda et al., 2011; Martín et al., 2011; Papageorgiou et al., 2016; Tete et al., 2019); and
in Spain, 7.5 ng L-1, in a drinking water treatment plant (Boleda et al., 2011). Samples
from the river Danube also reported a concentration of 0.04 to 0.7 ng L-1 (Martín et al.,
2011). In Pretoria, South Africa, the concentrations of simvastatin found for WWTP
influents, effluents and in Apies River, were 11.7±3.2 µg L-1, 2.65±0.8 µg L-1 and
1.585±0.3 µg L-1, respectively (Tete et al., 2019). Particularly at the national level, the
maximum level found in effluents from WWTPs in Portugal was 369.8 ng L-1 (Salgado
et al., 2010). The continuous widespread of this pharmaceutical, which may have

of
toxicological effects in organisms, constitutes a major concern, that was assessed in this
study.

ro
The main goal of this work was to evaluate the toxic effects in Danio rerio
resulting from an acute [120 hours post-fertilization (hpf)] and chronic [60 days post-
-p
fertilization (dpf)] exposure to SIM in ecologically relevant concentrations through
behavioural analysis (erratic and purposeful swimming, total distance and swimming
re
time), biochemical markers of oxidative stress (determination of the activity of the
enzymes SOD, CAT and GPx), biotransformation (GSTs) and lipid peroxidation
(TBARS), and histological assessment (sex determination and assessment of gonadal
lP

developmental stages).
na
ur
Jo

6
Material and methods

Chemicals

Simvastatin (SIM) ([(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-

yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl] 2,2-dimethylbutanoate;
CAS: 79902-63-9; purity ≥ 97 %) was acquired from Sigma-Aldrich (Schnellforf,
Germany). All reagents were purchased from Sigma-Aldrich, except for Bradford
reagent, purchased from Bio-Rad UK. The chemicals used for histological analyses were
ethanol absolute (CAS: 64-17-5), acquired from AGA – Álcool e Géneros Alimentares
S.A.; Appliclear (Xylene substitute) (CAS: 64742-49-0), Bouin liquor for clinical

of
diagnosis, and Histofix decalcifier 1 for clinical diagnosis, all were purchased from
PanReac AppliChem ITW Reagents; Hematoxylin solution modified acc. to Gill III,

ro
Certistain (Eosin y) (CAS: 15086-94-9) and DPX new (non-aqueous mounting medium
for microscopy) were acquired from Merck-Millipore; Hydrochloric acid standard
solution 1M was purchased from Fluka Analytical (Sigma-Aldrich). All other chemicals
-p
(namely those used for buffers preparation) were obtained either from Sigma-Aldrich or
Merck-Millipore.
re
Two stock solutions of SIM were prepared in ultrapure water (Milli-Q-Water), one with
lP

a concentration of 4 mg L-1, which was used to prepare a second stock solution, with a
final concentration of 4 µg L-1. Exposure media were prepared by successive dilution of
the stock solutions, with water provided from Tecniplast ZebTEC Zebrafish Facility
na

recirculating system.
ur

Test organisms
Jo

Zebrafish, Danio rerio, is a freshwater fish species, which constitutes an alternative

animal model in several areas of biological sciences, including Toxicology and
Ecotoxicology, allowing it to be considered as a model organism for vertebrate biology
studies (Dooley & Zon, 2000). This species is ideal not only for the assessment of
alterations in the morphological characteristics of its embryos (malformations), but also
for the evaluation of behavioural responses, and biochemical determinations (Dahm,
2006).

7
Individuals of the test species (Danio rerio) used in this study were kept in a Tecniplast
ZebTEC Zebrafish Facility recirculating system under controlled conditions (OECD,
2013a). Water temperature was 26 ± 1°C, pH was 7.5 ± 0.5, conductivity was 750 ± 50
µS cm-1, and dissolved oxygen was equal or above 95% of saturation. The culture water
was filtered and purified (by activated carbon and reverse osmosis) tap water, to which
“Instant Ocean Synthetic Sea Salt” (Spectrum Brands, USA) was added to regulate the
conductivity to 800 µS cm-1. The photoperiod cycle was upheld at 16h:8h (light: dark).
The adult individuals were fed twice daily during the week, once on the weekend, with
an artificial diet [GEMMA Micro 500, acquired from Skretting Zebrafish (Maine, United
States)]. To protect eggs from predation by adult individuals, marbles were placed in the

of
bottom of the aquarium for refuge. After the removal of the marbles, eggs were collected,
rinsed with water and malformations or absence of cleavage were identified under a
stereomicroscope (Nikon, SMZ 1500); malformed eggs were immediately discarded.

ro
Concentrations of chemicals
-p
The concentrations to which test organisms were exposed, in both acute and chronic
exposures, were 92.45, 184.9, 369.8, 739.6, and 1479.2 ng L-1. The highest concentration
re
was directly prepared from the stock solution, and submitted to successive dilution, as
mentioned before. The intermediate concentration value was based on the maximum level
lP

found in effluents (369.8 ng L-1) from WWTPs in Portugal (Salgado et al., 2010), taking
also into account the levels found in WWTP influents (11.7±3.2 µg L-1), effluents
(2.65±0.8 µg L-1) and Apies River (1.585±0.3 µg L-1), all located in Pretoria, South Africa
na

(Tete et al., 2019).

Acute exposure
ur

The method used in acute exposure was a modified version of the fish embryotoxicity test
(OECD, 2013a). In the present study, eggs from three different breeding aquariums (with
Jo

matured males and females) were transferred to 96-well plates, each well containing 300
μL of each test solution and one egg, seven replicates/plates (16 eggs per replicate, with
a total of 112 eggs) for each concentration and control group, without SIM. For the
measurement of the behavioural (120 hpf) and biomarker activities (after behavioural
analysis), the larvae were exposed during 120 h. According to Carlsson et al. (2013), these
experimental conditions, i.e., the modified fish embryotoxicity test conditions, in terms

8
of duration and method of exposure (96-well plates), do not affect exposed organisms,
and has no effects on obtained results.

Exposure media were newly prepared everyday (approximately 24h) with water retrieved
from the Tecniplast ZebTEC Zebrafish Facility recirculating system, including for the
control group, and partially renewed by approximately 80%, according to the OECD
guidelines (OECD, 2011; OECD, 2013a; OECD, 2013b).

Behavioural test

The period of exposure was extended to 120 hpf to allow the complete development of
the swim bladder of fish. In that way, we could assure the total validity of the locomotor

of
activity test using the Zebrabox (Viewpoint, Lyon, France) tracking system under
stabilized temperature (26±1 °C). Some investigations have performed behavioural

ro
evaluations using Zebrabox before and after 120 hpf, in larvae and adult individuals
(Miscevic et al., 2012; Chen et al., 2016). The larvae were observed for the identification
-p
of morphological alterations or deformities that could compromise their locomotory
capacity, being removed from the test if such conditions were observed, mostly
re
characterized by coagulation of the embryos, with no specific association with SIM
concentration, in the order of 5 to 10% in the exposed larvae (Şişman et al., 2008). The
lP

test was conducted in a cycle of four alternating periods (5 min light; 5 min dark; 5 min
light; 5 min dark), for twenty minutes. The alternate light and dark cycles are justified by
the natural behaviour of D. rerio, which varies from light to dark conditions (Blaser and
na

Peñalosa, 2011); normally developing larvae have visual motor reflex, increasing their
activity in the light, and decreasing in the dark (Emran et al., 2010). Data outputs were
obtained at the end of each cycle and the following parameters were calculated: erratic
ur

and purposeful swimming, total distance travelled (sum of erratic and purposeful
swimming) and swimming time.
Jo

After the motor activity test, pools of 20 larvae were allocated into Eppendorf microtubes
and kept at -80 °C (5 replicates, with a total of 100 larvae per concentration). For the
biomarker analysis, the larvae were homogenized in 1 mL of phosphate buffer (50 mM,
pH = 7.0, with Triton X-100 0.1 %) by sonication (Branson S-250A), on ice, during 30
seconds, and centrifuged (refrigerated centrifuge Thermo Fisher Scientific - Heraeus

9
mod. Megafuge 8R) at 15.000 g during 10 min at 4°C. After centrifugation, the remaining
pellets were discarded, and the supernatants were collected and stored at -80 °C.

Superoxide dismutase activity quantification

The SOD activity quantification (total, copper-zinc (Cu-Zn) SOD, and manganese (Mn)
SOD) was based on the method described by Flohé & Otting (1984). A xanthine-xanthine
oxidase system produced superoxide radicals. The result of the reaction of these radicals
with cytochrome c represented SOD’s total activity. Potassium cyanide inhibited the
activity of Cu-ZnSOD, allowing the quantification of the isolated activity of MnSOD.
The enzymatic activity in all of these cases was spectrophotometrically followed at λ =

of
550 nm and was then expressed in mmol min-1 mg-1 of protein.

Catalase activity quantification

ro
CAT activity quantification was based on the method by Aebi (1984). The decomposition
-p
of H2O2 was catalyzed by CAT. This decomposition was spectrophotometrically followed
at λ = 240 nm, by the decrease of absorbance. The enzymatic activity was then expressed
in μmol min-1 mg-1 of protein.
re
Glutathione peroxidase activity quantification
lP

The GPx activity quantification was based on the method proposed by Flohé & Günzler
(1984). The reduction of GSSG to its reduced form, GSH, by glutathione reductase,
through the oxidation of NADPH (ε = 6.2 mM cm-1), was spectrophotometrically
na

followed at λ = 340 nm. GPx selenium-dependent activity was measured using H2O2 as a
substrate, whereas GPx total activity (selenium-dependent + non-dependent) was
ur

measured using cumene hydroperoxide as a substrate. The enzymatic activity was then
expressed in mmol min-1 mg-1 of protein.
Jo

Glutathione S-transferases activity quantification

GSTs activity quantification was based on the method described by Habig et al. (1974).
The conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH was catalyzed by
GSTs, which led to the formation of a thioether (ε = 9.6 mM cm-1). This formation was
spectrophotometrically followed at λ = 340 nm, by the increase of the absorbance. The
enzymatic activity was then expressed in μmol min-1 mg-1 of protein.

10
Thiobarbituric acid reactive substances quantification

The TBARS quantification was based on the protocol described by Buege & Aust (1978).
In the presence of thiobarbituric acid (TBA) and heat (water bath at approximately 100
°C), MDA-like compounds reacted with TBA, which led to the formation of a coloured
end product. This formation was spectrophotometrically quantified at λ = 535 nm.
TBARS concentrations, expressed as MDA equivalents, were then calculated in μmol
min-1 mg-1 of protein.

Protein determination

The quantification of protein of all samples was based on the protocol by Bradford (1976).

of
The conjugation of the Bradford reagent with the total soluble protein led to the formation
of a coloured and stable complex, which was spectrophotometrically measured at λ = 595

ro
nm. Protein standards were prepared with bovine γ-globulin in a concentration of 1 mg
mL-1.

