Swaiacka Et Al, 2021

Environmental Pollution 273 (2021) 115891
Contents lists available at ScienceDirect
Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol
Review
Toxic effects of NSAIDs in non-target species: A review from the

perspective of the aquatic environment*

Klaudia Swiacka b
, Alicja Michnowska b, Jakub Maculewicz a, *, Magda Caban a,
Katarzyna Smolarz b
a sk, Wita Stwosza 63, 80-308, Gdan
Department of Environmental Analysis, Faculty of Chemistry, University of Gdan sk, Poland
b
Department of Marine Ecosystems Functioning, Institute of Oceanography, University of Gdansk, Av. Piłsudskiego 46, 81-378, Gdynia, Poland
a r t i c l e i n f o a b s t r a c t
Article history: The presence of pharmaceuticals in the aquatic environment, both in marine and freshwater reservoirs, is
Received 28 May 2020 a major concern of global environmental protection. Among the drugs that are most commonly used,
Received in revised form NSAIDs tend to dominate. Currently, being aware of the problem caused by drug contamination, it is
20 August 2020
extremely important to evaluate the scale and the full spectrum of its consequences, from short-term to
Accepted 15 October 2020
long-term effects. The influence on non-target aquatic animals can take place at many levels, and the
Available online 20 October 2020
effects can be seen both in behaviour and physiology, but also in genetic alterations or reproduction
disorders, affecting the development of entire populations. This review summarises all the advances
Keywords:
Pharmaceuticals
made to estimate the impact of NSAIDs on aquatic animals. Multicellular animals from all trophic levels,
Contaminants of emerging concern inhabiting both inland waters, seas and oceans, have been considered. Particular attention has been paid
Bioindicators to chronic studies, conducted at low, environmentally-relevant concentrations, to estimate the real ef-
Fish fects of the present pollution. The number of such studies has indeed increased in recent years, allowing
Bivalves for a better insight into the possible consequences of pharmaceutical pollution. It should be stressed,
Crustaceans however, that our knowledge is still limited to a few model species, while there are many groups of
organisms completely unexplored regarding the effects of drugs. Therefore, the main aim of this paper
was to summarise the current state of knowledge on the toxicity of NSAIDs in aquatic animals, also
identifying important gaps and major issues requiring further analysis.
© 2020 Elsevier Ltd. All rights reserved.
1. Introduction biotransformation in aerobic conditions and photolysis occurring in

surface waters (Koumaki et al., 2017). On the other hand, NSAIDs
Non-steroidal anti-inflammatory drugs (NSAIDs) are currently appear resistant to hydrolysis (Ton ski et al., 2019) and are unlikely
at the leading position among contaminants present in the envi- to undergo sorption (Koumaki et al., 2017; Elizalde-Vela zquez et al.,
ronment. According to a meta-analysis carried out by Santos et al. 2020). However, despite moderate compound-dependent trans-
(2010), NSAIDs accounted for more than 15% of all pharmaceuti- formation rates, these drugs are being constantly recorded in the
cals detected in the environment and were the most frequently environment. This is mainly due to the fact that their inflow to
detected of all groups. NSAIDs are in high demand and their con- surface waters is constant and degradation is compensated by
sumption in many countries remains high or even grows. They are continuous supply of subsequent doses.
used in enormous quantities not only by humans, but also veteri- The continuous inflow of NSAIDs from sewage treatment plants
nary medicine, especially in mass animal farming (Elizalde- poses a threat to aquatic fauna, which is constantly exposed to
Velazquez et al., 2020; Thalla and Vannarath, 2020; Bereketoglu these biologically active compounds (Fent et al., 2006; Wang et al.,
et al., 2020). NSAIDs are characterised by relatively low environ- 2018; Kolpin et al., 2002; Miarov et al., 2020). However, information
mental stability and are easily degradable, especially through concerning the toxicity of NSAIDs is highly limited, especially for
invertebrates, whereas much more data is available for fish
(Serrano et al., 2015; Parolini et al., 2009; Munari et al., 2018; Zanuri
*
This paper has been recommended for acceptance by Maria Cristina Fossi. et al., 2017). Although NSAIDs have been manufactured and used
* Corresponding author. for decades, they have only recently started to be studied and tested
E-mail address: [email protected] (J. Maculewicz).
https://doi.org/10.1016/j.envpol.2020.115891
0269-7491/© 2020 Elsevier Ltd. All rights reserved.
K. Swiacka, A. Michnowska, J. Maculewicz et al. Environmental Pollution 273 (2021) 115891
for their potential toxic effects on aquatic organisms. While the Jaimes et al., 2018; MPereira et al., 2017; NPaíga et al., 2016; OK’or-
mechanism of action of these compounds is well known in humans eje et al., 2016; PKermia et al., 2016; RMadikizela and Chimuka
and other vertebrates, in aquatic invertebrates, which are chroni- (2016); SRoberts and Thomas (2006); TTogola and Budzinski
cally exposed to these substances (often in trace level), these (2008); UKasprzyk-Hordern et al., 2008; WLoos et al., 2013; XMcE-
mechanisms are almost unknown. Hence, there is still a high un- neff et al., 2014; YNgubane et al., 2019.
certainty about the risks they pose to aquatic environment. In order to understand effects of NSAIDs on non-target aquatic
Today NSAIDs are regularly detected in water bodies around the species, various experiments were conducted and, apart from
world. This includes coastal marine and oceanic waters, but also short-term toxicity, some studies describe the results of experi-
freshwater reservoirs such as rivers and lakes (Bonnefille et al., mental work in which chronic exposure of model aquatic organ-
2018; Balakrishna et al., 2017; Arpin-Pont et al., 2016). The range isms to environmentally relevant NSAIDs levels were performed.
of concentrations recorded in the environment most often varies These results highlighted that even low concentrations of NSAIDs,
from several dozen to several hundred ng/L. However, the highest at continuous exposure, are toxic and contribute to the disruption
observed concentrations reach even several mg/L (Fig. 1.). Thus, for of many physiological processes to both model groups, vertebrates
the sake of simplicity, exposure experiments are usually assumed to and invertebrates (Mezzelani et al., 2018; Xu et al., 2019; Hayashi
be environmentally relevant at concentrations up to several mg/L et al., 2008; Lee et al., 2011).
(e.g. Aguirre-Martínez et al., 2013a; Gutie rrez-Noya et al., 2020; The aim of this work was to review the effects of NSAIDs in non-
Chen et al., 2014; Parolini et al., 2012). target aquatic animals (polychaetes, crustaceans, molluscs, fish and
References: AGonza lez-Alonso et al., 2017; BWu et al., 2010; others). Within this review the toxic effects of the most commonly
C
Borecka et al., 2015; Vidal-Dorsch et al., 2012; EYang et al., 2011;
D
detected NSAIDs in the aquatics: diclofenac (DCF), ibuprofen (IBU),
F
Fang et al., 2012; GKallenborn et al., 2018; HComeau et al., 2008; ketoprofen (KET), acetylsalicylic acid (ASA), indomethacin
I
Loli
c et al., 2015; JPereira et al., 2016; KAli et al., 2017; LRivera- (INDMT), naproxen (NPX) and mefenamic acid (MA) are described.
Fig. 1. Maximum concentrations of NSAIDs (ng/L) detected in aquatic ecosystems worldwide. Asterisks indicate data obtained from freshwater reservoirs.
2
In the individual subsections, the results of experimental studies all three phases of xenobiotic biotransformation. Stancova et al.
performed for individual taxonomic groups are discussed. All major (2015) investigated gene expression of antioxidant enzymes in
groups of marine and freshwater animals have been taken into liver and intestines of zebrafish exposed for 14 days to NPX con-
account, excluding single-celled organisms. Hence, a summary of centrations as high as 1 and 100 mg/L. Only a few studies tested the
the current state of knowledge on the toxicity of NSAIDs in marine toxic effects of NPX using fish. No effect of NPX on the transcrip-
animals is presented here, also identifying important gaps and tional changes of CYP1A genes in both organs or on the expression
major issues requiring further analysis. levels of superoxide dismutase (SOD) and glutathione peroxidase
(GPx) were found. However, it was observed that NPX was able to
2. Toxic effects of NSAIDs in aquatic organisms upregulate the activity of GST by affecting glutathione S-transferase
P2 (GST P2) mRNA in the intestine. The effect was found to be dose-
2.1. Fish dependent. In addition, significant effect of NPX on catalase (CAT)
gene expression was found only in the intestines. In Zivna et al.