Chronic exposure
-p
re
Chronic exposure was based on OECD guidelines (OECD, 2011; OECD, 2013b), with
some modifications. In the present study, seven hundred and twenty eggs were transferred
lP

into twenty-four Petri dishes (with 15 mL of medium each), thirty eggs per plate, at the
beginning of the experiment. At 96 hpf, larvae were transferred into the exposure
aquariums (1 L in each aquarium). Twenty-five larvae were assigned per aquarium, two
na

replicates per concentration, including a control group (unexposed individuals). The

aquaria were randomly distributed in the exposure room, under wide spectrum fluorescent
bulbs. The exposure period ended at 60 dpf (chronic exposure) according to the OECD
ur

guideline (OECD, 2011). Fish were fed twice daily during the week, and once during the
weekend, with synthetic rations, particularly GEMMA Micro 150 (9 to 30 dpf), GEMMA
Jo

Micro 300 (30 to 60 dpf), acquired from Skretting Zebrafish (Maine, United States),
according to Martins et al. (2016). The exposure media was partially renewed by
approximately 80%, according to the OECD guidelines and as mentioned before for the
acute exposure (OECD, 2011; OECD, 2013a; OECD, 2013b). The water used to prepare
the exposure media was under controlled conditions (water temperature = 26 ± 1°C, pH
= 7.5 ± 0.5, conductivity = 750 ± 50 µS cm-1 and dissolved oxygen saturation ≥ 95%).

11
Post hatch survival was above or equal to 70% in all treatment groups, in agreement with
the requirements of the adopted OECD guideline (OECD, 2011).

Fish sacrifice

After the chronic exposure, five specimens from each aquarium were euthanized by
immersion into a solution of the anaesthetic MS-222 (200 to 300 mg L-1), according to
the OECD guideline (OECD, 2011) until there was no observable opercular movement
and the specimens could not swim. Experiments took into consideration the AVMA
Guidelines for the euthanasia of animals, and also the Portuguese animal welfare law
(Decreto-Lei 113/2013). This facility is certified by the Portuguese veterinarian authority

of
and the experiments were conducted according to guidelines set up by the animal welfare
commission of the University of Aveiro.

ro
Sample processing

-p
Before the dissection, individuals were measured for length (±0.01 mm) and weight
(±0.01 mg) (Table 1). Fish were dissected, the head and tail were removed and discarded
for the incorporation of Bouin solution into the entire organism, and the specimens (five
re
per replicate) were held in plastic tissue cassettes, followed by immersion into Bouin
solution for 24h. After being chemically fixed in Bouin, the specimens held in tissue
lP

cassettes were decalcified (24h), dehydrated through an increasing series of alcohols (70
%, 80 %, 90 % and 100 %, one hour each), cleared with xylene (2 h), impregnated in
paraffin wax (56 to 58 °C), and sectioned (5 to 7 µm) using a microtome (Leica, Reichert-
na

Jung 2030). These sections were stained with hematoxylin-eosin, mounted with DPX in
coverslips and analyzed by light microscopy (Olympus CX41).
ur

Sex determination and identification of the gonadal developmental stages were made
according to OECD (2010). Female individuals were classified into six categories, such
Jo

as Juvenile (only oogonia), Stage 0 – Undeveloped (oogonia to perinucleolar oocytes),

Stage 1 – Early development (pre-vitellogenic follicles, mostly perinucleolar to cortical
alveolar), Stage 2 – Mid-development (≥ 50% of follicles are early and mid-vitellogenic),
Stage 3 – Late development (mostly late vitellogenic follicles), Stage 4 – Late
development/hydrated (mostly late vitellogenic and mature follicles), and Stage 5 – Post-
ovulatory (mostly spent follicles). Male individuals were also classified into five stages,
namely Juvenile (exclusively spermatogonia), Stage 0 - Undeveloped (immature phases

12
present, with no spermatozoa), Stage 1 – early spermatogenic (immature phases
predominant, with some observable spermatozoa), Stage 2 – mid-spermatogenic
(spermatocytes, spermatids and spermatozoa present in similar quantities), Stage 3 – late
spermatogenic (mature sperm predominant), and Stage 4 – spent (loose connective tissue
with remains of sperm) (OECD, 2010). Immature juvenile fish in which sex and gonadal
staging were difficult to assess were excluded from the analysis.

Statistical analyses

Data from sex ratios and maturation stages were evaluated for independence, comparing
each treatment group with the control, using Fisher’s exact test (FET) and Chi-square

of
(χ2), respectively. On the other hand, data from behavioural and biochemical testing were
evaluated for equal variance (Levene test) and normality (Shapiro-Wil test), before the

ro
statistical analyses, with attempted transformations to the data, when needed. Behavioural
results were compared by One-way analysis of variance (ANOVA) on Ranks, and in some
cases by One-Way ANOVA, when data transformations made it possible. On the other
-p
hand, biochemical results were only compared by or One-way ANOVA (Murphy et al.,
2003). These statistical analyses were followed by a Dunnett or Dunn multi-comparison
re
test, after One-way ANOVA or One-way ANOVA on Ranks, respectively, to identify
significant differences between the treatments and the control group. All statistical
lP

methods followed Zar (2010) procedures. The adopted level of significance for all
statistical methods was 0.05. Data are presented as mean and standard error. The analyses
were performed with the software SPSS 24.0.
na

Results
ur

Behavioural results

Erratic swimming
Jo

According to the results obtained in terms of erratic swimming by fish exposed to SIM,
there were significant differences between exposed groups and the control animals, for
both (light and dark) periods. During the first light cycle (0 to 300s), there was a
significant decrease in erratic swimming, except for the organisms exposed to the second
highest concentration (739.6 ng L-1), compared with control treatment (One-Way
ANOVA on Ranks followed by a Dunn’s test: H5=91.651, p<0.001). During the first dark

13
period (300 to 600s), there was a significant increase, particularly in animals exposed to
the intermediate (369.8 ng L-1) and highest concentrations (1479.2 ng L-1), compared with
control (One-Way ANOVA on Ranks followed by a Dunn’s test: H5=67.317, p<0.001).
During the second light cycle (600 to 900s), there was a significant decrease of this
parameter in fish exposed to the lowest concentration (92.45 ng L-1), followed by a
significant increase in those exposed to the highest concentration (1479.2 ng L-1),
comparing to the control (One-Way ANOVA on Ranks followed by a Dunn’s test:
H5=35.1, p<0.001). In the final dark period (900 to 1200s), there was a significant
decrease, namely in animals exposed to the lowest (92.45 ng L-1) and second highest
concentrations (739.6 ng L-1), followed by a significant increase in individuals subjected
to the highest concentration (1479.2 ng L-1), compared with control (One-Way ANOVA

of
on Ranks followed by a Dunn’s test: H5=101.299, p<0.001) (Fig. 1A).

ro
Purposeful swimming

Considering purposeful swimming by fish exposed to SIM, there were significant

-p
differences between the exposed groups and the control fish, for both periods. During the
first light cycle (0 to 300s), there was a significant increase, except for the organisms
re
exposed to the lowest (92.45 ng L-1) and highest (1479.2 ng L-1) concentrations, compared
with control (One-way ANOVA followed by a Dunnett’s test: H5=111.148, p<0.001).
lP

During the first dark period (300 to 600s), there was a significant decrease in the
individuals subjected to the lowest concentration (92.45 ng L-1), followed by a significant
increase in those exposed to the second highest concentration (739.6 ng L-1), compared
na

with control animals (One-Way ANOVA on Ranks followed by a Dunn’s test: H5=
59.682, p<0.001). During the second light cycle (600 to 900s), there was a significant
decrease in organisms exposed to the lowest concentration (92.45 ng L-1), followed by a
ur

significant increase in those subjected to the two highest concentrations (739.6 and 1479.2
ng L-1), compared with control (One-Way ANOVA on Ranks followed by a Dunn’s test:
Jo

H5=86.617, p<0.001). In the final dark period (900 to 1200s), there was a significant
increase of this parameter in individuals exposed to the three highest concentrations
(369.8, 739.6 and 1479.2 ng L-1), compared with control (One-Way ANOVA on Ranks
followed by a Dunn’s test: H5=61.514, p<0.001) (Fig. 1B).

Total distance travelled

14
Considering the total distance travelled by fish exposed to SIM, there were significant
differences found between the exposed individuals and the control group in all the light
and dark periods. During the first light cycle (0 to 300s), there was a significant increase,
particularly in the individuals exposed to the lowest (92.45 ng L-1), intermediate (369.8
ng L-1) and second highest concentration (739.6 ng L-1), compared with control (One-
Way ANOVA on Ranks followed by a Dunn’s test: H5=57.018, p<0.001). During the first
dark period (300 to 600s), there was a significant decrease in the organisms exposed to
the lowest concentration (92.45 ng L-1), followed by a significant increase in those
exposed to the two highest concentrations (739.6 and 1479.2 ng L-1), compared with
control fish (One-Way ANOVA on Ranks followed by a Dunn’s test: H5=82.244,

of
p<0.001). During the second light cycle (600 to 900s), there was a significant decrease in
the individuals exposed to the lowest concentration (92.45 ng L-1), followed by a
significant increase in those exposed to the second highest concentration (739.6 ng L-1),

ro
compared with control (One-Way ANOVA on Ranks followed by a Dunn’s test:
H5=75.903, p<0.001). During the second and final dark period (900 to 1200s), there was
-p
a significant increase in the organisms exposed to the two highest concentrations (739.6
and 1479.2 ng L-1), compared with control (One-Way ANOVA on Ranks followed by a
re
Dunn’s test: H5=60.931, p<0.001) (Fig. 2A).

Swimming time
lP

According to the results of the swimming time measured in fish exposed to SIM, there
were significant differences between treatment groups and the control, in the light and
na

dark cycles. During the first light cycle (0 to 300s), there was a significant decrease in
organisms exposed to the intermediate (369.8 ng L-1) and second highest concentration
(739.6 ng L-1), compared with control (One-Way ANOVA on Ranks followed by a
ur

Dunn’s test: H5=18.426, p=0.002). During the first dark period (300 to 600s), there was
a significant decrease in the organisms exposed to the two lowest concentrations (92.45
Jo

and 184.9 ng L-1), followed by a significant increase in the two highest concentrations
(739.6 and 1479.2 ng L-1), compared with control (One-Way ANOVA on Ranks followed
by a Dunn’s test: H5=99.196, p<0.001). During the second light cycle (600 to 900s), no
significant differences were found between animals from the exposed groups and those
from the control. In the final dark cycle (900 to 1200s), there was a significant decrease
in individuals exposed to the lowest concentration (92.45 ng L-1), followed by a
significant increase in those subjected to the two highest concentrations (739.6 and 1479.2
15
ng L-1), compared with control (One-Way ANOVA on Ranks followed by a Dunn’s test:
H5=92.713, p<0.001) (Fig. 2B).

Biochemical results

Superoxide dismutase activity

According to the results obtained in terms of Cu-ZnSOD activity in fish exposed to SIM,
there was a significant decrease, except for the individuals exposed to the lowest
concentration (92.45 ng L-1), compared with control fish (One-way ANOVA followed by
a Dunnett’s test: F5.15=37.728, p<0.001). MnSOD activity in the same individuals
revealed no significant differences between treatment groups and the control (Fig. 3A).

of
Catalase activity

ro
Considering the results obtained in terms of the CAT activity in fish exposed to SIM,
there were no significant differences were found among all experimental groups (One-
way ANOVA: F5.12=3.784, p=0.027) (Fig. 3B).
-p
re
Glutathione peroxidase activity

According to the results of total GPx activity measured in fish exposed to SIM, there was
lP

a significant decrease, except for animals subjected to the lowest concentration (92.45 ng
L-1), compared with control organisms (One-way ANOVA followed by a Dunnett’s test:
F5.15=8.567, p<0.001). Selenium-dependent GPx activity reported a significant increase
na

in organisms exposed to the lowest concentration (92.45 µg L-1), compared with control
animals (One-way ANOVA followed by a Dunnett’s test: F5.13=16.685, p<0.001) (Fig.
3C).
ur
Jo

16
Glutathione S-transferases activity

Considering the GSTs activity of fish exposed to SIM, there was a significant decrease in
animals of all the treatment groups, compared with control fish (One-way ANOVA
followed by a Dunnett’s test: F5.18=17.955, p<0.001) (Fig. 4).

Thiobarbituric acid reactive substances levels

The results obtained in terms of TBARS levels in fish exposed to SIM, evidenced a
significant decrease, except for the individuals exposed to the lowest concentration (92.45
ng L-1), compared with control fish (One-way ANOVA followed by a Dunnett’s test:
F5.13=12.107, p<0.001) (Fig. 5).

of
Sex determination

ro
Taking into consideration the visual inspection of the HE glass slides of gonads, no
significant differences were found, in terms of sex determination (i.e. the percentage of
-p
males vs females), between the experimental groups and the control (FET, p=1, for 92.45
and 184.9, separately compared with the control; FET, p=0.301 for 369.8 ng L-1,
re
compared with the control; FET, p=0.565 for 739.6 ng L-1, compared with the control;
FET, p=0.085 for 1479.2 ng L-1, compared with the control), although some type of
lP

feminization appears to occur in individuals exposed to higher SIM concentrations (Fig.

6).
na

Gonadal developmental stages

Only three of the six stages of maturation before mentioned were identified in females
exposed to SIM, namely Stage 0 – Undeveloped (Fig. 7A), Stage 1 – Early development
ur

(Fig. 7B), Stage 2 – Mid-development (Fig. 7C). Regarding the recorded developmental
stages, no significant differences in the percentage of individuals in each stage were found
Jo

between treatment groups, compared with the control (χ2=3.214, df=1, p=0.073, for 92.45
ng L-1, compared with the control; χ2=1.556, df=2, p=0.459 for 184.9 ng L-1, compared
with the control; χ2=1.440, df=2, p=0.487 for 369.8 ng L-1, compared with the control;
χ2=2.667, df=1, p=0.102 for 739.6 ng L-1, compared with the control; χ2=0, df=1, p=1 for
1479.2 ng L-1, compared with the control) (Fig. 8A).

17
Only three of the five stages of maturation mentioned above were identified in male
individuals exposed to SIM, namely Stage 0 – Undeveloped (Fig. 7D), Stage 1 – Early
spermatogenic (Fig. 7E) and Stage 2 – Mid-spermatogenic (Fig. 7F). Taking into
consideration the observation of male developmental stages in individuals exposed to
SIM, no significant differences were found between treatment groups and fish from the
control treatment (χ2=0.194, df=2, p=0.907 for 92.45 ng L-1, compared with the control;
χ2=2.333, df=2, p=0.311 for 184.9 ng L-1, compared with the control; χ2=1.875, df=2,
p=0.392 for 369.8 ng L-1, compared with the control; χ2=0.833, df=2, p=0.659 for 739.6
ng L-1, comparing with the control) (Fig. 8B).

A summary of the major findings, and a potential relationship among the here analysed

of
parameters, is represented in Fig. 9.

ro
Discussion

Behavioural assessment
-p
According to the results obtained for erratic swimming, observed in individuals
exposed to SIM, the general patterns observed consisted in a decrease in the first light
re
period, an increase in the first dark cycle, and a decrease in the second light and dark
periods, compared with the control group. Regarding the results obtained in terms of
lP

purposeful swimming in individuals exposed to SIM, the overall patterns reported were
an increase in the first light period, a decrease in the first dark cycle and an increase in
the second light and dark periods, compared with control organisms. Overall, there was a
na

decrease in erratic swimming and an increase in purposeful swimming. Moreover, erratic

and purposeful swimming seemed to have a proportionally inverse relation. Campos et
ur

al. (2016) also reported decreasing movement in zebrafish embryos exposed to SIM (0.3
nM to 10 µM). In our study, the decrease in erratic movements and the increase in
purposeful movements suggest a reduction of anxiety-like behaviour. Some
Jo

pharmaceuticals with antioxidant properties, such as SIM, are known to decrease erratic
movements of individuals, such as in this case, through the reduction of stress and
anxiety-like behaviour (Bouayed, 2011; Hassan et al., 2014; De Carvalho et al., 2019).

Regarding the results of total distance travelled in individuals exposed to SIM, the
overall pattern suggests an increase in the first light and dark cycles, relatively similar
values in the second light period, and an increase in the second dark cycle, compared with

18
the control group. According to the results obtained for swimming time in individuals
exposed to SIM, the general patterns observed were a decrease in the first light cycle, an
increase in the first dark period, relatively similar values in the second light cycle and an
increase in the second dark period, compared with control. Thus, exposed individuals
showed an increase in total distance travelled, as well as increased swimming time, which
means that more distance was travelled while they spent more time swimming. This
indicates that SIM could have caused an increase in the overall activity of the organisms
exposed. As mentioned before, SIM can increase the β-oxidation of fatty acids (Park et
al., 2016). The increasing degradation of fatty acids leads to a higher input of energy in
the individuals, leading to more locomotion activity (Bhagavan & Ha, 2015). Overall,

of
data from behavioural analysis suggests that SIM potentiates the locomotion of
individuals, leading also to a decrease in erratic swimming.

ro
Moreover, when exposed to alternating light and dark periods, zebrafish larvae
presented increased activity in light periods, and decreased activity in the dark cycles, in
-p
agreement with the results obtained by Emran et al. (2010). In this assay, through the
analysis of electroretinograms, the authors concluded that visual responsiveness can be
re
partially related to circadian rhythms, where it seems that larvae can anticipate night and
day. At 120 hpf (exactly when the behavioural analysis was performed in our assay),
larvae had just started to eat, and required conserving energy. This is a mechanism that
lP

could explain the increase in activity during light periods, and decrease in the dark cycles
(Prober et al., 2006).
na

In all of the behavioural endpoints assessed, a decrease in activity is noticeable

from the first light cycle (0 to 300s) to the second (600 to 900s). This may imply a
habituation phenomenon, i.e., a form of learning, where repeated stimulus leads to
ur

attenuated responsiveness, or when a considerable amount of time can decrease the

response to an alteration in terms of light or darkness, such as in this case (Basnet et al.,
Jo

2019). In a different study, the authors found that 8 different behavioural endpoints
resulted in habituation, occurring independently of each other, with various molecular
mechanisms. One of the mechanisms underlying this process seems to be signalling
pathways, through dopamine and/or serotonin receptors (Randlett et al., 2019).

Additionally, there was a discrepancy between the observed effects when animals
were exposed to lower and higher doses in terms of behavioural assessment. Dahl et al.

19
(2006) studied growth-related sublethal endpoints in harpacticoid copepods exposed to
SIM. In this study, it was suggested that differences between effects in low and high
concentrations can be due to different ecotoxicological modes of action of SIM, i.e.,
effects at higher concentrations seem to be related to energy-mediated processes, while
effects in terms of endocrine disruption seem to affect the individuals in lower
concentrations.

Biochemical assessment

According to the results obtained for SOD activity, there were no significant differences
in MnSOD activity, while Cu-Zn SOD activity was significantly decreased, except for

of
individuals exposed to 92.45 ng L-1, comparing to the control. Cunha et al. (2016) also
reported a decrease in SOD activity in zebrafish embryos exposed to SIM. The decreasing

ro
activity of Cu-Zn SOD can be due to the antioxidant properties and synergism of SIM
with antioxidants. It has been hypothesized that the mechanisms underlying these
properties of SIM may be due to the inhibition of oxidant formation (NADPH-oxidase)
-p
and the increase in bioavailability of nitric oxide that can neutralize radicals (Stoll et al.,
2004). The increase in antioxidant defences, reduces significantly the quantity of ROS,
re
leading to a decrease in antioxidant enzymes activity (Strzyżewski et al., 2013). Statins
can decrease the production of superoxide anion through a direct effect on the enzyme’s
lP

structure and consequently its function, thus reducing SOD activity (Delbosc et al., 2002;
Wassmann et al., 2001). Decreased SOD activity can also be a response to increased
production of H2O2 and O2-, by autooxidation of excess glucose and nonenzymatic
na

glycation of proteins (El-Missiry, 2012). Moreover, high levels of hydroxyl radicals and
H2O2 can lead to partial inactivation of SOD (Thorpe et al., 2013), contributing to the
reduction of its activity.
ur

Regarding the results obtained in terms of CAT activity in individuals exposed to

Jo

SIM, no significant differences were found, compared to control fish. The results from
this study are not in agreement with previously reported data, where exposure to SIM
caused an increase in CAT activity, in humans (Kaminsky et al., 2010; Piechota-
Polanczyk et al., 2012). In this case, and considering the absence of increase of CAT
activity, the formation of H2O2 seems to not have been favoured by the exposure to SIM.
The antioxidant properties of SIM allied to the absence of production of H2O2, related to
the inhibition of SOD activity, can explain the obtained results. The lack of significant

20
results in our study can also be due to the duration of exposure used in our assay, justifying
the contradictions concerning other studies from the literature, which indicated an
increase in CAT activity, conducted in humans (De Sotomayor et al., 2009; Piechota-
Polanczyk et al., 2012). It was also suggested by Federici et al. (2007) that the absence of
significant effects may be due to the capacity of the here used organism to use other
antioxidant enzymes to avoid oxidative stress, namely GPx, to counteract H2O2 levels.