Fish are aquatic vertebrates most widely used for toxicity (2013) the exposure of D. rerio to ASA at following concentrations
studies of various contaminants. Their well-known physiology and 40, 120 and 250 mg/L resulted in oxidative stress induction (an
behaviour are crucial for their employment in such tests. In addi- increase of GST, CAT and GPx activities). Guiloski et al. (2015) tested
tion, fish play an important role in the environment, determining toxicity of DCF on freshwater fish Hoplias malabaricus using anti-
the biological structure of habitats as well as modifying prey oxidant enzymes assay. The following DCF concentrations: 0.2; 2
communities. Fish are also an important food source for numerous and 20 mg/kg were injected into the peritoneum of each individual
aquatic organisms (Jones, 1988). For toxicological studies, species (n ¼ 10) at 12 doses, resulting in an increase in activity of SOD, GPx
such as Danio rerio and Oryzias latipes, easy to grow and maintain, and glutathione (GSH). This study revealed that DCF interferes with
are most often employed. An additional advantage of these species metabolic pathways and thus causes oxidative stress in fish.
is their short generation time and frequent reproduction (Hallare Reduction of GST activity in H. malabaricus liver was also observed,
et al., 2004; Flippin et al., 2007). This attribute is of great value in possibly indicating a decrease of liver metabolic rate. On the con-
toxicity tests at early stages of development. Some toxicological trary, some studies highlighted an increase in GST activity in fish
tests are also performed on economically important fish such as after DCF exposure. For example, in Stepanova et al. (2013) GST
Cyprinus caprio, Oncorhynchus mykiss and Rhamdia quelen (Guiloski activity increased in juvenile C. caprio exposed to DCF at concen-
et al., 2017). tration of 3 mg/L. Guiloski et al. (2017) also investigated the toxic
effect of DCF on R. quelen. Fish were exposed to 0.2, 2 and 20 mg/L of
2.1.1. Cyto & genotoxicity DCF for 21 days. In all treatment groups a significant reduction of
Islas-Flores et al. (2017) observed that already a short-term SOD, as well as increase activity of GSH and GST were observed. In a
exposure (12, 24, 48, 72, and 96 h) to DCF (17.6 mg/L), IBU group treated with 2 mg/L reduction in CAT and ethoxyresorufin- O-
(7.10 mg/L) and their mixture induced various cyto- and genotoxic deethylase (EROD) activities were noted. At low concentrations,
effects in C. carpio. Significant increase in the number of micro- DCF also disrupted antioxidant defence systems in the liver and
nuclei was observed in fish exposed to DCF at 72 h and in fish gonads. Enzymes belonging to the CYP450 family (e.g. EROD) play a
exposed to mixture of DCF and IBU at 24, 48 and 72 h. DNA damage crucial role in biotransformation of most xenobiotics including
(comet assay), measured as the intensity of the tail DNA relative to NSAIDs (Gomez et al., 2011; Whyte et al., 2000; Xu et al., 2019).
the head, was observed in C. carpio exposed to DCF, IBU and their These results may therefore suggest that R. quelen is able to
mixture at all exposure time. Exposure to DCF/IBU mixture resulted metabolise DCF to less complex compounds. Similarly, Nunes et al.
in the highest levels of DNA damage. Moreover, alterations in the (2020) observed an increased GST activity in Solea senegalensis
specific activity of caspase-3 due to IBU exposure at 24 h were exposed to 0.5, 1 and 2 mg/L of DCF for 28 days. Interestingly, this
observed. Pandey et al. (2017) evaluated the DNA damage in study verified whether the drug’s effect depends on its uptake
Oreochromis niloticus chronically exposed to DCF at concentrations route. In fish exposed to DCF diluted in water, no effect on CAT
of 0.17, 0.34 and 0.68 mg/L for 60 days. Similarly to the results activity was seen, while a decrease in CAT activity was observed
obtained by Islas-Flores et al. (2017), also in this study DCF at sub- only in those individual fed with polychaetes previously exposed to
lethal concentrations caused DNA damage as revealed by comet DCF. Stepanova et al. (2013) observed reduction in lipid peroxida-
assay. Rocco et al. (2010) also noted genotoxic effect of IBU (92 ng/L) tion (LPO) in D. rerio exposed to DCF. This is a very notable obser-
in zebrafish seen as DNA fragmentation in erythrocytes, loss of DNA vation, indicating a potential positive effect of the drug (reduction
integrity, apoptosis and genomic alterations. In contrast, Guiloski of cell membrane damage due to oxidative lipid degradation). LPO
et al. (2017) did not observe genotoxic effect in Rhamdia quelen may contribute to the cell damage and dysfunction. LPO was
exposed to 0.2, 2, and 20 mg/L of DCF for 21 days as no DNA damage assessed by measuring the level of thiobarbituric-acid-reactive-
in blood and liver cells or increased frequencies of micronuclei substances (TBARS) in fish tissue. However, LPO is more
were found. Ghelfi et al. (2016) also did not reported DNA damage frequently examined in invertebrates such as crustaceans and
in R. quelen exposed to DCF at concentrations of 0.2, 2 and 20 mg/L mussels than in fish (Mezzelani et al., 2016, 2018; Quinn et al., 2011;
for 96 h. Munari et al., 2016). Similarly, Ghelfi et al. (2016) showed that DCF
caused reduction in LPO in juvenile R. quelen. Feito et al. (2012) also
2.1.2. Gene expression & metabolism disorders observed LPO reduction in juvenile zebrafish acutely exposed
Numerous studies indicate that NSAIDs interfere with metabolic (90 min) to DCF at environmentally relevant concentration
pathways in fish. It has been observed that NSAIDs disturb the (0.03 mg/L). Unfortunately, very little is known about the mode of
expression of different genes as well as the activity of various action of NSAIDs inside the body of aquatic organisms, so the
metabolic and protective enzymes. Gro € ner et al. (2015) observed a mechanism behind a decrease in LPO after DCF exposure is not yet
significant increase in the expression of key enzymes involved in explained.
xenobiotic biotransformation phases I, II, III (cytochrome P450 1A
[CYP1A], glutathione S-transferase [GST] and multi-drug resistance 2.1.3. Endocrine disruption
protein [MRP]) in O. niloticus exposed to environmental concen- Various authors highlighted that pharmaceuticals belonging to
tration (4 nM) of DCF. The results clearly indicated that DCF induces NSAIDs act like endocrine disruptors in fish. Gravel and Vijayan
3
(2006) were the first to reveal disruption of cortisol production in concerning the effect of IBU on the reproduction of O. latipes were
O. mykiss exposed to IBU. Such effects were previously observed in obtained by Flippin et al. (2007). Due to exposure to IBU, fish
fish only after exposure to organic pollutants and heavy metals spawned less frequently and more eggs per brood was produced.
(Hontela, 2005). Corcoran et al. (2012) demonstrated that IBU With an increase of drug concentration, fish reproduction was less
significantly induced CYP2K expression in C. carpio hepatocyte frequent. The highest number of eggs per day (twice as high as in
cultures. The influence of selected NSAIDs (DCF, IBU, ASA, MA and the control) was observed in fish exposed to 100 mg/L IBU. In the
NPX) on steroid hormones and expression of 21 genes from second part of this experiment zebrafish was exposed to 1e100 mg/
hypothalamicepituitaryegonadal (HPG) axis in D. rerio was also L IBU, and as a consequence reduced egg production was observed.
evaluated by Ji et al. (2013). Fish were exposed for 14 days to each Delayed hatching was also observed in the offspring group not
NSAIDs at concentrations of 10, 100 and 1000 mg/L. IBU, MA and ASA directly exposed to IBU (the parents were exposed). This may be a
(with the greatest effect of IBU) affected transcription of most of the consequence of the reduced gametes quality or the horizontal
tested genes from HPG axis and increased the transcription level of transfer of IBU (its deposition into eggs). Delay in time, slower
genes involved in the acceleration of gametogenesis, maturation of hatching and increased frequency of malformation after parental
oocytes in females and spermatogenesis in males. As a conse- exposure suggests trans-generational effects of IBU. In contrast, in
quence, this may lead to disruption of gonadotropin production. On Morthorst et al. (2013) IBU did not affect cumulative egg production
the other hand, Han et al. (2010) observed that IBU affected the in D. rerio exposed to the drug concentrations: 21, 201 or 506 mg/L
production of estradiol (E2), which was higher in fish exposed to for 7 days. Gutie rrez-Noya et al. (2020) also investigated terato-
this pharmaceutical. Apart from estrogenic effect, the compound genic effects and embryotoxicity of IBU on C. carpio. Fish oocytes
also affected androgens by decreasing the level of testosterone (T) and embryos were exposed to IBU at environmentally relevant
and both effects were dose dependent. Flippin et al. (2007) also concentrations: 1.5, 3, 4.5, 6, 7.5, 9 and 11.5 mg/L for 96 h post
showed abnormal E2 and T levels in O. latipes due to IBU (1 mg/L). fertilization (hpf). Teratogenic effect was revealed by the higher
Moreover, in Han et al. (2010) the highest concentration of IBU mortality of oocytes and delayed hatching. Embryotoxicity was
caused significant increase in vitellogenin (VTG) content indicating shown as the delay of embryos development and embryos mal-
its endocrine disrupting effect. VTG, a key protein product involved formations included yolk deformation, hypopigmentation and
in egg yolk formation in females, is an important marker in eco- pericardial swelling. Zhang et al. (2020), studied teratogenic effect
toxicological studies, thus observed changes in VTG gene expres- of two selected NSAIDs, IBU and DCF, on cardiovascular develop-
sion or VTG content may indicate their endocrine disrupting nature. ment in D. rerio embryos at concentrations: 0.04, 0.2, 1, 5 and 25 mg/
€ ner et al. (2015) an impact of DCF on the expression
Similarly, in Gro L for 56 hpf. It was observed that both drugs disrupted cardiac
of VTG genes in O. niloticus was observed. Hong et al. (2007) also physiology of embryos. IBU increased the heart rate and the average
highlighted significant effects of DCF at environmentally relevant ventricular stroke volume in the last hour of exposure, resulting in
concentration (1 mg/L) on VTG gene expression in O. latipes. Fish up-regulation of cardiac outputs, while DCF increased heart rate in
were exposed to DCF for 12 h, 1, 2 and 4 days. In contrast, Morthorst a dose-dependent manner. David and Pancharatna (2009) obtained
et al. (2013) did not observe any significant changes in VTG content similar results with D. rerio embryos in which cardiac physiology
in D. rerio exposed to several IBU concentrations 21, 201 or 506 mg/L was affected by IBU. IBU at concentration 10 mg/L and higher lead to
for 7 days. Kwak et al. (2018) tested the endocrine disrupting po- cardias abnormalities such as irregular heartbeat and pericardial
tential of NPX in O. latipes. Endocrine disruption was revealed by edema. Another important study evaluating the ecotoxic potential
significant increase in the expression of VTG and E2 receptors of NSAID (DCF in particular) on embryos Salmo trutta f. fario was
genes. Xu et al. (2019) also tested endocrine disrupting potential of done by Schwarz et al. (2017). Fish embryos were exposed to DCF
trace level of NPX in zebrafish. The experiment lasted 60 days and concentrations of 0.1, 0.5, 1, 10, 100 mg/L over 127 days. However,
consisted of exposure concentrations 0.1, 1, 10 and 100 mg/L. Here, embryotoxicity of DCF was not observed in this study. DCF did not
the authors assessed the toxic effect of NPX on thyroid gland by affect the level of stress proteins, lipid peroxidation, hatching,
measuring thyroid hormones level (triiodothyronine and mortality and development of embryos. Unlike Zhang et al. (2020),
thyroxine) in tested individuals. The levels of triiodothyronine and no effect of DCF on heart rate was observed in embryos. Yokota et al.
thyroxine were significantly lowered in fish exposed to NPX, with (2017) also investigated the effect of DCF on reproduction in
dose-dependent reduction of triiodothyronine. Additionally to that, O. latipes. The authors also verified whether the observed abnor-
the level of transthyretin (TTR) in serum was also significantly malities are permanent or reversible when O. latipes are returned to
affected by this pharmaceutical. Since TTR is a transport protein clean water (without DCF). Mating pairs of fish were exposed for 14
responsible for moving thyroid hormones into different tissues, days to 7.1, 37, and 78 mg/L DCF and significant reduction of
changes in its level together with lowering the levels of thyroid fecundity and fertility was observed in fish exposed to 37 and
gland hormones suggest significant thyroid-related disorder in 78 mg/L DCF. Furthermore, during depuration phase gradual resto-
exposed zebrafish. As thyroid hormones in fish play an important ration of reproductive capacity of O. latipes was observed. In further
role in development and reproduction (Blanton and Specker, 2007), study by Yokota et al. (2018) teratogenic properties of DCF were
disruption in their synthesis and/or transportation may ultimately investigated using medaka fish as models. Fertilized fish eggs were
lead to the dysfunction of these life-crucial processes and thus exposed to the following DCF concentrations: 0.608, 2.15, 7.29, 26.5
affect further population development. and 94.8 mg/L for 90 days post-fertilization (dpf). As in Schwarz
et al. (2017), also within the above-mentioned experiment DCF
2.1.4. Reproduction disruption and teratogenic effect did not affect the success and time of hatching. Moreover, no effect
Han et al., 2010 tested the effect of IBU on reproduction pro- of DCF on post-hatch mortality, growth of hatched larvae and their
cesses and early life stages of O. latipes. For this purpose the chronic behaviour was noted. The only observed teratogenic effect caused
experiment (two stages) was conducted. Fish eggs (until hatching, by DCF was mandibular defect in fish larvae exposed to 7.29 mg/L
first stage) and fry (second stage) were exposed to IBU at doses of and above. Study by Xia et al. (2017) is another one investigating
0.01, 0.1, 1, 10, 100, or 1000 mg/L for 132 day post-hatch (dph). IBU the effect of NSAIDs (IBU and DCF) on early life stages of D. rerio.
affected the spawning of O. latipes. Adult fish exposed to IBU pro- Zebrafish embryos were exposed to 5, 50 and 500 mg/L starting
duced significantly more eggs per brood than fish from the control, from 6 up to 120 hpf. Only the highest DCF and IBU concentrations
but hatching (time to hatch) was delayed. Similar results delayed hatching. Moreover, it was shown that tested
4
pharmaceuticals affected expression of selected genes involved in condition (CI) of O. latipes was verified. The condition of fish was
neural function and development in embryos. A similar effect was not affected by IBU, but an observed adverse effect included
observed in Hallare et al. (2004), where delayed hatching was decreasing the survival of adult fish in a time and dose dependent
observed only in embryos exposed to 1000 and 2000 mg/L DCF. manner. Schwarz et al. (2017) also observed increased mortality in
Chen et al. (2014) in turn, showed teratogenic effect of DCF at juvenile stages of S. trutta f. fario (about 6 months post hatch) due to
environmental concentration (3.78 mM) in zebrafish. In exposed DCF. Fish were exposed to concentrations 0.1, 1, 10, 100, 200 mg/L for
embryos various morphological abnormalities including shorter 25 days, but only 100 mg/L and above caused significant mortality of
body length, pericardial edema, liver atrophy, muscle degeneration juveniles. Similarly, in Kwak et al. (2018) NPX significantly
and abnormal pigmentation were observed. Furthermore, DCF decreased the survival of juvenile O. latipes, which were exposed to
altered the expression and regulation of genes involved in funda- the following concentrations: 0.005, 0.05, 0.5, 5, and 50 mg/L until
mental life-supporting processes like cardiac differentiation and 30 dph. Morthorst et al. (2013) did not observe the effect of IBU on
body patterning. DCF concentrations above 10.13 mM was lethal to GSI in D. rerio exposed to 21, 201 and 506 mg/L for 7 days. Likewise,
all embryos. In Zivna et al. (2013) the toxicity of ASA on the early life Guiloski et al. (2015) did not observe the effect of DCF on GSI in H.
stages of C. carpio was performed. C. carpio embryos were exposed malabaricus. However, the reduction of liver size (lower HIS) in H.
to 0.004, 0.04, 0.4, 4 and 20 mg/L of ASA for 34 days. Results of this malabaricus due to injection of DCF (0.2; 2 and 20 mg/kg) to the
study showed an increased rate of hatching, stimulation of devel- peritoneum was found. On the other hand, Flippin et al. (2007)
opment (early ontogeny), morphological abnormalities (increased observed inhibition of gonadal growth (lower GSI) in O. latipes
body pigmentation and axial and/or lateral curvature of the spine), exposed to IBU at1 mg/L. Lee et al. (2011) did not observe any effect
lesions (increased mucous cell numbers and the dermo-epidermal of DCF on HIS in O. latipes exposed for three months to concen-
changes) and oxidative stress (reduction in GPx, CAT and gluta- trations range 1e10,000 mg/L DCF. In this experiment, however, DCF
thione reductase (GR) activities). However, there are no other was added to the ambient water instead of being injected as in
studies concerning ASA embryo-toxicity in fish. Guiloski et al. (2015). Different way of application may affect the
toxicity of DCF and thus mitigate the effects observed in fish.
2.1.5. Morphological and tissue alterations
Only a few studies investigated the effect of NSAIDs on tissue 2.1.7. Behavioural changes
condition in fish. Han et al. (2010) did not observe any effect of IBU Another interesting effect observed in fish treated with non-
on the gonads, liver and kidney in adult medaka fish which were steroidal drugs includes behavioural anomalies. Schwarz et al.
already exposed as eggs for 90 dph to the IBU concentration range (2017) reported increased aggression with increase DCF concen-
0.01e100 mg/L. Contrary, in Schwarz et al. (2017) the toxic effect of tration in juvenile S. trutta f. fario. The symptoms of aggression
DCF was revealed in juvenile stages of S. trutta f. fario, in which included bites and, as a consequence, wounds observed already at
particular lesions in gills, liver and kidney were observed. Mohebi 10 mg/L. On the other hand, more frequent wounds in fish may be
Derakhsh et al. (2020) also observed histopathological changes in due to their weaker condition, as a result of exposure to DCF and
C. carpio gills, liver and kidney due to DCF. In this study, C. carpio thus less capacity for self-defence. Ajima et al. (2015) also noted
was exposed to 1.25, 2.5 and 5 mg/L of DCF over 21 days. Observed behavioural changes in R. quelen treated with 25 mg/L DCF. Those
lesions included necrosis of epithelial cells of kidney tubules, included respiratory disorders, loss of balance and irregular
epithelial hyperplasia and lamellar fusion in gills, as well as swimming. Xia et al. (2017) revealed that IBU and DCF affected
degeneration, vacuolation and necrosis of hepatocytes in liver. locomotion in zebrafish larvae and decreased their spontaneous
Similarity, Schwaiger et al. (2004) noted numerous alterations in movement. In addition, IBU disrupted free swimming of larvae
rainbow trout organs (gills and kindey). O. mykiss were exposed to during dark period (Fig. 2). Behavioural changes observed in larvae
DCF concentrations 1e500 mg/L for 28 days. Observed lesions and juvenile fish exposed to NSAIDs may be related to other effects
included accumulation of protein droplets in tubular renal cells, caused by these drugs e.g. increased mortality, morphological
vacuolisation and necrosis of tubular epithelial cells as well as changes, nervous and reproductive disorders or stress (Xia et al.,
proliferation of the renal interstitial tissue, necrotic changes of the 2017).