Some studies demonstrate that the exposure to SIM leads to a decrease in GPx
total activity, in very distinct animal models, such as rodents (mg kg-1) (Srinivasa Rao et
al., 2012) and humans (10 mg day-1 during 8 months) (Ungureanu et al., 2003). The
significant decrease in GPx total activity corroborates the above-mentioned results in

of
terms of the antioxidant properties of SIM (Strzyżewski et al., 2013), which may
contribute to a reduction of the activity of antioxidant enzymes, including GPx.

ro
Moreover, while CAT is present in the peroxisomes, GPx is located in the cytoplasm.
Such as in the case of MnSOD, mostly found in the mitochondria, CAT activity was not
-p
significantly different from the individuals of the control group, which suggests that SIM
did not act in the mitochondria to generate oxidative stress (Modesto & Martinez, 2010).
re
Moreover, the significant increase in selenium-dependent GPx activity in individuals
exposed to the lowest concentration of SIM could represent an increase in H2O2 levels.
However, in higher concentrations, the two mechanisms mentioned before, i.e. the
lP

inhibition of NADPH-oxidase and the increase of nitric oxide levels, could have lead to
a decrease in ROS, including H2O2, which in turn produced non-significant results in GPx
na

selenium-dependent activity above this concentration.

According to the obtained results, individuals exposed to SIM experienced a

significant decrease in GSTs activity, compared with control organisms. Apart from
ur

Cunha et al. (2016) and Wang et al. (2019), that reported an increase in GSTs activity in
zebrafish embryos and adult Mugilogobius abei after exposure to SIM, to the best of our
Jo

knowledge, there are no other studies that investigated this enzyme’s activity in aquatic
organisms exposed to SIM. However, other studies have reported similar results, where
GSTs levels decreased, namely in human patients with pancreatic damage, when treated
with SIM (Matalka et al., 2013; Prokop’eva & Gulyaeva, 2000). There is evidence that in
phase 2 of metabolism, SIM undergoes glucuronidation in aquatic organisms, i.e.,
glucuronic acid conjugation by UDP-glucuronosyltransferases (UGTs) (Prueksaritanont
et al., 2002; Solé & Sanchez-Hernandez, 2015). So, SIM cannot preferentially be
21
metabolized in D. rerio via GSTs. Alternatively, GSTs can also prevent the interaction
of xenobiotics with nucleic acids and proteins, also protecting against oxidative stress and
damage (Dzoyem & Eloff, 2014; Rahman, 2007; Smith et al., 2013). The lack of
modification of GSTs levels reinforces the previously made assumption, concerning the
putative absence of clear pro-oxidative effects. In that way, as it was mentioned before
for SOD and GPx enzymes activity, the antioxidant properties of SIM could have led to
the hereby observed results.

Considering the results obtained for lipid peroxidation, there was a significant
decrease in this parameter in individuals exposed to SIM in levels of 184.9, 369.8, 739.6
and 1479.2 ng.1 L, compared with control. As far as we know, there are only a few studies

of
that investigated the relationship between SIM exposure and lipid peroxidation, being in
the context of treatment of a specific condition with statins and conducted only in humans

ro
and rodents. Some of these studies reported a decrease in lipid peroxidation in humans,
after oral administration of SIM, at dosages of 10 mg kg-1 (Mohamadin et al., 2011) and
-p
administration of 20 mg day-1 during 4 to 12 weeks (Broncel et al., 2006). The decrease
in lipid peroxidation in this assay allow inferring the decrease in ROS levels, through the
re
alteration in the process of interaction of free radicals with polyunsaturated fatty acids,
leading to a decrease in MDA-like compounds. Compounds with antioxidant properties,
such as SIM, have been shown to lead to a decrease in oxidative damage linked to lipid
lP

peroxidation (Venturini et al., 2010; Golbidi et al., 2011).

Histological assessment
na

The number of studies about the toxicity of SIM in aquatic species in terms of
endocrine disruption is scarce (Dahl et al., 2006; Neuparth et al., 2014), and none of them
ur

was performed with D. rerio. According to the results obtained in terms of sex
determination in individuals exposed to SIM, no significant differences were found
Jo

between treatment groups and the control. However, a feminization response is evident
for the three highest concentrations, compared with the control. A few studies reported
sexual development disruption in Gammarus locusta (Neuparth et al., 2014) and
harpacticoid copepods (Dahl et al., 2006) when exposed to SIM. HMG-CoA reductase
inhibitors, such as statins, are thought to affect sex hormone and LDL biosynthesis (Ser
et al., 2010). SIM’s inhibition of HMG-CoA reductase leads to a decrease in the
production of LDL (Vandeputte et al., 2007; Rang et al., 2007). This could result in a

22
decrease in sex hormones, such as androgens, leading to a decrease in the number of
males (Pradhan & Olsson, 2016). Sex ratio data interpretation is complex and it can be
influenced by a variety of factors, such as chemical (EDCs) and non-chemical (genetic
factors, food supply, growth rate, temperature and stress) (Green et al., 2015).
Considering the lack of significant results in terms of sex determination, and the
differences from the results of this assay when compared to others in terms of the
development of individuals exposed to SIM, we can assume that major differences in
terms of development alterations may be due to the distinct levels tested, and in terms of
sensitivity of individuals. Development changes may only be attained when SIM is
present in high levels, such as in the mentioned studies from the literature, where SIM
was used in the mg L-1 range, well above the ecologically relevant concentrations to which

of
zebrafish were exposed in our assay (ng L-1 range). Moreover, differences between the
tested class of species and those used in the above-mentioned investigations could also

ro
lead to differences in terms of endocrine disruption.

-p
According to the maturation stages, no significant differences were found between
treatment groups in both female and male individuals, compared with the control. As
re
mentioned before, SIM can decrease LDL levels, leading to a decrease in sex hormones.
Estrogen levels can alter gonadotropin secretion, which regulates oocyte maturation, and
in turn can lead to less developed organisms, such as in the case of the higher
lP

concentrations of SIM used in our assay (Marques et al., 2000). Previous investigations
have shown that statins can interfere with reproductive development (Bustan et al., 2017;
na

Leite et al., 2014). Given the lack of results in terms of gonadal maturation phases in
females and males exposed to SIM, we can assume that SIM did not have any effect in
terms of reproductive disruption. Although the concentrations used in this assay were
ur

based on the maximum level found in effluents from WWTPs in Portugal, the range used
was at ng L-1, which compared to previous studies, might have been a low dosage to
Jo

produce endocrine disruption effects. Moreover, SIM does not seem to have reproductive
disruption effects, in terms of sex ratio and maturation stages. However, the statistical
meaning of both sex determination and gonadal staging should be looked upon carefully
in this study because of the low number of replicates and sample size used, two factors
that may have biased the obtained results.

Moreover, there is a decrease in length and weight of female and male individuals,
related directly to increasing concentrations of SIM. SIM exposure at ng L-1 range in
23
Gammarus locusta also caused alterations in growth and maturation (Neuparth et al.,
2014). HMG-CoA reductase inhibition, and therefore the decrease in the production of
cholesterol, and decrease in triglycerides level, can lead to a disruption in growth and
reproduction, as they are essential in fish adulthood (Barros et al., 2018).

Considering all the above-mentioned results, in terms of behaviour, biochemical

assessment, and reproductive disruption, in individuals exposed to SIM, we can conclude
that this compound, in these particular conditions, caused alterations in the first two
determined biomarkers, namely behaviour, and biochemical parameters. In terms of
behavioural alterations, SIM does not seem to have a well-defined mechanism of action.
Locomotion changes seem to be potentially due to alterations in energy deficits

of
(biosynthesis of ATP) and neuronal changes. On the other hand, the biochemical
assessment revealed decreasing activity in several enzyme activities. The antioxidant

ro
properties of SIM might explain this phenomenon, since the presence of an antioxidant
drug, may counteract the overproduction of ROS. Reproductive disruption through
-p
changes in sex ratio and maturation stages of individuals exposed to SIM did not reveal
any significant differences, compared with the control group. The ability of SIM to lower
re
LDL levels should reduce the production of sex hormones, such as estrogen and androgen,
which ultimately should lead to a lower number of juvenile females, through the decrease
in estrogen, responsible for the maintenance of femaleness in adulthood. However, this
lP

outcome was not reported. Moreover, a lower concentration of estrogen and androgen
should also lead to a decrease in ovarian and testicular differentiation, which should be
na

reflected in terms of maturation stages of individuals. Again, effects of this nature were
not observed after exposing D. rerio to SIM under the proposed conditions.

Furthermore, the reduction of anxiety-like behaviour evidenced by a decrease in

ur

erratic swimming can suggest a correlation with the antioxidant properties of SIM, that
in turn, reduced the activity of the biochemical markers assessed. CNS is highly
Jo

susceptible to damage due to free radicals. Thus, oxidative stress can affect vision, muscle
and brain activity (Nallasamy et al., 2012). The oxidative effects of specific substances,
namely drugs (e.g. paracetamol) has been related to significant chenges in enzymes
involveed in the regulation of the CNS function, such as cholinesterases, in fish, such as
Anguilla anguilla (Nunes et al., 2015). This effect was not restricted to fish, since it was
also observed for marine mollusks, Mytilus galloprovincialis (Solé et al. (2010), and the
crustacean Daphnia magna (Daniel et al., 2019). It is thus posssible to suggest the
24
establishment of a relationship between oxidative stress and neurotoxicity, considering
that ChE activity may be altered due to ROS interference (Delwing-de Lima et al., 2010;
Oliveira et al., 2015). The effects of paracetamol are not limited to its pro-oxidant effects,
which may result in cholinesterasic inhibition. In fact, paracetamol, most probably by
impairing neuronal function regulated by cholinesterases, may affect the behaviour of fish
as well, as shown to occur by Matus et al. (2018), in Phaloceros harpagos, and by
Nogueira et al. (2019), in Danio rerio. Antioxidants lead to a reduction in oxidative stress,
which can be related to the behavioural results obtained in this assay. Oxidative stress can
damage lipids, proteins and DNA, which can have an impact on the degree of maturation
of gonadal stages. The antioxidant properties of SIM seemed to decrease oxidative stress

of
enzymes activity, compared with the control.

Moreover, with increasing SIM concentration, an intensification of the toxic

ro
effects (biochemical and behavioural) was somewhat expected. However, this trend did
not occur in this case. Instead, there seems to be a U-shaped curve in terms of behavioural
-p
and biochemical results, a conditions already described and known as hormesis. Hormesis
has been reported in various toxicological, biochemical and pharmacological assays
re
(Calabrese & Baldwin, 2001). It represents the direct stimulation or overcompensation
towards a disruption of homeostasis. Endogenous and synthetic agonists, such as SIM,
can directly stimulate biological processes in a dose range, inhibiting those same
lP

processes when in other (namely higher) levels (Calabrese & Baldwin, 2001). In chronic
exposures, especially at low concentration, a U-shaped effect seems to be a common
na

pattern elicited by some EDCs (Barros et al., 2018). The initial direct inhibition of the
antioxidant enzymes may represent a disruption in homeostasis; the antioxidant effects of
SIM were supressed at higher doses, and SIM became prooxidant in higher doses. Barros
ur

et al. (2018) suggested that the endogenous feedback mechanism for homeostasis in terms
of cholesterol levels, due to the exposure of individuals to SIM can explain non-
Jo

monotonic responses, like the ones evidenced in this assay (Combelles et al., 2009). In
fact, EDCs seem to contribute for an extensive array of biological responses, that are not
exclusively related to reproduction and development. As summarized by Diamanti-
Kandarakis et al. (2009), EDCs act in a vast number of biologically critical parameters,
including metabolism, that are likely to be deleteriously affected by these chemicals.