pillar cells in gills, dilation of the capillary walls, proliferation of
interlamellar and chloride cells in gills, inflammatory reactions and 2.2. Molluscs
epithelial lifting. Furthermore, DCF has contributed to an increase
in the frequency of necrosis of respiratory epithelial cells in gills. Molluscs, especially bivalves and gastropods, are generally
Yokota et al. (2017) in turn, observed morphological abnormalities considered as good bioindicators in environmental studies. Species
in O. latipes exposed to DCF concentrations 7.1, 37, and 78 mg/L for 14 characterised by sessile lifestyle and either filtering or deposit
days. In female fish from all treatment groups swollen abdomen feeding behaviour are in particular susceptible to exposure to
was observed. Moreover, the lower jaw defect was noted in males contaminants present in the ambient environment and as a
which were exposed to DCF concentrations 37 and 78 mg/L. During consequence to their accumulation in the tissues and potential
depuration phase (14 days) the swelling was significantly reduced. toxicity (Yusof et al., 2004; Wołowicz et al., 2005). Specific molec-
However, the mandibular defect was not reversed during or after ular or/and cellular targets are also present in this group of or-
the depuration phase. The authors of this study were the first to ganisms, thus most of environmental pollutants, including
reveal that DCF causes skeletal deformities in fish that appear to be pharmaceuticals, will potentially induce the toxic effect in this non-
irreversible. target group. Hence, relatively high responsivity of molluscs to
contamination along with their simultaneous stress resistance
2.1.6. Effect on condition and survival make them good models for ecotoxicological research. Adaptability
To assess the condition and development of individual organs or of bivalves and gastropods also provides great advantage of using
the whole body, different indexes are used, which are usually these organisms in laboratory-based toxicity testing (Krieger et al.,
calculated as the ratio of organ weight to whole body weight or 1981; Blaise et al., 2006; Renault, 2015). Different bivalve species
body weight to length. Examples of such indexes are: condition were used in pharmaceutical studies with Mytilidae family being
index (CI), gonadosomatic index (GSI) or hepatosomatic index recognized as the most popular model organisms used in both
(HIS). In study by Han et al. (2010) the effect of IBU on the general laboratory and environmental investigations. Hence, Mytilidae are
5
Fig. 2. The most common toxic effects of NSAIDs in aquatic organisms.
not included in this review as impact of NSAIDs on this group was evaluated by exposure of haemolymph cells for 1 h to 0.2, 0.5,

recently discussed in Swiacka et al. (2019). 0.8 mM DCF, and 0.2, 1, 3 mM IBU. Both drugs revealed acute cyto-
genotoxic effect to treated haemocytes resulting in irreversible
2.2.1. Cyto & genotoxicity DNA damage and a decrease of LMS. DCF showed dose-related
Cyto- and/or genotoxicity assessment of NSAIDs exposure in trend in DNA fragmentation and caused significant DNA damage
molluscs is one of the most investigated aspects of this subject. The at the lowest concentration tested, whereas IBU was found to be
most used markers include lysosomal membrane stability (LMS) less genotoxic. Significant decrease in LMS was achieved only at the
considered as one of the most valid biomarkers for general chem- highest exposures with DCF and IBU. Severe chronic effect of IBU in
ical pollution that is included in many biomonitoring programmes D. polymorpha was later described in Parolini et al. (2011a). In this
worldwide (ICES, 2015), DNA damage and/or fragmentation, study mussels were exposed for 96 h to environmentally relevant
apoptotic cells number and micronuclei (MN). Reduced LMS reveals concentrations of IBU (1, 9, 35 nM). Apoptotic cells number
cytotoxic effect of xenobiotics based on lysosomal membrane increased along with both time and concentration of exposure and
permeability and therefore straitened endocytosis, excretion of the same trend was revealed for MN count indicating clear DNA
hydrolytic enzymes into cytosol and possibly altered immunolog- damage to mussels haemocytes caused by environmental concen-
ical functions of cells in molluscs (Moore 1990; Lin and Steichen tration of IBU and dose-related decrease of LMS. In contrary to this
1994; ICES, 2015). DNA damage is likely considered as a conse- study, environmental concentration of DCF (0.3, 1, 3 nM) was not
quence of oxidative stress (Steinert 1999; De Almeida et al., 2007) sufficient for induction of significant cytogenotoxic effects in
provoked by exposure to various contaminants, whereas increased D. polymorpha haemocytes after mussels were exposed for 96 h, as
MN number reflects on exposure to chemicals of clastogen or described in Parolini et al. (2011b). No significant effect on
aneuploidogen characteristics (Hummelen et al., 1992; Bolognesi apoptosis and MN formation was shown. Treated cells only
and Fenech 2012). IBU and DCF were proved to have cyto- and/or demonstrated mild decrease in LMS in the end of exposure to
genotoxic effect on several mollusc taxa in acute and chronic highest concentration tested.
toxicity experiments although there is no data available consid- In study by Quinn et al. (2011) DCF injection (1000 mg/L) into
ering other NSAIDs in this matter. body of D. polymorpha also did not resulted in significant DNA
In Parolini et al. (2009) in vitro cytogenotoxicity of DCF and IBU damage suggesting only mediocre cytogenotoxic effect of DCF on
on haemocytes of zebra mussel, Dreissena polymorpha was mussels. Parolini and Binelli (2012) demonstrated that mixture of
6
two NSAIDs, DCF and IBU with paracetamol (PAR, not considered as genes associated with arachidonic acid pathway, apoptosis and
NSAID) has cytogenotoxic effect on D. polymorpha haemocytes. peroxisomal-activated receptors. It was also shown that the highest
Mussels exposed for 96 h to three environmentally relevant levels concentration of IBU resulted in up-regulation of GST, sulfo-
of drugs concentration (low: 0.1 mg/L DCF, 0.1 mg/L IBU, 0.5 mg/L PAR; transferase and CYTP450 genes confirming their key role in drug
medium: 0.5 mg/L DCF, 1 mg/L IBU, 1 mg/L PAR; high: 1.5 mg/L DCF, metabolism and detoxification. Similar effect of IBU exposure (1
9 mg/L IBU, 13 mg/L PAR) showed cytogenotoxic effect in haemocytes and 100 mg/L) on gene expression was also observed in Pacific
in response to each treatment. Decrease of LMS, DNA damage oysters, Crassostrea gigas, for overall time of 7 days (Serrano et al.,
expressed as increase in MN and apoptotic cells counts all indicate 2015). Exposed oysters showed significant differences in gene
that mixture of DCF, IBU and PAR has cytogenotoxic effect on transcription in gills tissue. In all treatments, significant up-
D. polymorpha cells. In Aguirre-Martínez et al. (2013a) Manila regulation was observed for CYTP450 genes, as well as genes
clams, Ruditapes philippinarum, exposed to environmentally rele- associated with GST, cyclooxygenase (COX) proteins and fatty acid
vant IBU concentrations (0.1, 5, 10, and 50 mg/L) also responded with binding protein. This clearly suggests activation of detoxifying and
decrease in LMS in haemolymph starting from 10 mg/L treatment. antioxidative mechanisms. Differential transcription of CYTP450
35 days exposure to IBU revealed dose-effect trend, significant for was observed between two exposures, suggesting that IBU produce
lowest drug concentrations. Decrease of LMS as result of hormesis-like effect associated with those genes. Up-regulated
R. philippinarum exposure to IBU (14 dayse0.1, 5, 10 and 50 mg/L genes of GST isoforms indicate the role of this enzyme in IBU-
IBU) was also observed in another study by Aguirre-Martínez et al. induced detoxifying metabolism in bivalves. This, along with sig-
(2016). Conversely, no significant increase was observed in DNA nificant decrease of GR activity and lack of significant changes in
damage during this experiment, moreover DNA damage was GPx levels indicates that antioxidative response in C. gigas could be
significantly reduced in clams exposed to two highest concentra- based rather on GSH protein than on enzymatic pathway itself.
tions as opposed to control which can be explained by highly Unspecific up-regulation of COX genes transcription after 1 day
increased activity of antioxidants (discussed in chapter 2.2.2). exposure to 100 mg/L can be explained by compensation reaction to
Similar effect of decreased DNA damage between control and COX inhibition in this treatment.
exposed bivalves was observed in gills of brown mussels, Perna In contrary, inhibition of COX activity similar to human response
perna in response to DCF exposure (20, 200, 2000 ng/L) for 48 h to NSAIDs was observed in freshwater Elliptio complanata. Gagne
what was noted in two highest treatments (Fontes et al., 2018). et al. (2005) tested effect of increasing IBU concentrations (10, 20
However, DNA damage was greater in digestive glands of mussels and 50 mM) for 30 days of exposure, by direct intramuscular in-
exposed to highest concentration of DCF in comparison to control. jection of the drug. Treated mussels showed almost 4-fold decrease
Exposed mussels also revealed significant decrease in LMS under all of COX activity in gills and 1.4-fold drop in gonadal tissues when
treatments and differential effect on DNA damage which decreased compared to control. Inhibition of COX occurred along with in-
under two highest 48 h exposures. However, DNA damage crease in synaptosome transport activity. This response is also
increased in lowest treatment, probably due to distinct enzymatic present in human treated with this NSAIDs. Serotonergic effect of
reactions to oxidative stress. Different exposure approach using IBU was tested in gonad ganglia resulting in 2.4- fold and 1.4-fold
IBU-containing sediment (0.02, 0.15, 1.51 ng/g dw) also resulted in boost of dopamine and serotonin reuptake, respectively. IBU-
decreased LMS in haemolymph of charru mussel, Mytella char- induced COX activity inhibition was also observed in a study by
ruana, in response to two highest treatments (Pusceddu et al., Aguirre-Martínez et al. (2018) in which freshwater clams Corbicula
2018). fluminea were exposed to 0.1, 5, 10 and 50 mg/L IBU during 21 days
Matozzo et al. (2012) discovered that Manila clams exposed to experiment. Significant decrease in COX activity occurred in gonads
IBU (100, 500, 1000 mg/L) for 7 days showed a decrease in total at two highest concentrations tested along with increase in mito-
haemocyte count and LMS when treated with highest IBU con- chondrial electron transport (MET) (also in MET/total lipids ratio)
centration, and significant increase in haemocytes proliferation in and decrease of AChE activity in the last treatment. These results
response to two highest treatments. Highest concentration also indicate that IBU affects energy status and neurotransmitting
induced an increase in lactate dehydrogenase activity in haemo- pathway of treated clams, but simultaneously no significant effect
lymph indicating damage of haemocytes. Due to lack of innate of selected concentrations of IBU on spawning process was
immunity in bivalves and crucial role of haemocytes in immune observed in dopamine and VTG levels.
response, these results may suggest immunosuppressing effect of Brown mussels, P. perna also expressed COX inhibition in gills
IBU exposure. Boisseaux et al. (2017) revealed that DCF induced tissue in response to 48 and 96 h DCF (20, 200, 2000 ng/L) exposure
moderate immune response in freshwater gastropod, Lymnea (Fontes et al., 2018) but with simultaneous increase of COX activity
stagnalis, by exposure to environmentally relevant (1, 10 mg/L) and in digestive gland after 96 h exposure to middle concentration
therapeutic (100, 1000 mg/L) concentrations of this drug for 3 days. tested. Toxic cellular and physiological effect of DCF was proved by
DCF induced slight increase in phagocytosis capacity of haemocytes alterations of enzymes levels taking part in detoxifying mecha-
and affected positively their density in comparison to control. nisms such as EROD (increase after 96 h exposure to two highest
Simultaneously, no significant effect of DCF on cell viability of concentrations), antioxidative agents like GST (significant inhibi-
haemocytes was observed. tion under all treatments), GPx (inhibition at the middle concen-
tration after 48 h, and at highest 96 h treatment), LPO (enhanced by
2.2.2. Gene expression & metabolism disorders all exposures) and AChE (induced activity at highest 48 h exposure).
Available data suggests that toxic effects of NSAIDs in molluscs Antioxidative and choline neurotransmitter response was also
are mostly based on induction of oxidative stress and detoxifying revealed as an effect of 96 h ASA exposure (5, 18, 75, 300, 1200 ng/L)
mechanisms assessed by changes in genes expression and activity in sea snail Gibbula umbilicalis as described in Gime nez and Nunes
of specific enzymes of known function. In Milan et al. (2013) Manila (2019). Snails expressed significant increase of CAT and AChE ac-
clams exposed to 100 and 1000 mg/L IBU for 7 days revealed tivity showing clear dose-effect trend, along with stable increase in
changes in lysosome activity, acetylcholinesterase (AChE) and SOD LPO levels, but no significant changes to GST activity suggesting
activity in digestive gland but with no clear time-dose-effect trend only moderate oxidative stress induced by drug exposure.
observed. However, gene expression analysis presented in the Induction of oxidative stress and affecting detoxification
study showed that IBU induce multiple transcriptional changes for mechanisms by various NSAIDs in molluscs were also assessed by
7
measurement of activities of enzymes participating in various toxicity to R. philippinarum larvae in association to water acidifi-
biochemical pathways. Such effect was observed in D. polymorpha cation. DCF itself (0.5 mg/L) was able to significantly affect normal
in Parolini et al. (2011a) after 96 h exposure for 1, 9, 35 nM IBU. In development of the larvae in terms of damaged shells and pro-
exposed mussels increased activity levels of SOD, CAT, GPx and GST lapsed tissues in 96 h long experiment, but its toxic effect was even
clearly indicated drug-based induction of oxidative stress leading more apparent under reduced pH conditions. Co-exposure of DCF
to latter DNA damage and cytological changes (as discussed in and acidified water (pH ¼ 7.8) resulted in alterations of shell length/
chapter 2.2.1). Similar mechanism of action was observed for DCF height ratio of the treated larvae. Interestingly, DCF and DCF co-
exposure in D. polymorpha (Quinn et al. (2011). Direct injection of exposure did not affect larval survival which was significantly
1000 mg/L DCF into mussel body and 96 h exposure resulted in altered by pH itself. DCF (31.25, 62.5, 125, 250, 500, 1000 mg/L)
significant increase in GST activity, LPO expression and meth- exposure also significantly inhibited fertilization rate and embryo-
allothioneins (MTs) alterations (also evident within 24 h exposure), larval development in P. perna in all treatments, affecting embryo-
suggesting chronic effects of DCF exposure associated with induc- larval development of the mussels at highest concentration. IBU
tion of antioxidative and detoxifying mechanisms in mussel tissues. exposure was revealed to have severe effect on embryo-larval
It is worth noting that in this study no significant change in DNA development of P. perna as was observed after 48 h in Pusceddu
damage and endocrine system (assessed with alkali-labile phos- et al. (2018). Organisms were exposed to sediment fractions con-
phate method) was observed suggesting that DCF has only medi- taining IBU concentrations of 1.5, 15, 150.8, 1508 ng/g dw. Signifi-
ocre toxic effect on treated bivalves. In study by Aguirre-Martínez cant toxic effect was observed starting from second lowest
et al. (2016) Manila clams, R. philippinarum exposed for 14 days to treatment to result in complete inhibition of development at the
0.1, 5, 10 and 50 mg/L IBU expressed a dose-effect increase of GPx final exposure. Long-term experiment described in Pounds et al.
activity and LPO. GST and EROD also revealed significant raise in (2008), with 21 days exposure to 0.32, 1, 3.2 and 10 mg/L IBU
activity in comparison to control clams. Dibenzylfluorescein deal- resulted in IBU-induced effect in reduced hatching starting from
kylase (DBF) activity corresponding to phase I of detoxifying second lowest exposure to complete lack of hatching under final
pathway was negatively altered only in the two highest exposures. treatment.
Altered enzymatic activities presented in this study indicate IBU- In contrary to IBU and DCF, INDMT exposure did not significantly
induced activation of detoxifying mechanisms, altered anti- altered fertility indexes of freshwater snail, Physella acuta exposed
oxidative pathways and neurotoxic effect expressed as significant to 0.5, 5, 50 and 500 mg/L INDMT for 7 days (Lo pez-Doval et al.,
increase of AChE in 0.1 mg/L IBU treatment and latter decrease of its 2012). Drug exposure was shown to only affect biomass of eggs
activity. clutches at highest concentration, with no effects on survival,
Trombini et al. (2019) also provided evidence of IBU and DCF number of eggs nor clutches, therefore suggesting no significant
effect on enzymatic profile of digestive gland and gills of effect of INDMT on this snail in water.
R. philippinarum. Clams exposed for overall 22 days to 15 mg/L of
both drugs showed significant changes in activity of antioxidative 2.2.4. Effect on condition and survival
and detoxifying enzymes, but trends were not evident in most Condition and mortality were chosen as NSAIDs toxicity
cases. DCF had minimal effect on gills, whereas highly affected SOD, markers only in a few studies. In study by Quinn et al. (2011) it was
CAT, GR, GST and LPO levels in digestive gland in first week of revealed that DCF exposure (1000 mg/L body injection, 96 h expo-
exposure. IBU exposure resulted in enhanced SOD, GR and LPO sure) did not induce any changes to condition factor of
levels in both tissues and in increased GPx activity in digestive D. polymorpha, although it is important to take into the account the
gland. Clams recovered their oxidative states in the middle of timespan of experiment and possibility that condition factor would
exposure with DCF, and at the end of IBU treatment. MTs assay also change after longer period of drug exposure. In Pounds et al. (2008)
proved that NSAIDs-induced oxidative stress occurs mostly in first freshwater juvenile snails, Planorbis carinatus exposed for 72 h to
stage of exposure and is further balanced due to antioxidant ac- 0.1, 1, 10 and 100 mg/L IBU reacted to acute toxicity by increased
tivity of specific biomolecules. Effect of both drugs on choline mortality from 20 to 40% at 0.1 and 10 mg/L treatment, to 100% at
neurotransmitter was confirmed with significant AChE activity in- highest exposure. Long-term experiment presented in the same
crease in digestive gland (DCF exposure) and gills (IBU and DCF). study (21 days exposure to 0.32, 1, 3.2 and 10 mg/L IBU) revealed
Costa et al. (2019) used climate change vs. pharmaceuticals that IBU-treated snails are characterized with decrease in wet
approach to determine changes induced with DCF exposure in as- weight which reflects reduced condition in tested animals.
sociation with different temperature and pH treatments. Clams
R. philippinarum and Ruditapes decussatus were first acclimated to 2.2.5. Behavioural changes
different pharmaceuticals and temperature conditions (control: pH Behaviour-based assessment of NSAIDs toxicity is lacking in
8.1, 17 C, climate change scenario: pH 7.7, 20 C) and thereafter molluscs although positive impact of NSAID was revealed in
treated with zero or 1 mg/L DCF for 7 days. Both species expressed _
Zbikowska et al. (2017) where freshwater gastropod, Planorbarius
different response to treatment. In Manila clams, the impact of DCF corneus treated with KET (100 mg/g), showed inhibition of symp-
was shown only in reduced respiration rate, increase in glycogen toms of behavioural fever, observed as altered choice of environ-
content in control conditions and decrease of protein concentra- mental temperature as a consequence of influenced thermal
tions in both treatments. In R. decussatus DCF induced enhance- preference, induced with injection of lipopolysaccharide. Moreover,
ment of SOD, CAT and GPx in both exposures with only synergetic snails first injected with KET then with pyrogen expressed symp-
DCF-temperature-pH effect observed for both species in GST ac- toms with approximate 20 h delay. These results suggest that KET
tivity confirming drug-based induction of metabolic pathway of may undergo processes in lower animals similar to those known in
detoxification and antioxidant response in clams. humans resulting in antipyretic effect of this drug.
2.2.3. Reproductive and teratogenic effects 2.3. Crustaceans

Several studies revealed that DCF and IBU exposure can have
detrimental effects on reproductive success of bivalves and snails Crustaceans are among the key groups of organisms in both
affecting important traits associated with hatching, larval devel- marine and freshwater ecosystems, forming crucial link in the
opment and survival. Study by Munari et al. (2016) revealed DCF trophic chain between both plankton and benthic communities.