Conclusions

25
 Ecotoxicological data for lipid-regulating drugs, such as SIM, are still lacking,
and these drugs have only been confusingly characterized in terms of modes of action and
consequences in aquatic organisms. This assay provided information about the
ecotoxicity of SIM, in both embryonic and juvenile stages of D. rerio, Results from this
study demonstrate that zebrafish early-life stages and juvenile individuals can serve as
model organisms in ecotoxicological assays (Scholz et al., 2008; Lammer et al., 2009) to
study the toxic effects of statins of therapeutic use. Overall, zebrafish larvae responded to
SIM through hyperactivity, and with the reduction of the antioxidant defences. In terms
of reproductive disruption, no significant alterations were reported after the here-defined
exposure conditions, regarding sex determination and maturation stages of individuals.

of
The results here obtained, which were attained at concentrations of ecological relevance
of this pharmaceutical, can suggest deleterious effects in non-target individuals, thereby
compromising the physiology of non-target, environmentally exposed fish. Further

ro
investigations with longer periods of exposure of individuals to simvastatin, should be
relevant to determine the role of this lipid-lowering drug in the reproductive disruption.
-p
re
Credit author statement
lP

Daniela Rebelo - methodology development, validation, formal analysis, investigation, data

curation, Writing - original draft preparation.

Alberto Teodorico Correia - methodology development, validation, formal analysis,

investigation, data curation, Writing – review & eediting.
na

Bruno Nunes - conceptualization, methodology development, validation, resources, writing -

review & editing, supervision, project administration, funding acquisition.

Declaration of interests
ur

The authors declare that they have no known competing financial interests or personal
Jo

relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

26
Bruno Nunes is hired by “ECO-R-pharmplast - Ecotoxicity of realistic combinations of
pharmaceutical drugs and microplastics in marine ecosystems”, Fundação para a Ciência
e a Tecnologia, FCT (reference POCI-01-0145-FEDER-029203). This research was
financially supported by CESAM (UIDB/50017/2020+UIDP/50017/2020), by CIIMAR
(UIDB/04423/2020+UIDP/04423/2020), by FCT/MCTES through national funds
(PIDDAC), and by the co-funding by the FEDER, within the PT2020 Partnership
Agreement and Compete 2020.

References

Aebi, H. (1984). Catalase in vitro. Methods in Enzymology 105:121–126. doi:

of
10.1016/s0076-6879(84)05016-3

Ayala, A., Muñoz, M. F., & Argüelles, S. (2014). Lipid peroxidation: production,

ro
metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-
nonenal. Oxidative Medicine and Cellular Longevity. doi: 10.1155/2014/360438
-p
Banerjee, S., Ghosh, J., & Sil, P. C. (2016). Drug Metabolism and Oxidative Stress:
Cellular Mechanism and New Therapeutic Insights. Biochemistry & Analytical
re
Biochemistry s3 doi:10.4172/2161-1009.1000255
lP

Barros, S., Montes, R., Quintana, J. B., Rodil, R., André, A., Capitão, A., Soares, J.,
Santos, M., Neuparth, T. (2018). Chronic environmentally relevant levels of
Simvastatin induces non-monotonic responses in Zebrafish (Danio rerio). Aquat
na

Toxicol. 201:47-57. doi: 10.1101/289694

Basnet, R., Zizioli, D., Taweedet, S., Finazzi, D., & Memo, M. (2019). Zebrafish Larvae
ur

as a Behavioural Model in Neuropharmacology. Biomedicines 7(1):23. doi:

10.3390/biomedicines7010023
Jo

Betteridge, D. J. (2000). What is oxidative stress? Metabolism: Clinical and Experimental

49(2 Suppl 1):3–8. doi: 10.1016/S0026-0495(00)80077-3

Bhagavan, N. V., & Ha, C.-E. (2015). Lipids I: Fatty Acids and Eicosanoids. Essentials
of Medical Biochemistry 269–297. doi: 10.1016/B978-0-12-416687-5.00016-6

Blaser, R. E., Peñalosa, Y. M. (2011). Stimuli Affecting Zebrafish (Danio rerio) Behavior

27
 in the light/dark Preference Test. Physiol Behav 104(5):831-7. doi:
10.1016/j.physbeh.2011.07.029

Blondet, N. M., Messner, D. J., Kowdley, K. V., & Murray, K. F. (2018). Mechanisms of
Hepatocyte Detoxification. Physiology of the Gastrointestinal Tract, 981–1001. doi:
10.1016/B978-0-12-809954-4.00043-8

Bogers, R. (2008). PhD final thesis - Markers of endocrine disruption in fish. Wageningen
University. Retrieved from http://edepot.wur.nl/122056

Boleda, M. R., Galceran, M. T., & Ventura, F. (2011). Behaviour of pharmaceuticals and
drugs of abuse in a drinking water treatment plant (DWTP) using combined

of
conventional and ultrafiltration and reverse osmosis (UF/RO) treatments.
Environmental Pollution 159(6):1584–1591. doi: 10.1016/j.envpol.2011.02.051

ro
Boxall, A. B. A. (2004). The environmental side effects of medication. EMBO Reports
5(12):1110–1116. doi: 10.1038/sj.embor.7400307 -p
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram
re
quantities of protein utilizing the principle of protein-dye binding. Analytical
Biochemistry 72(1–2):248–254. https://doi: 10.1016/0003-2697(76)90527-3
lP

Bouayed, J. (2011). Relationship Between Oxidative Stress and Anxiety: Emerging Role
of Antioxidants Within Therapeutic or Preventive Approaches. In Anxiety
na

Disorders. InTech. doi: 10.5772/19214

Broncel, M., Koter-Michalak, M., & Chojnowska-Jezierska, J. (2006). The effect of

statins on lipids peroxidation and activities of antioxidants enzymes in patients with
ur

dyslipidemia. Przeglad Lekarski 63(9):738–742. PMID: 17479860

Jo

Buege, J. A., & Aust, S. D. (1978). [30] Microsomal lipid peroxidation. Methods in
Enzymology, 52:302–310. doi:10.1016/S0076-6879(78)52032-6

Bustan, A. A., Jawad, A. M., & Zagidullin, N. (2017). The Effect of Two Types of Statins
(Rosuvastatin and Atorvastatin) on the Fertility of Male and Female Mice. British
Journal of Medicine & Medical Research 19(12):1-11. doi:
10.9734/BJMMR/2017/31150

28
Calabrese, E. J., & Baldwin, L. A. (2001). U-Shaped Dose-Responses in Biology,
Toxicology, and Public Health. Annual Review of Public Health 22(1):15–33. doi:
10.1146/annurev.publhealth.22.1.15

Campos, L. M., Rios, E. A., Guapyassu, L., Midlej, V., Atella, G. C., Herculano-Houzel,
S., Benchimol, M., Mermelstein, C., Costa, M. L. (2016). Alterations in zebrafish
development induced by simvastatin: Comprehensive morphological and
physiological study, focusing on muscle. Experimental Biology and Medicine
241(17):1950–1960. doi: 10.1177/1535370216659944

Carlsson, G., Patring, J., Kreuger, J., Norrgren, L., & Oskarsson, A. (2013). Toxicity of

of
15 veterinary pharmaceuticals in zebrafish (Danio rerio) embryos. Aquatic
Toxicology 126:30–41. doi: 10.1016/j.aquatox.2012.10.008

ro
Chavan-Gautam, P., Rani, A., & Freeman, D. J. (2018). Distribution of Fatty Acids and
Lipids During Pregnancy. Advances in Clinical Chemistry 84:209–239. doi:
10.1016/bs.acc.2017.12.006
-p
Chen, J., Tanguay, R. L., Xiao, Y., Haggard, D. E., Ge, X., Jia, Y., Zheng, Y., Dong, Q.,
re
Huang, C., Lin, K. (2016). TBBPA exposure during a sensitive developmental
window produces neurobehavioral changes in larval zebrafish. Environmental
lP

Pollution 216:53–63. doi: 10.1016/j.envpol.2016.05.059

Combelles, C. M. H., Gupta, S., & Agarwal, A. (2009). Could oxidative stress influence
na

the in-vitro maturation of oocytes? Reproductive BioMedicine Online 18(6):864–

880. doi: 10.1016/S1472-6483(10)60038-7
ur

Cunha, V., Santos, M. M., Moradas-Ferreira, P., & Ferreira, M. (2016). Simvastatin
effects on detoxification mechanisms in Danio rerio embryos. Environmental
Jo

Science and Pollution Research 23(11):10615–10629. doi: 10.1007/s11356-016-

6547-y

Dahl, U., Gorokhova, E., & Breitholtz, M. (2006). Application of growth-related

sublethal endpoints in ecotoxicological assessments using a harpacticoid copepod.
Aquatic Toxicology 77(4):433–438. doi: 10.1016/J.AQUATOX.2006.01.014

Dagneaux, D., Boschetti, F., Babcock, R., & Vanderklift, M. (2009). Detecting general

29
 patterns in fish movement from the analysis of fish tagging data. 18 th World IMACS
/ MODSIM Congress. Retrieved from http://mssanz.org.au/modsim09

Dahm, R. (2006). The Zebrafish Exposed: “See-through” mutants may hold the key to
unraveling the mysteries of embryonic development. American Scientist. Sigma XI,
The Scientific Research Honor Society 94(5): 446-453. doi: 10.2307/27858837

Daniel D., Dionísio R., Alkimin G. D., Nunes B. (2019). Acute and chronic effects of
paracetamol exposure on Daphnia magna: how oxidative effects may modulate
responses at distinct levels of organization in a model species. Environmental
Science and Pollution Research 26(4):3320-3329. doi: 10.1007/s11356-018-3788-y.

of
Davidson, M. H. (2002). Combination Therapy for dyslipidemia. Clinical Lipidology: A
Companion to BraunWald’s Heart Disease 352–362. doi: 10.1016/B978-

ro
141605469-6.50033-0

-p
De Carvalho, T. S., Cardoso, P. B., Santos-Silva, M., Lima-Bastos, S., Luz, W. L., Assad,
N., Kauffmann, N., Passos, A., Brasil, A., Bahia, C. P., Moraes, S., Gouveia, A., De
Jesus Oliveira Batista, E., Oliveira, K. R. M. H., Herculano, A. M. (2019). Oxidative
re
Stress Mediates Anxiety-Like Behaviour Induced by High Caffeine Intake in
Zebrafish: Protective Effect of Alpha-Tocopherol. Oxidative Medicine and Cellular
lP