8
Simple, short life-cycle animals such as cladocerans are used as strong oxidative stress in H. azteca, as confirmed by the wide range
basic models in the toxicity testing of various pollutants. However, of markers used. In Lucero et al. (2015) H. azteca was used as a
in recent years there has been an increasing number of more in- model organism to investigate the effect of NPX through an artifi-
depth analyses, where not only simple toxicological parameters cial sediment enriched with the drug. Two concentrations of NPX
are determined, but also more subtle, sublethal effects of drugs at were used in this study - 76.60 mg/kg and 399.28 mg/kg, and
biochemical, physiological or behavioural levels. Therefore, this exposure lasted 48 h. Biomarker analyses showed statistically sig-
chapter presents the current state of knowledge on sublethal, long- nificant increase in SOD and CAT activity as well as decrease in GPX
term effects of NSAIDs in crustaceans. activity. The results showed unequivocally that the sediment con-
taining NPX induces oxidative stress in H. azteca. Toxic effects of the
2.3.1. Cyto & genotoxicity drug administered through sediments have also been studied in
In the study by Go mez-Olivan et al. (2014) the genotoxic Maranho et al. (2015a), where Ampelisca brevicornis was used as a
response after 48 and 96 h was assessed, showing that all tested model species. The concentrations of IBU in the sediment were
pharmaceuticals (9.7 mg/L DCF; 2.9 mg/L IBU and 0.018 mg/L NPX) 0.05, 0.5, 5, 50 and 500 ng/g, while the exposure time was 10 days.
increase prevalence of DNA damage in D. magna cells. Similarly, a The applied biomarkers showed activation of detoxification and
statistically significant increase in DNA damage levels was observed antioxidant defence system. What is important, at environmental
in work by Lucero et al. (2015). The results showed that the sedi- concentrations of IBU, a statistically significant increase in DBF, GST
ment containing NPX induces genotoxicity in H. azteca. In turn, and GPX activity as well as an increase in LPO level were observed.
Aguirre-Martínez et al. (2013b) observed an increase in DNA Nieto et al. (2013) assessed the effect of NSAIDs (DCF and IBU) in
damage in cells obtained from gills and hepatopancreas of crab the freshwater shrimp Atyaephyra desmarestii. The tested concen-
C. maenas exposed to IBU for 28 days at environmentally relevant trations were 14.6 and 47.24 mg/L for osmoregulation measure-
concentrations of 0.1, 5, 10 and 50 mg/L. ments and 13.3 and 70.6 mg/L for respiration and nutrition
measurements, for DCF and IBU respectively. For IBU, no effects
2.3.2. Gene expression & metabolism disorders were observed in the studied parameters, whereas for DCF, a
Studies performed by Heckmann et al. (2006, 2008) show a decrease in respiration under reduced oxygen content conditions
strong link between a rapid, acute transcriptomic response and a was observed. In another study (Nieto et al., 2016), chronic toxicity
chronic phenotypic response in D. magna exposed to IBU at con- at concentrations of 70, 10 and 10 mg/L for IBU, DCF and carba-
centrations of 0, 20, 40 and 80 mg/L. It has been suggested that, in mazepine was assessed. It was shown that none of the tested
the first stage, IBU deregulates eicosanoid metabolism and then as a compounds had any effect on animal nutrition or osmoregulation,
result of signal transduction through disturbances in peroxisome either individually or in the mixture. However, in the case of oxygen
proliferator activated receptors or calcium signalling pathways, it consumption, a positive correlation was observed between the
leads to deregulation of the endocrine system and oogenesis. This temperature increase and the effect of drugs, whose action was
effect is confirmed by the observed fertility problems (Heckmann more intense at 25 C.
et al., 2006, 2008). In a study by Wang et al. (2016) a significant Effects of NSAIDs were also analysed on crabs. Eades and Waring
increase in GST concentration after 6 h of exposure to IBU was (2010) analysed the effect of environmental concentrations of DCF
observed, but its activity decreased with a longer time. It was also (10 and 100 ng/L) on Carcinus maenas in two different salinities - 35
observed that IBU increased the activity of SOD enzyme catalyzing and 17.5 PSU. It was shown that low drug concentrations had a
processes related to removal of reactive oxygen species. Generally, significant effect both on haemolymph osmolality and osmolality
the induction of all three examined antioxidant enzymes was capacity in both salinities, providing valuable information on po-
observed (GST, SOD, CAT), which may be an adaptive mechanism to tential osmoregulation disturbances in crabs exposed to environ-
pollution. Increase in MDA concentration was also observed, sug- mental concentrations of DCF. In turn, Augirre-Martinez et al.
gesting ongoing oxidative degradation of lipids and indicating that (2013) has determined the effect of chronic exposure to IBU in
ROS were accumulated, causing damage to cell membranes. This C. maenas. Crabs were exposed to the drug for 28 days at envi-
study confirms that the toxic effects of IBU are manifested by dis- ronmentally relevant concentrations of 0.1, 5, 10 and 50 mg/L.
turbing the expression of genes and enzymes associated with Biomarker analyses were performed separately in gills, livers,
detoxification and antioxidative processes. Similar effects were muscles and gonads. In this study, the most evident changes were
observed in the work by Go mez-Olivan et al. (2014). The experi- observed in gills and hepatopancreas - increased activity of EROD,
ments were carried out for 48 h at 9.7 mg/L DCF; 2.9 mg/L IBU and DBF, GST, GPX, as well as increased levels of LPO. Additionally, in all
0.018 mg/L NPX. It was observed that all the studied drugs caused concentrations except 0.1 mg/L, a statistically significant decrease in
statistically significant increase in lipid peroxidation level, sug- LMS was observed. These studies confirmed that C. maenas can
gesting increased ROS production. On the other hand, only in the serve as a bioindicator and is sensitive to IBU. In turn, the study by
case of IBU an increase in carbonyl content proteins (PCC) was Oskarsson et al. (2012) examined the effect of DCF and IBU on the
recorded. In the case of SOD activity an increase was observed for physiology of Gammarus spp. The examined parameters included
IBU and NPX, while a decrease was observed for DCF. In the case of respiration rate, ammonia excretion and O:N ratio. The organisms
CAT an increase was observed for all compounds (DCF, IBU and were exposed to drugs at three concentrations: 10, 100 and
NPX), but it was statistically significant only for IBU and NPX. 1000 mg/L, but no statistically significant changes were observed in
In Novoa-Luna et al. (2016), the Hyalella azteca amphipod was any of these concentrations.
used to evaluate the oxidative stress induced by wastewater from
the NSAIDs factory. A statistically significant increase in hydroper- 2.3.3. Reproduction disruption and teratogenic effect
oxide content and lipid peroxidation level was observed, which In recent study by Grzesiuk et al. (2020) the influence of chronic,
may result from the formation of ROS. Significant effect on PCC was multi-generational exposure to IBU was tested on D. magna em-
also noted, but it was strongly suggested that it could be related to bryos. An environmentally relevant concentration (4 mg/L) of IBU
the presence of NaClO in the wastewater. In addition, an increase in was used during the exposure of five consecutive generations of
SOD, CAT and GPx activity was observed, which constitute an D. magna, with the potential for recovery tested on the sixth one.
antioxidant defence system, necessary for the removal of ROS. In- No statistically significant differences between the first and fifth
dustrial wastewater from NSAID-manufacturing plant caused generation were observed, but the analysis of the sixth generation
9
showed that exposed individuals were characterised by faster population growth rates was observed for D. magna at 30 mg/L. The
growth rate, both those re-treated with the drug and those trans- NOEC for reproduction was 10 mg/L for D. magna and 0.37 mg/L for
ferred to clean water, as compared to control organisms. Moreover, M. macrocopa, which is much higher than that recorded in the
although most of the parameters were not affected by IBU, the environment. Similar effects of IBU were observed in a paper by
morphological observations provided important information. In Han et al. (2010), where the NOEC for survival of D. magna was
the first generation exposed to pharmaceutical, 20%, 70% and 20% of 33.3 mg/L and the Lowest Observed Effect Concentration (LOEC) for
deformed offspring were observed, while in the fifth generation reproduction was 1.23 mg/L.
deformations affected as many as 80%, 90% and 50% of individuals. Nevertheless, Wang et al. (2016) also observed a decreasing
Among the morphological changes noted, the most important ones growth and reproductive capacity with increasing IBU concentra-
were the lack of eyes in neonates, as well as deformed antennules tion, which confirms potential negative effects of chronic exposure
and carapace. Such deformations may have a significant impact on to this drug. The authors suggested that such effects are caused by
the functioning of organisms, as antennas are their main locomo- the influence on the arachidonic acid metabolism. However, they
tory organ. It has been suggested that these effects may be due to might also result from the increased energy expenditure on
disruption of lipid membranes by IBU (Jaksch et al., 2015), or may detoxification, which reduces the amount of available energy for
result from disorders associated with eicosanoids, playing impor- growth and egg production. On the other hand, De Oliveira et al.
tant role in vitalogenesis and ovulation in invertebrates. This study (2016) observed no influence of DCF on the parameters related to
as the first provides important information on the long-term, population growth.
multi-generational effects of IBU in aquatic organisms. Mennillo et al. (2018) assessed the chronic toxicity of a racemic
On the other hand, tests performed by Hayashi et al. (2008) mixture of KET and its enantiomer dexketoprofen in C. dubia. In the
included a 10-day exposure to IBU at 20, 40 and 80 mg/L, as well range 1e1000 mg/L, a statistically significant effects on reproduction
as 10-day recovery in clean water. During the exposure, both at 20 at the highest concentration was observed, which was stronger for
and 40 mg/L, a decrease in reproduction was observed, while at dexketoprofen. The results suggest that this enantiomer may be
80 mg/L it was completely inhibited. However, after transfer to more toxic to crustaceans, but the effective concentrations are
clean water, organisms from all concentrations recovered and their many times higher than those recorded in the environment.