Longevity. doi: 10.1155/2019/8419810

De Sotomayor, M. Á., Pérez-Guerrero, C., Herrrera, M. D., Jimenez, L., Marín, R.,
na

Marhuenda, E., & Andriantsitohaina, R. (2009). Improvement of age-related

endothelial dysfunction by simvastatin: effect on NO and COX pathways. British
Journal of Pharmacology 146(8):1130–1138. doi: 10.1038/sj.bjp.0706420
ur

Delbosc, S., Morena, M., Djouad, F., Ledoucen, C., Descomps, B., & Cristol, J. P. (2002).
Jo

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are able to

reduce superoxide anion production by NADPH oxidase in THP-1-derived
monocytes. Journal of Cardiovascular Pharmacology 40(4):611–617. doi:
10.1097/00005344-200210000-00015

Delwing-de Lima, D., Wollinger, L.F., Casagrande, A.C.M., Delwing, F., da Cruz, J.G.P.,
Wyse, A.T.S., Magro, D.D.–D. (2010) Guanidino compounds inhibit
acetylcholinesterase and butyrylcholinesterase activities: effect neuroprotector of

30
 vitamins E plus C. International Journal of Developmental Neuroscience 28(6):465–
473. doi: 10.1016/j.ijdevneu.2010.06.008

Diamanti-Kandarakis, E., Bourguignon, J.-P., Giudice, L.C., Hauser, R., Prins, G.S.,
Soto, A.M., R. Zoeller, R.T., Gore, A.C. (2009). Endocrine-Disrupting Chemicals:
An Endocrine Society Scientific Statement. Endocr Rev. 30(4):293–342. doi:
10.1210/er.2009-0002

Djordjević, V. B. (2004). Free Radicals in Cell Biology. International Review of

Cytology 237:57–89. doi: 10.1016/S0074-7696(04)37002-6

Dooley, K., & Zon, L. I. (2000). Zebrafish: a model system for the study of human

of
disease. Current Opinion in Genetics & Development 10(3):252–256. doi:
10.1016/S0959-437X(00)00074-5

ro
Dzoyem, J. P., & Eloff, J. N. (2014). Biochemical Parameters in Toxicological Studies in
-p
Africa: Significance, Principle of Methods, Data Interpretation, and Use in Plant
Screenings. Toxicological Survey of African Medicinal Plants 659–715. doi:
10.1016/B978-0-12-800018-2.00023-6
re
Ebele, A. J., Abou-Elwafa Abdallah, M., & Harrad, S. (2017). Pharmaceuticals and
lP

personal care products (PPCPs) in the freshwater aquatic environment. Emerging

Contaminants. KeAi Communications Co 3(1):1-16. doi:
10.1016/j.emcon.2016.12.004
na

El-Missiry, M. A. (2012). Antioxidant Enzyme. Mansoura University, Egipt. doi:

10.5772/2895
ur

El-Saad, A. M. A., & Elgerbed, M. S. (2010). Dimethoate-induced hepatotoxicity in rats

and the protective roles of vitamin E and N-acetylcysteine. Egypt. J. Exp. Biol.
Jo

(Zoo.) 6(2):219-230. ISSN: 2090 - 0511

Emran, F., Rihel, J., Adolph, A. R., & Dowling, J. E. (2010). Zebrafish larvae lose vision
at night. Proceedings of the National Academy of Sciences, 107(13), 6034–6039.
doi: 10.1073/pnas.0914718107

Federici, G., Shaw, B., & Handy, R. (2007). Toxicity of titanium dioxide nanoparticles

31
 to rainbow trout (Oncorhynchus mykiss): Gill injury, oxidative stress, and other
physiological effects. Aquatic Toxicology 84(4):415–430. doi:
10.1016/j.aquatox.2007.07.009

Flohé, L., & Günzler, W. A. (1984). Assays of glutathione peroxidase. Methods in

Enzymology 105:114–121. doi: 10.1016/S0076-6879(84)05015-1

Flohé, L., & Otting, F. (1984). Superoxide dismutase assays. Methods in Enzymology
105:93–104. doi: 10.1016/s0076-6879(84)05013-8

Gracia-Lor, E., Sancho, J. V, Serrano, R., & Hernández, F. (2012). Occurrence and
removal of pharmaceuticals in wastewater treatment plants at the Spanish

of
Mediterranean area of Valencia. Chemosphere 87(5):453–462. doi:
10.1016/j.chemosphere.2011.12.025

ro
Green, C., Brian, J., Kanda, R., Scholze, M., Williams, R., & Jobling, S. (2015).
-p
Environmental concentrations of anti-androgenic pharmaceuticals do not impact
sexual disruption in fish alone or in combination with steroid oestrogens. Aquatic
Toxicology 160:117–127. doi: 10.1016/j.aquatox.2014.12.022
re
Golbidi, S., Laher, I., & Ebadi, S. A. (2011). Antioxidants in the Treatment of Diabetes.
lP

Current Diabetes Reviews 7:106–125. doi: 10.2174/157339911794940729

Habig, W. H., Pabst, M. J., & Jakoby, W. B. (1974). Glutathione S-transferases. The first
na

enzymatic step in mercapturic acid formation. J Biol Chem. 249(22):7130–7139.

PMID: 4436300

Halliwell, B. (2009). Free Radicals and Antioxidants: A Personal View. Nutrition

ur

Reviews 52(8):253–265. doi: 10.1111/j.1753-4887.1994.tb01453.x

Jo

Hassan, W., Barroso Silva, C., Mohammadzai, I. U., Teixeira da Rocha, J., & Landeira-
Fernandez, J. (2014). Association of Oxidative Stress to the Genesis of Anxiety:
Implications for Possible Therapeutic Interventions. Current Neuropharmacology
12(2):120–139. doi: 10.2174/1570159x11666131120232135

Holth, T. F., Nourizadeh-Lillabadi, R., Blaesbjerg, M., Grung, M., Holbech, H., Petersen,
G. I., Aleström, P. & Hylland, K. (2008). Differential gene expression and

32
 biomarkers in zebrafish (Danio rerio) following exposure to produced water
components. Aquatic Toxicology 90(4):277–291. doi:
10.1016/J.AQUATOX.2008.08.020

Jelic, A., Gros, M., Ginebreda, A., Cespedes-Sánchez, R., Ventura, F., Petrovic, M., &
Barceló, D. (2011). Occurrence, partition and removal of pharmaceuticals in sewage
water and sludge during wastewater treatment. Water Research 45(3):1165–1176.
doi: 10.1016/j.watres.2010.11.010

Jemec, A., Drobne, D., Tišler, T., & Sepčić, K. (2010). Biochemical biomarkers in
environmental studies—lessons learnt from enzymes catalase, glutathione S-

of
transferase and cholinesterase in two crustacean species. Environmental Science and
Pollution Research 17(3):571–581. doi: 10.1007/s11356-009-0112-x

ro
Lammer, E., Carr, G. J., Wendler, K., Rawlings, J. M., Belanger, S. E., & Braunbeck, T.
(2009). Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a

Physiology Part C:
-p
potential alternative for the fish acute toxicity test? Comparative Biochemistry and
Toxicology & Pharmacology 149(2):196–209. doi:
re
10.1016/j.cbpc.2008.11.006

Leite, G. A. A., Rosa, J. de L., Sanabria, M., Cavariani, M. M., Franci, J. A. A., Pinheiro,
lP

P. F. F., & Kempinas, W. D. G. (2014). Delayed reproductive development in

pubertal male rats exposed to the hypolipemiant agent rosuvastatin since prepuberty.
Reproductive Toxicology, 4493–103. doi: 10.1016/j.reprotox.2014.01.004
na

Lumb, A. B. (2017). Oxygen Toxicity and Hyperoxia. Nunn’s Applied Respiratory

Physiology 341-356. doi: 10.1016/b978-0-7020-6294-0.00024-1
ur

Lykkesfeldt, J. (2007). Malondialdehyde as biomarker of oxidative damage to lipids

Jo

caused by smoking. Clinica Chimica Acta 380(1–2):50–58. doi:

10.1016/j.cca.2007.01.028

Kaczala, F., & Blum, S. E. (2015). The Occurrence of Veterinary Pharmaceuticals in the
Environment: A Review. Current Analytical Chemistry 12(3):169–182. doi:
10.2174/1573411012666151009193108

Kaminsky, Y., Suslikov, A., & Kosenko, E. (2010). Specific and Pronounced Impacts of

33
 Lisinopril and Lisinopril Plus Simvastatin on Erythrocyte Antioxidant Enzymes. The
Journal of Clinical Pharmacology 50(2):180–187. doi: 10.1177/0091270009344854

Kasprzyk-Hordern, B., Dinsdale, R. M., & Guwy, A. J. (2009). The removal of

pharmaceuticals, personal care products, endocrine disruptors and illicit drugs
during wastewater treatment and its impact on the quality of receiving waters. Water
Research 43(2):363–380. doi: 10.1016/J.WATRES.2008.10.047

Marques, P., Skorupskaite, K., George, J. T., & Anderson, R. A. (2000). Physiology of
GNRH and Gonadotropin Secretion. Endotext. MDText.com, Inc.

Martín, J., Buchberger, W., Alonso, E., Himmelsbach, M., & Aparicio, I. (2011).

of
Comparison of different extraction methods for the determination of statin drugs in
wastewater and river water by HPLC/Q-TOF-MS. Talanta 85(1):607–615. doi:

ro
10.1016/j.talanta.2011.04.017

-p
Martins, S., Monteiro, J. F., Vito, M., Weintraub, D., Almeida, J., & Certal, A. C. (2016).
Toward an Integrated Zebrafish Health Management Program Supporting Cancer
and Neuroscience Research. Zebrafish, 13(S1):S-47-S-55. doi:
re
10.1089/zeb.2015.1198
lP

Matalka, I. I., Mhaidat, N. M., & Fatlawi, L. A. (2013). Antioxidant activity of

simvastatin prevents L-arginine-induced acute toxicity of pancreas. International
Journal of Physiology, Pathophysiology and Pharmacology 5(2):102–108.
na

PMID: 23750308

Matus, G. N., Pereira, B. V. R., Silva-Zacarín, E. C. M., Costa, M. J., dos Santos, A. A.
ur

C., Nunes, N. (2018). Behavior and histopathology as biomarkers for evaluation of

the effects of paracetamol and propranolol in the neotropical fish species
Jo

Phalloceros harpagos. Environmental Science and Pollution Research

25(28):28601-28618 doi: 10.1007/s11356-018-2839-8.

Metzler, M. (2006). Endocrine Disruptors Part I. Springer Berlin Heidelberg. ISBN:

3540482091, 9783540482093

Miscevic, F., Rotstein, O., & Wen, X.-Y. (2012). Advances in Zebrafish High Content
and High Throughput Technologies. Combinatorial Chemistry & High Throughput

34
 Screening 15(7):515–521. doi: 10.2174/138620712801619140

Modesto, K. A., & Martinez, C. B. R. (2010). Roundup® causes oxidative stress in liver
and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus
lineatus. Chemosphere 78(3):294–299. doi: 10.1016/j.chemosphere.2009.10.047

Mohamadin, A. M., Elberry, A. A., Gawad, H. S. A., Morsy, G. M., & Al-Abbasi, F. A.
(2011). Protective effects of simvastatin, an HMGCoA reductase inhibitor, against
oxidative damage in experimental diabetic rats. International Journal of PharmTech
Research 3(3):1780–1795. doi:10.1155/2011/167958

Monat-Descamps, C., & Deschamps, F. (2012). Nervous system disorders induced by

of
occupational and environmental toxic exposure. Open Journal of Preventive
Medicine 2(3):272–278. doi: 10.4236/ojpm.2012.23039

ro
Murphy, L. R., Kinsey, S. T., & Durako, M. J. (2003). Physiological effects of short-term
-p
salinity changes on Ruppia maritima. Aquatic Botany 75(4):293–309. doi:
10.1016/S0304-3770(02)00206-1
re
Nallasamy, P., Zhu, H., Misra, H. P., Li, Y., & Jia, Z. (2012). Reactive oxygen species
and oxidative stress in spinal cord injury - Updated experimental and clinical
lP

evidence. Systems Biology of Free Radicals and Antioxidants (2451–2467).

Springer, Berlin, Heidelberg. doi: 10.1007/978-3-642-30018-9_191
na

Neuparth, T., Martins, C., Santos, C. B. de los, Costa, M. H., Martins, I., Costa, P. M., &
Santos, M. M. (2014). Hypocholesterolaemic pharmaceutical simvastatin disrupts
reproduction and population growth of the amphipod Gammarus locusta at the ng/L
ur

range. Aquatic Toxicology 155:337–347. doi: 10.1016/J.AQUATOX.2014.07.009

Nikolaou, A., Meric, S., Fatta, D. (2007). Occurrence patterns of pharmaceuticals in water
Jo

and wastewater environments. Analytical and Bioanalytical Chemistry 387:1225-

34. doi: 10.1007/s00216-006-1035-8

Nogueira A. F., Pinto G., Nunes B. (2019). Embryonic development, locomotor behavior,
biochemical and epigenetic effects of the pharmaceutical drugs paracetamol and
ciprofloxacin in larvae and embryos of Danio rerio when exposed to environmental
realistic levels of both drugs. Environmental Toxicology 34(11):1177-1190. doi:

35
 10.1002/tox.22819.

Nunes B., Verde M. F., Soares A. M. V. M. (2015). Biochemical Effects of the

Pharmaceutical Drug Paracetamol on Anguilla anguilla. Environmental Science and
Pollution Research 22(15):11574-84. doi: 10.1007/s11356-015-4329-6

OECD. (2010). Test No. 123: Guidance Document on the Diagnosis of Endrocrine-
related Histopathology in Fish Gonads. OECD Guidelines for the Testing of
Chemicals. Paris, France

OECD. (2011). Test No. 234: Fish Sexual Development Test. OECD Guidelines for the
Testing of Chemicals. Paris, France

of
OECD. (2013a). Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD

ro
Guidelines for the Testing of Chemicals. Paris, France

OECD. (2013b). Test No. 210: Fish, Early-life Stage Toxicity Test. OECD Guidelines
for the Testing of Chemicals. Paris, France
-p
re
Oliveira, L.L.D., Antunes, S.C., Gonçalvez, S., Rocha, O., Nunes, B. (2015) Evaluation
of ecotoxicological effects of drugs on Daphnia magna using different enzymatic
biomarkers. Ecotoxicology and Environmental Safety 119:123-131. doi:
lP

10.1016/j.ecoenv.2015.04.028

Omar, W. A., & Mahmoud, H. M. (2017). Risk assessment of polychlorinated biphenyls

na

(PCBs) and trace metals in River Nile up- and downstream of a densely populated
area. Environmental Geochemistry and Health 39(1):125–137. doi: 10.1007/s10653-
016-9814-4
ur

Örn, S. (2006). PhD thesis - The zebrafish as a model organism for evaluation of
Jo

endocrine disrupters. Department of Biomedical Sciences and Veterinary Public

Health, Swedish University of Agricultural Sciences.

Paíga, P., Correia, M., Fernandes, M. J., Silva, A., Carvalho, M., Vieira, J., Jorge, S.,
Silva, J., G., Freire, C., Delerue-Matos, C. (2019). Assessment of 83 pharmaceuticals
in WWTP influent and effluent samples by UHPLC-MS/MS: Hourly variation.
Science of the Total Environment 648:582–600. doi:

36
 10.1016/j.scitotenv.2018.08.129

Papageorgiou, M., Kosma, C., & Lambropoulou, D. (2016). Seasonal occurrence,

removal, mass loading and environmental risk assessment of 55 pharmaceuticals and
personal care products in a municipal wastewater treatment plant in Central Greece.
Science of The Total Environment 543:547–569. doi:
10.1016/J.SCITOTENV.2015.11.047

Park, H. S., Jang, J. E., Ko, M. S., Woo, S. H., Kim, B. J., Kim, H. S., Park, H. S., Park,
I. S., Koh, E. H., Lee, K. U. (2016). Statins increase mitochondrial and peroxisomal
fatty acid oxidation in the liver and prevent non-alcoholic steatohepatitis in mice.

of
Diabetes and Metabolism Journal 40(5):376–385. doi: 10.4093/dmj.2016.40.5.376

Piechota-Polanczyk, A., Goraca, A., Demyanets, S., Mittlboeck, M., Domenig, C.,

ro
Neumayer, C., Wojta, J., Nanobachvili, J., Huk, I., Klinger, M. (2012). Simvastatin
Decreases Free Radicals Formation in the Human Abdominal Aortic Aneurysm Wall
-p
via NF-κB. European Journal of Vascular and Endovascular Surgery 44(2):133–137.
doi: 10.1016/J.EJVS.2012.04.020
re
Pradhan, A., & Olsson, E. (2016). Regulation of zebrafish gonadal sex differentiation.
AIMS Molecular Science 3(4):567–584. doi: 10.3934/molsci.2016.4.567
lP

Prober, D. A., Rihel, J., Onah, A. A., Sung, R.-J., & Schier, A. F. (2006).
Hypocretin/Orexin Overexpression Induces An Insomnia-Like Phenotype in
na

Zebrafish. Journal of Neuroscience 26(51):13400–13410. doi:

10.1523/JNEUROSCI.4332-06.2006
ur

Prokop’eva, N. V., & Gulyaeva, L. F. (2000). Glutathione S-transferase activity in the

liver in acute pancreatitis at various terms of disease and during treatment with
Jo

inducers. Bulletin of Experimental Biology and Medicine 129(5):458–459. doi:

10.1007/BF02439801

Prueksaritanont, T., Subramanian, R., Fang, X., Ma, B., Qiu, Y., Lin, J. H., Pearson, P.
G., Baillie, T. A. (2002). Glucuronidation of statins in animals and humans: A novel
mechanism of statin lactonization. Drug Metabolism and Disposition 30(5):505–
512. doi: 10.1124/dmd.30.5.505

37
Rahman, K. (2007). Studies on free radicals, antioxidants, and co-factors. Clinical
Interventions in Aging 2(2):219–236. PMID: 18044138

Rang, H. P., Ritter, J. M., Flower, R. J., & Henderson, G. (2007). Rang and Dale’s
pharmacology. Churchill Livingstone, Philadelphia. ISBN: 9780702040740

Ray, P. D., Huang, B.-W., & Tsuji, Y. (2012). Reactive oxygen species (ROS)
homeostasis and redox regulation in cellular signaling. Cellular Signalling
24(5):981–990. doi: 10.1016/j.cellsig.2012.01.008

Randlett, O., Haesemeyer, M., Forkin, G., Shoenhard, H., Schier, A. F., Engert, F., &
Granato, M. (2019). Distributed Plasticity Drives Visual Habituation Learning in

of
Larval Zebrafish. Current Biology 29(8):1337-1345. doi: 10.1016/j.cub.2019.02.039

ro
Salgado, R., Noronha, J. P., Oehmen, A., Carvalho, G., & Reis, M. A. M. (2010). Analysis
of 65 pharmaceuticals and personal care products in 5 wastewater treatment plants
-p
in Portugal using a simplified analytical methodology. Water Science and
Technology 62(12):2862–2871. doi: 10.2166/wst.2010.985
re
Scholz, S., Fischer, S., Gündel, U., Küster, E., Luckenbach, T., & Voelker, D. (2008).
The zebrafish embryo model in environmental risk assessment—applications
lP

beyond acute toxicity testing. Environmental Science and Pollution Research

15(5):394–404. doi: 10.1007/s11356-008-0018-z
na

Ser, J. R., Roberts, R. B., & Kocher, T. D. (2010). Multiple interacting loci control sex
determination in lake Malawi Cichlid fish. Evolution 64(2):486–501. doi:
10.1111/j.1558-5646.2009.00871.x
ur

Sharma, M. (2019). Behavioural responses in effect to chemical stress in fish: A review

Madhu Sharma. International Journal of Fisheries and Aquatic Studies, 7(1):1-5.
Jo

Sharma, P., Jha, A. B., Dubey, R. S., & Pessarakli, M. (2012). Reactive Oxygen Species,
Oxidative Damage, and Antioxidative Defence Mechanism in Plants under Stressful
Conditions. Journal of Botany 2012:1–26. doi: 10.1155/2012/217037

Şişman, T., İncekara, Ü., & Yıldız, Y. S. (2008). Determination of acute and early life
stage toxicity of fat‐ plant effluent using zebrafish (Danio rerio). Environmental

38
 Toxicology 23(4):480–486. doi: 10.1002/tox.20366

Smith, G. S., Walter, G. L., & Walker, R. M. (2013). Clinical Pathology in Non-Clinical
Toxicology Testing. Haschek and Rousseaux’s Handbook of Toxicologic Pathology
565–594. doi: 10.1016/B978-0-12-415759-0.00018-2

Solé, M., & Sanchez-Hernandez, J. C. (2015). An in vitro screening with emerging

contaminants reveals inhibition of carboxylesterase activity in aquatic organisms.
Aquatic Toxicology 169:215–222. doi: 10.1016/j.aquatox.2015.11.001

Solé, M., Shaw, J.P., Frickers, P.E., Readman, J.W., Hutchinson, T.H. (2010) Effects on
feeding rate and biomarker responses of marine mussels experimentally exposed to

of
propranolol and acetaminophen. Analytical Bioanalytical Chemistry 396:649-656.
doi: 10.1007/s00216-009-3182-1

ro
Speijer, D., Manjeri, G. R., & Szklarczyk, R. (2014). How to deal with oxygen radicals
-p
stemming from mitochondrial fatty acid oxidation. Philosophical Transactions of the
Royal Society B: Biological Sciences, 369(1646). doi: 10.1098/rstb.2013.0446
re
Srinivasa Rao, A., Ahmed, M. F., & Ibrahim, M. (2012). Hepatoprotective activity of
Melia azedarach leaf extract against simvastatin induced hepatotoxicity in rats.
lP