reproduction-related parameters returned to levels close to control. A study by Prameswari et al. (2017) examined the effect of COX
This may confirm that the effect of IBU on the synthesis of eicosa- inhibitors, including ASA, on the reproduction of another crab,
noids is similar in invertebrates and vertebrates, and the effects Oziothelphus senex senex. The study showed that the administration
associated with reproduction are completely reversible. Similar of ASA causes a statistically significant induction of ovarian devel-
findings regarding inhibition of reproduction were reported by opment, while the administration of COX inhibitors, both alone and
Heckmann et al. (2007), who investigated the effect of IBU on the together with ASA, causes a statistically significant slowdown in
life history traits and population dynamics of D. magna during 14 their development. Although the exact mechanism of action of ASA
days of exposure. The concentrations of 20, 40 and 80 mg/L of IBU and COX on regulation of sexual development of crustaceans re-
were used. A delayed onset and reduced fecundity were observed quires more research, it seems that they play a key role in their
in treated cladocerans. Total reproduction and number of offspring reproduction.
per female were statistically significantly reduced in all concen-
trations, and in 80 mg/L there was no reproduction at all. There was 2.3.4. Influcence on condition and survival
a strongly negative correlation between IBU concentration and As crustaceans and in particular cladocerans are the main
reproduction, and 14-day EC50 was determined at 13.4 mg/L. aquatic organisms used for the determination of toxicity endpoints,
Additionally, it was observed that the population structure changed there are many studies to determine the impact of NSAIDs on the
dramatically - at 80 mg/L the population consisted of adult in- survival of these animals. For example, a study by Cleuvers (2004)
dividuals only, while the percentage of adult individuals relative to tested the acute toxicity of NSAIDs for D. magna. Three compounds -
control was 2x higher for 20 mg/L and 5x higher for 40 mg/L. It was IBU, DCF and NPX - were tested. EC50 in the immobilization test
therefore observed that IBU may have a significant impact on the (48h) was 108, 68 and 174 mg/L for IBU, DCF and NPX. In contrast,
reproduction of cladocerans and disturb their population structure. the test with IBU and DCF mixture showed that although toxicity to
In Lee et al. (2011), the chronic toxicity test of DCF showed a D. magna should be predicted using a simple concept of addition of
decrease in the number of young per female and number of young concentrations, in practice it was higher than expected. When
per brood in D. magna. A negative correlation between DCF con- concentrations of EC20/2 and EC50/2 were used in the mixture,
centration and population growth was observed. In the case of immobilization reached 50 and 80%. These studies show that
M. macrocopa a delay in reproduction was noted at 50 mg/L and the compounds with similar effects administered in the mixture can
number of young per female and the number of young per brood give synergistic effects and consequently lead to increased toxicity.
were also decreased at the same concentration. A decrease in In the study Du et al. (2016), the acute toxicity of two NSAIDs - DCF
population growth rate was also observed with increasing DCF and IBU in D. magna was assessed. LC50 of DCF ranged from 82.3 at
concentration. Additionally, a decrease in survival was observed for 24-h exposure to 2.00 mg/L at 21-day exposure. For IBU, on the
both species. It is worth noting, however, that the No Observed other hand, these values ranged from 116 to 3.94 mg/L at 24 h and
Effect Concentration (NOEC) for reproduction was 8.3 and 16.7 mg/ 21 days.
L for D. magna and M. macrocopa, respectively, but these concen- In general, for cladocerans such as D. magna or M. macrocopa,
trations are several orders of magnitude higher than those NSAIDs usually have similar effects on survival, with EC50 or LC50
currently recorded in the environment. Similarly, in the study by values ranging from several to several hundred mg/L. However, in
Kwak et al. (2018) a decrease in the number of young per female case of higher crustaceans such as crabs or shrimps this kind of
and per brood for D. magna was observed in the chronic exposure tests are missing.
experiment after 21 days at 30 mg/L of DCF. Additionally, the On the other hand, in the work by Grzesiuk et al. (2018) the
growth presented as body length was also disturbed at 30 mg/L. In authors investigated whether IBU can affect crustaceans by moving
the case of M. macrocopa a decrease of these two indicators was up the trophic chain. Interestingly, it was noted that D. magna fed
observed at concentrations >1.1 mg/L (per female) and >3.3 mg/L on algae that had been cultured with IBU had higher survival rates,
(per brood) respectively. A statistically significant decrease in faster growth rate and matured faster. The algae were cultured in
10
deficiency of inorganic phosphorus, which may cause thickening of (SA), on H. diversicolor was studied. However, the study covered
the cell wall, which in turn makes it difficult to digest by crusta- only acute toxicity expressed as effects on selected biomarkers. The
ceans. However, the presence of IBU made the digestion easier, environmentally relevant concentrations of salicylic acid (SA) were
which may be related to the effect of drug on cell membranes, used (50, 75, 112.5, 168.75 and 253.125 mg/L). For CAT, statistically
reducing their thickness and elasticity. Therefore, cladocerans had significant differences were observed only in the concentration of
easier access to nutrients, which resulted in faster growth and 50 mg/L. Also for GPx and selenium-dependent GPx statistically
higher survival. significant changes were observed and the activity increased with
SA concentration. For AChE, a decrease in activity was observed.
2.3.5. Behavioural changes Compared to Gomes et al. (2019), a stronger pro-oxidative effect of
In the study by De Lange et al. (2006), the effect of a wide range SA was observed. Nevertheless, neither ASA nor SA have shown to
of IBU concentrations on the behaviour of a freshwater crustacean - be toxic to polychaetes. Thus, given the current concentrations
Gammarus pulex was investigated. Locomotive behaviour was recorded in ecosystems, it appears that the compounds do not pose
tested 30 min after exposure for concentrations ranging from 1 ng/L a serious risk to these species. However, it should be stressed that in
to 1 mg/L. A statistically significant decrease in total locomotor the environment they may be exposed to multiple NSAIDs at once,
activity was observed at the concentration of 10 ng/L. However, at which may increase their toxicity through synergistic action. In
higher concentrations of the drug, the organisms retained mobility addition, there is still a need for chronic research, better reflecting
similar to control. Although this study suggests that even envi- the real situation, where the inflow of these compounds to eco-
ronmental concentrations of IBU may disturb the functioning of systems is practically constant.
small crustaceans, no concentration-related correlation was Also in the study by Maranho et al. (2014) H. diversicolor was
observed. In order to obtain more insightful information, these used as a model. Polychaetes were exposed to IBU, which was
studies have been extended with further parameters (De Lange administered by adding the drug to the sediment. In the 14-day
et al., 2009) obtained by using multispecies freshwater bio- test, the environmentally relevant concentrations - 0.05, 0.5, 5, 50
monitor (MFB) technique. The authors observed that the action of and 500 ng/g - were used. In this study, an increase in AChE activity
the drug is bipolar depending on the concentration - in case of low and LPO correlated with the increase in drug concentration. For
concentrations increased ventilation was observed, while higher DBF, GST and GR enzymes, on the other hand, a decrease in activity
concentrations resulted in increased locomotion. was observed with an increase in IBU concentration, but the dif-
ferences were not statistically significant. Conversely, no changes in
2.4. Polychaeta GPX and EROD activity were observed. Although no major enzy-
matic changes were observed in the study, increased levels of LPO
Polychaetes are another group of animals increasingly used in and DNA damage indicate a potentially significant negative effect of
ecotoxicology. These organisms form a very important element of IBU on polychaetes. In another, identical experiment conducted by
benthic communities, both freshwater and marine. Many poly- the same team (Maranho et al., 2015b) the influence of IBU on
chaete species are endobenthic animals, living in close contact with biomarkers related to cellular energy status, inflammatory pro-
contaminated sediments. As very actively moving, they are one of cesses and neuroendocrine effects was assessed. It was observed
the main bioturbators, influencing the habitat quality of many other that the highest IBU concentrations had significant effect on energy
organisms. Therefore, analyses using polychaetes may show the status, expressed as MET activity and total lipids content. Addi-
potential impact of various pollutants, including NSAIDs, on benthic tionally, the influence of all concentrations on COX activity inhibi-
communities, which are exposed to drugs deposed both in water tion and positive correlation of mortality with drug concentration
and sediments. was shown.