Journal of Applied Pharmaceutical Science 2(7):144–148. doi:

10.7324/JAPS.2012.2721
na

Stoll, L. L., McCormick, M. L., Denning, G. M., & Weintraub, N. L. (2004). Antioxidant
effects of statins. Drugs of Today 40(12):975. doi: 10.1358/dot.2004.40.12.872573

Strzyżewski, K. W., Pioruńska-Stolzmann, M., Majewski, W., Kasprzak, M., &

ur

Strzyżewski, W. (2013). Effect of Surgical Treatment on Lipid Peroxidation

Parameters and Antioxidant Status in the Serum of Patients with Peripheral Arterial
Jo

Disease. Disease Markers 35(6):647–652. doi: 10.1155/2013/530946

Tete, V. S., Nyoni, H., Mamba, B. B., & Msagati, T. A. M. (2019). Occurrence and spatial
distribution of statins, fibrates and their metabolites in aquatic environments.
Arabian Journal of Chemistry 13(2):4358-4373. doi: 10.1016/j.arabjc.2019.08.003

Thorpe, G. W., Reodica, M., Davies, M. J., Heeren, G., Jarolim, S., Pillay, B.,

39
 Breitenbach, M., Higgins, V. J., Dawes, I. W. (2013). Superoxide radicals have a
protective role during H2O2 stress. Molecular Biology of the Cell 24(18):2876–2884.
doi: 10.1091/mbc.e13-01-0052

Ungureanu, D., Filip, C., Artenie, A., & Artenie, R. (2003). Evaluation of simvastatin
antioxidant effects. Revista Medico-Chirurgicala a Societatii de Medici Si
Naturalisti Din Iasi 107(1):66–71. PMID: 14755972

Vandeputte, M., Dupont-Nivet, M., Chavanne, H., & Chatain, B. (2007). A polygenic
hypothesis for sex determination in the European sea bass Dicentrarchus labrax.
Genetics 176(2):1049–1057. doi: 10.1534/genetics.107.072140

of
Venturini, C. D., Merlo, S., Souto, A. A., Fernandes, M. da C., Gomez, R., & Rhoden, C.
R. (2010). Resveratrol and red wine function as antioxidants in the nervous system

ro
without cellular proliferative effects during experimental diabetes. Oxidative
Medicine and Cellular Longevity 3(6):434–441. doi: 10.4161/oxim.3.6.14741
-p
Voss, J.-U., Roller, M., Brinkmann, E., & Mangelsdorf, I. (2005). Nephrotoxicity of
organic solvents: biomarkers for early detection. International Archives of
re
Occupational and Environmental Health 78(6):475–485. doi: 10.1007/s00420-005-
0611-0
lP

Wang, C., Ku, P., Nie, X., Bao, S., Wang, Z., & Li, K. (2019). Effects of simvastatin on
the PXR signaling pathway and the liver histology in Mugilogobius abei. Science of
na

the Total Environment 651(1):399–409. doi: 10.1016/j.scitotenv.2018.09.133

Wassmann, S., Laufs, U., Bäumer, A. T., Müller, K., Ahlbory, K., Linz, W., Itter, G.,
ur

Rösen, R., Böhm, M., Nickenig, G. (2001). HMG-CoA reductase inhibitors improve
endothelial dysfunction in normocholesterolemic hypertension via reduced
Jo

production of reactive oxygen species. Hypertension 37(6):1450–1457. doi:

10.1161/01.HYP.37.6.1450

Zar, J. (2010). Biostatistical Analysis (5th ed.). New Jersey: Pearson Prentice Hall.

Figure Captions

40
Fig. 1: (A) Erratic swimming and (B) Purposeful swimming for acute exposed
individuals, in cycles of light and dark periods. Data are Mean±SE (n=20 fish/treatment).
* Stands for significant differences among treatments, compared with the control, during
the respective light or dark period (Dunnett’s test in the first light cycle and Dunn’s test
in the other periods, p<0.05).

of
*
Erratic swimming (mm)

ro
* *
* *
*
* *
-p
* *
re
lP

B
na

*
Purposeful swimming (mm)

* *
*
ur

* *
*

* *
Jo

*
*

41
 Fig. 1

Fig. 2: (A) Total distance traveled and (B) Swimming time for acute exposed individuals,
in cycles of light and dark periods. Data are Mean±SE (n=20 fish/treatment). * Stands for
significant differences among treatments, compared with the control, during the
respective light or dark period (Dunn’s test, p<0.05).

of
A *

ro
*

** * **
* -p
re
* *
lP
na
ur
Jo

42
 B

**
** **

* *

of
ro
Fig. 2

-p
re
Fig. 3: (A) SOD activity, i.e., Cu-ZnSOD (grey) and MnSOD activity (black), (B) CAT
activity, and (C) GPx activity, i.e., selenium dependent activity (grey) and total activity
lP

(black) in acute exposed individuals. Data are Mean±SE (n=20 fish/treatment). * Stands
for significant differences among treatments, compared with control (Dunnett’s test,
p<0.05).
na
ur

Fig. 2
Jo

43
A
14

(mmol min -1 mg -1 protein)

12
Cu-ZnSOD
10
SOD activity

activity
8

6 MnSOD
activity
4

2
* * * *
0
0 92.45 184.9 369.8 739.6 1479.2
Simvastatin concentration (ng L-1)

of
B 16

14

ro
(µmol min -1 mg -1 protein)

12
CAT activity

10

6
-p
re
4

2
lP

0
0 92.45 184.9 369.8 739.6 1479.2
Simvastatin concentration (ng L-1)
na

C
250
(mmol min-1 mg -1 protein)

GPx
200
ur

* * * selenium
* *
GPx activity

dependent
150 activity
Jo

GPx total
100
activity

50

0
0 92.45 184.9 369.8 739.6 1479.2
Simvastatin concentration (ng L-1)

Fig. 3
44
Fig. 4: GSTs activity in acute exposed individuals. Data are Mean±SE (n=20
fish/treatment). * Stands for significant differences among treatments, compared with
control (Dunnett’s test, p<0.05).

350

300
(µmol min -1 mg -1 protein)

of
250
GSTs activity

ro
200
* *
*
150
*
100

50
-p
re
0
0 92.45 184.9 369.8 739.6 1479.2
Simvastatin concentration (ng L-1)
lP

Fig. 4
na

Fig. 5: TBARS in acute exposed individuals. Data are Mean±SE (n=20 fish/treatment). *
ur

Stands for significant differences among treatments, compared with control (Dunnett’s
test, p<0.05).
Jo

45
 0.08

0.07
(µmol min -1 mg -1 protein)
0.06 *
0.05
*
* *
TBARS

0.04

0.03

0.02

0.01

0.00
0 92.45 184.9 369.8 739.6 1479.2
Simvastatin concentration (ng L-1)

of
Fig. 5

ro
-p
Fig. 6: Sex ratio (percentage of males and females) in chronically exposed individuals
(n=10 fish/treatment).
re
lP
na
ur
Jo

Fig. 7: Histological architecture in terms of maturation stages of females and males in

chronically exposed individuals (n=20 fish/treatment). (A) Stage 0 - Undeveloped in
females at 369.8 ng L-1, with perinucleolar oocytes (arrow); (B) Stage 1 – Early
development in females at 92.45 ng L-1, with cortical alveolar oocyte (arrow); (C) Stage

46
2 – Mid-development in females at 369.8 ng L-1, with mid-vitellogenic follicles (arrow)
(D) Stage 0 – Undeveloped in males at 92.45 ng L-1, with primary spermatocyte (arrow);
(E) Stage 1 – Early spermatogenic in males of the control group, with immature phases
predominant (circle) and observable spermatozoa (arrow); and (F) Stage 2 – Mid-
spermatogenic in males at 739.6, with spermatids (arrowhead) and spermatozoa (arrow).
X200 Magnification. Hematoxylin-Eosin.

of
ro
-p
re
lP
na

Fig. 8: (A) Female and (B) Male developmental stages in chronically exposed individuals
(n=10 fish/treatment).
ur
Jo

47
 of
Fig. 9: Summary of major findings and putative relantioships between parameters.
Antioxidant properties of SIM seem to decrease oxidative stress in the exposed

ro
organisms, which can alter vision, muscle and brain activity and lead to a reduction of
erratic swimming in D. rerio. – represents non-significant results, ↑ represents an increase
and ↓ represents a decrease in the determined biomarkers, compared with the control,
-p
considering acute exposed individuals.
re
lP
na

Behavioural assessment Biochemical assessment

SOD GPx
Concentra Purposeful TBA
ur

Erratic swimming Total distance Swimming time

tion swimming RS
(ng L-1) CA Seleniu GST
Cu- M T m Tot s
Jo

300 600 300 600 900 300 600 900 300 600 900
0a 900 a 0 a 0a 0a Zn n depen al
a a a a a a a a a a a
300 1200 300 300 300 dent
600 900 600 900 1200 600 900 1200 600 900 1200
s s s s s
s s s s s s s s s s s

92.45 ↓ - ↓ ↓ - ↓ ↓ - ↑ ↓ ↓ - - ↓ - ↓ - - - ↑ - ↓ -

184.9 ↓ - - - ↑ - - - - - - - - ↓ - - ↓ - - - ↓ ↓ ↓

48
369.8 ↓ ↑ - - ↑ - - ↑ ↑ - - - ↓ - - - ↓ - - - ↓ ↓ ↓

739.6 - - - ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑ - ↑ ↓ - - - ↓ ↓ ↓

1479.2 ↓ ↑ ↑ ↑ - - ↑ ↑ - ↑ - ↑ - ↑ - ↑ ↓ - - - ↓ ↓ ↓

Table 1: Length and weight measurements of female and male individuals from the
different treatment groups, with respective Mean and SE (n=10 fish/treatment). Fig. 9

Females Males

of
Simvastatin
Length (mm) Weight (mg) Length (mm) Weight (mg)
concentration (ng L-1)
Mean SE Mean SE Mean SE Mean SE

ro
0 23.85 0.75 329.20 3.26 20.70 0.71 268.35 3.51
92.45 23.10 0.73 327.90 2.58 20.50 0.69 267.00 3.83
184.9 23.00 0.64 327.65 3.26 20.45 0.70 265.35 2.47
369.8
739.6
1479.2
-p
22.90 0.77 325.90 3.68
22.50 0.71 324.40 3.47
22.15 0.68 321.70 3.13
20.05 0.77 264.85 3.33
19.75 0.76 262.35 3.41
19.30 0.71 259.80 2.70
re
Table 1
lP
na
ur
Jo

49