2.4.1. Cyto & genotoxicity 2.4.3. Reproduction disruption and teratogenic effect
So far, the only work analysing the effects of NSAIDs on cyto or The influence of IBU and DCF on the reproduction of polychaetes
genotoxicity in polychaetes has been performed by Maranho et al. was described in Zanuri et al. (2017) using Arenicola marina as a
(2014). In this study IBU was found to cause concentration- biological model. The studied concentrations were 0.6e842 ng/L
dependent increase in DNA damage in Hediste diversicolor. A sta- DCF and 0.01e2370 ng/L IBU. It was observed that DCF reduced the
tistically significant increase in DNA damage was observed in in- swimming speed of sperm, the percentage of mobile sperm and the
dividuals exposed to concentrations of 5 and 500 ng/g of sediment. success of fertilization in the case of preincubated sperm, and the
effects were dependent on concentration and time. In turn, IBU was
2.4.2. Gene expression & metabolism disorders non-toxic to gametes of A. marina. Observed EC50 values of DCF
In the study by Nunes et al. (2020), the toxic effects of DCF in were well above environmental, but importantly, the NOEC and
marine polychaetes H, diversicolor were assessed by biomarker LOEC values were in the range or only slightly above environmental
analysis. The environmental concentrations of 0.5, 1 and 2 mg/L concentrations. Sperm mobility is an important parameter and its
were used in 28 days exposure. DCF had a statistically significant decrease may cause problems with oocyte fertilization, especially
effect on the activity of GSTs enzymes, causing its decrease. How- in case of low sperm density. This study shows that NSAIDs can
ever, no influence on AChE activity was observed. On the other pose a threat to the fertility of marine polychaetes and even con-
hand, in the study by Gomes et al. (2019) the acute toxicity as well centrations already recorded in the environment may affect their
as the chronic effect of ASA on H. diversicolor was determined. For reproduction.
this purpose, biomarkers of oxidative stress were employed. A total
of 6 concentrations were used - 2.5; 25 and 250 mg/L for acute, and 2.4.4. Morphological and tissue alterations
0.005; 0.1 and 2 mg/L for chronic toxicity testing. The exposure time So far only one work (Gomes et al., 2019) assessed the histo-
was 96 h for acute toxicity and 28 days for chronic toxicity. In the logical alterations caused by NSAIDs in polychaetes, but no signif-
case of acute toxicity, significant changes in CAT activity were icant changes were noted either in the case of acute or chronic
observed, positively correlated with concentration. However, no exposure. However, in the case of chronic exposure an increased
changes were observed in the chronic study. Moreover, in Nunes number of mucous cells has been observed. As it was suggested,
(2019) the effect of one of the metabolites of ASA, salicylic acid increased production of mucous cells may be one of the
11
mechanisms protecting the organisms against xenobiotics. invertebrates, although it seems that basic metabolic pathways in
terms of NSAIDs exposure may be very similar among various
2.5. Others aquatic taxa.
NSAIDs are known of induction of reactive oxygen species for-
In the study by Zanuri et al. (2017), environmental concentra- mation in various cell types in humans and similar effect is also
tions of DCF and IBU were used and their effect on the reproductive frequently observed in lower taxonomical groups. The most com-
success of the important benthic invertebrates - the starfish Asterias mon toxic effect of NSAID exposure in all discussed taxa consists of
rubens and the sea urchin Psammechinus miliaris was assessed. The induction of oxidative stress revealed as alterations in enzymatic
mobility of sperm and the success of insemination were tested. The activity of molecules known of antioxidant activity and/or changes
tested concentrations were 0.6e842 ng/L DCF and 0.01e2370 ng/L in transcription of genes associated with antioxidant/detoxifying
IBU. It was observed that DCF reduced the swimming speed of pathways. Induction of defence responses is always burdened with
sperm, the percentage of mobile sperm and the success of fertil- high energetic costs due to which overall energy metabolism and
ization in the case of preincubated sperm, and the effects were energetic components deposition may undergo significant changes.
dependent on concentration and time. Both studied compounds This means that long-term sustaining of enhanced detoxifying
showed much higher toxicity to P. billionis, while A. rubens proved mechanisms may have detrimental effect on other physiological
to be drug resistant. aspects of organism, such as reproduction. Several reproductive
On the other hand, a study by Yamindago et al. (2019) assessed traits were proved to be affected by NSAIDs exposure reflecting
the influence of NPX on Hydra magnipapillata - a freshwater polyp serious impact of this group of drugs on reproductive success,
e by investigating morphological and transcriptional changes significantly threatened also by teratogenic effect observed as a
caused by the drug. The acute toxicity test showed that the LC50 for consequence of drug exposure in variety of taxa. Oxidative stress is
H. magnipapillata was 52, 45 and 43 mg/L NPX at 24, 48 and 72 h. also considered as a main factor in induction of irreversible DNA
Morphological analyses showed that the concentration of 40 mg/L damage and cytological changes (such as LMS) observed in multiple
of NPX stimulated the contraction of the body column and tenta- studies reflecting cytogenotoxic properties of NSAIDs. Irreversible
cles, and these morphological changes were associated with a DNA damage observed as DNA fragmentation and/or genetic le-
decrease in food activity after 96 h. On the gene expression level, sions such as MN clearly indicate a vulnerability of NSAIDs-treated
changes in the genes encoding proteins involved in the body’s animals for genetic instability that may enhance the risk of cancer
response to stress factors through endoplasmic reticulum stress formation and even affect genomic stability of whole populations in
response were observed. Additionally, the influence of NPX on natural environment.
changes in the regulation of genes involved in tryptophan meta- Fish, as the most investigated group of organisms in assessment
bolism was observed, which plays an important role in serotonin of NSAIDs toxicity, besides already mentioned oxidative state al-
synthesis - serotonin levels are associated with stress and behav- terations, revealed disruption of reproduction-related endocrine
ioural changes. The influence on the regulation of genes associated processes in response to treatment of several drugs such as
with the Wnt signalling pathway has also been noted, which is increased estrogenicity, although the specific mechanisms
important in the context of embryogenesis and tissue homeostasis; responsible for gender-related changes induced by NSAIDs remain
at the same time it has both pro and anti-inflammatory properties. unknown. Altered endocrine system may provide serious health
Statistically significant differences in the number of differentially issues of animals in prolonged exposure to drugs resulting in sig-
expressed genes have also been shown, which play an important nificant changes in reproductive processes and therefore affecting
role in protection against oxidative DNA damage or instability. It stability of population. Unfortunately, endocrine system is not still
was also noted that NPX induced PLA2 and DDC expression after 6 fully investigated in aquatic invertebrates therefore data concern-
and 24 h. PLA2 plays an important role in the creation of inflam- ing NSAIDs impact on this aspect is very scarce. Well-known
mation and is one of the main targets in the treatment of inflam- physiology of fish is a great advantage in the interpretation of
mation with NSAIDs. In summary, both acute toxicity in benthic toxicity tests results. Conversely, in lower organisms, such as
polyp was assessed and the effect of NPX on morphological changes molluscs, crustaceans and other invertebrates, in-depth knowledge
and transcriptional responses was indicated. Genetic studies on physiological processes and biochemical pathways is often
showed potentially extensive harmful effects of NPX on the phys- missing. For example, target effects of discussed NSAIDs related to
iological and metabolic processes of H. magnipapillata. These effects COX inhibition were not proven in all bivalve studies, indicating a
were similar to the commonly known side effects of this drug. serious need for further investigation considering arachidonic acid
In addition, several current studies have assessed the potential pathway in lower invertebrates.
impact of NSAIDs on small plankton animals - rotifers (Gonz alez- Since many physiological aspects of lower invertebrates do not
Perez et al., 2016; Sarma et al., 2014). Also for these species, being present well-defined pathway or had not been fully recognized yet,
essential link in the trophic chain, a negative impact of DCF and IBU the toxicity effects of contaminants might not be adequately
was observed in parameters such as reproduction rate. Neverthe- assessed. Organ-dependent sensitivity to pharmaceuticals was
less, it is clear that much more research is needed on organisms highlighted in some organism groups. This indicates that under-
from beyond a few model animal groups. standing of biochemical response should be assessed alongside
with proper biomarker, careful selection of organ/tissue and choice
3. Conclusions and future perspectives of method in relevance to the tissue type. As most results discussed
here indicate a high potential of NSAID to affect the expression of
NSAIDs showed toxic effects in a variety of aquatic biota in both genes involved in detoxification, modern omics (transcriptomics,
acute and chronic exposures, with therapeutic and environmen- genomics) appear as the most relevant tools in ecotoxicological
tally relevant concentrations using different exposure types, studies, although due to lack of full transcriptomes for many species
including injection, water or sediment exposure. In general, studies such investigation may not always be possible.
discussed in this review confirm that exposure to NSAIDs adversely Among all described toxic properties of NSAIDs, the most
affects aquatic ecosystems due to disruption of metabolic homeo- worrying apply to teratogenic effects. Significant teratogenic, either
stasis of targeted animals. With no doubt, there is a significant morphology- or directly metabolism-based alterations have been
difference in state of knowledge about physiology of fish and lower observed as a result of NSAIDs exposure in all discussed taxonomic
12
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long-term exposure to NSAIDs would trigger adaptive mechanisms gastropod Lymnea stagnalis under short-term exposure to pharmaceuticals of
that would counteract changes induced by drugs in organisms. concern for immune systems: diclofenac, cyclophosphoamide and cyclosporine
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Declaration of competing interest clam species to diclofenac. Ecotoxicol. Environ. Saf. 189, 109899. https://doi.org/
10.1016/j.ecoenv.2019.109899.
David, A., Pancharatna, K., 2009. Developmental anomalies induced by a non-
The authors declare that they have no known competing selective COX inhibitor (ibuprofen) in zebrafish (Danio rerio). Environ. Tox-
financial interests or personal relationships that could have icol. Pharmacol. 27 (3), 390e395. https://doi.org/10.1016/j.etap.2009.01.002.
appeared to influence the work reported in this paper. De Almeida, E., Bainy, C., de Melo Loureiro, P., Martinez, R., Miyamoto, O., Onuki, J.,
Barbosa, F., Garcia, C., Prado, M., Ronsein, E., Sigolo, A., Brochini, B., Martins, M.,
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Appendix A. Supplementary data bivalves as indicators of environmental stress in the Brazilian marine envi-
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Physiol. Mol. Integr. Physiol. 146, 588e600. https://doi.org/10.1016/
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Environmental Pollution 273 (2021) 115891

Contents lists available at ScienceDirect

Toxic effects of NSAIDs in non-target species: A review from the

1. Introduction biotransformation in aerobic conditions and photolysis occurring in

Fig. 2. The most common toxic effects of NSAIDs in aquatic organisms.

2.2.3. Reproductive and teratogenic effects 2.3. Crustaceans

histopathological alterations and bioaccumulation in rainbow trout. Aquat. (4), 347e570.

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