Environmental Advances 7 (2022) 100164

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Environmental Advances
journal homepage: www.sciencedirect.com/journal/environmental-advances

Medicating the environment? A critical review on the risks of

carbamazepine, diclofenac and ibuprofen to aquatic organisms
Niña Sarah P. Batucan a, *, Louis A. Tremblay b, c, Grant L. Northcott d, Christoph D. Matthaei a
a
Department of Zoology, University of Otago, 340 Great King Street, Dunedin 9054, New Zealand
b
Cawthron Institute, Nelson 7010, New Zealand
c
School of Biological Sciences, University of Auckland, Auckland 1142, New Zealand
d
Northcott Research Consultants Limited, Hamilton 3200, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: Pharmaceuticals are an important class of micropollutants within the freshwater environment. There is sufficient
Pharmaceutical evidence of their impacts on exposed biota to warrant further research to better assess their risk. We reviewed
Acute ecotoxicological freshwater studies conducted from 2010-2020 on carbamazepine, diclofenac, and ibuprofen,
Chronic
three high-use pharmaceuticals frequently detected globally in surface waters. One hundred and thirteen studies
Endpoint
Biomarker
encompassing short-term (mean exposure duration 6.4 days) and long-term exposures (mean 37.8 days) were
Toxicity meta-analysis reviewed. Study designs were examined and critiqued using a qualitative analysis, and a quantitative analysis
compared toxicities (lowest observed effect concentration, LOEC) between the three pharmaceuticals in both
short- and long-term tests. Short-term tests were predominant (60% of studies), as reported in past related re­
views. Examination of experimental designs highlighted important limitations. Most studies had low replication
(n=3 per treatment) and provided no effect sizes for their findings, and 55% of studies did not supply mea­
surements of actual exposure concentrations. Thirty-five percent of studies detected clear effects (mostly nega­
tive) at environmentally relevant concentrations. In short-term studies, biomarkers were the most sensitive
endpoints; the same applied to community-level endpoints in long-term studies. The LOECs of all three phar­
maceuticals (0.05-10 μg L− 1) were similar in both short- and long-term studies. Future research should prioritise
long-term, environmentally relevant exposures, using a combination of biomarker and community-level end­
points. Due to the acute toxicity of pharmaceuticals, future studies should investigate how these contaminants
may modify communities and ecosystems, rather than single-species approaches. Finally, a comprehensive meta-
analysis would be pertinent when enough long-term, environmentally relevant studies with rigorous designs and
effect size information have accumulated.

1. Introduction
1.1. The challenge of micropollutants
The term ‘micropollutants’ describes organic contaminants detected
in the environment at concentrations ranging from ng–μg L− 1 and en­
compasses ‘emerging organic contaminants’ (Kümmerer, 2011; Daugh­
ton, 2016). There have been many scientific publications on the
prevalence of micropollutants in the environment since the early 2000s
(Kolpin et al., 2002; Daughton, 2016; Tiedeken et al., 2017). Thus, their
“emergent” status is more attributable to the recent rise of their recog­
nition as important contaminants whose environmental risks remain
largely unknown (Luo et al., 2014), and consequently their release into
receiving environments remains unregulated (Stewart et al., 2016;
Saggioro et al., 2018).
Micropollutants are ubiquitous contaminants in global aquatic eco­
systems and include fire retardants, musks, industrial solvents, pesti­
cides, personal care products, and pharmaceuticals among many others
(Schwarzenbach et al., 2006). Thousands of novel synthetic chemicals
are produced annually and may accumulate within the environment and
pose a risk to exposed biota (Bernhardt et al., 2017; Johnson et al.,
2020). Some well- characterised impacts include the decline of bird
populations in North America from the insecticide dichlorodiphenyl­
trichloroethane (DDT, Bailey et al., 1974), the feminisation of

* Corresponding author.
E-mail address: batni629@student.otago.ac.nz (N.S.P. Batucan).

https://doi.org/10.1016/j.envadv.2021.100164
Received 15 September 2021; Received in revised form 16 December 2021; Accepted 27 December 2021
Available online 28 December 2021
2666-7657/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
N.S.P. Batucan et al.
freshwater fish from synthetic estrogen, 17α-ethynylestradiol (Harries
et al., 1997; Kidd et al., 2007), the rapid decline of population of Asian
vultures in the Indian subcontinent caused by the consumption of car­
casses contaminated with the veterinary pharmaceutical diclofenac
(Oaks et al., 2004), and the decline in reproductive success of European
cetaceans linked to polychlorinated biphenyls (PCBs, Jepson et al.,
2016). Other cases include the disruption of bee populations and other
important insect pollinators from exposure to neonicotinoid insecticides
(Mogren and Lundgren, 2016), which were designed to minimise
toxicity to mammals and maximise toxicity to insect pests (Ensley,
2018).
1.2. Pharmaceuticals as freshwater contaminants
An important class of micropollutants are pharmaceuticals, which
enter freshwater bodies via discharge from wastewater treatment facil­
ities, agricultural and aquaculture activities, landfill leachate, and
groundwater contaminated by leaking sewage pipes (Ellis, 2006). These
micropollutants partition into the water, sediment, and biotic com­
partments (Inostroza et al., 2017). Trace amounts of pharmaceutical
contaminants have been detected in drinking water in several countries
including the USA, Germany, and Canada (Heberer et al., 2002; Kley­
wegt et al., 2011; Bexfield et al., 2019), but current evidence suggests
the risk they present to humans is negligible (WHO 2011; Kibuye et al.
2019). However, evidence is lacking on the safety of chronic low-dose
exposure, especially at sensitive life stages such as foetal development
and early childhood (Collier 2007).
Reviews on the occurrence and ecotoxicological effects of different
classes of pharmaceuticals (e.g. Santos et al., 2010; Godoy et al., 2015;
Silva et al., 2015) indicated the prevalence of acute over chronic
exposure studies. Consequently, environmental management and pol­
icies that address the potential environmental risk of pharmaceuticals
are mainly informed and shaped using acute toxicity data (Verlicchi
et al., 2012). Such assessments rely on the assumption that the effects
seen in acute exposures are comparable in both quality and quantity to
chronic, low-dose exposures (Kümmerer, 2009). Efforts have been made
to guide future research into generating information that underpins a
more accurate narrative on the real environment risk of micropollutants
(Boxall et al., 2012; Furley et al., 2018; Van den Brink et al., 2018; Gaw
et al., 2019). Building such a narrative is an on-going challenge in
ecotoxicology (Daughton, 2016).
In addition to inadequate risk assessment, there is growing concern
around the quality of ecotoxicological evidence in the scientific litera­
ture (Ågerstrand et al., 2011; Harris et al., 2014; Hanson et al., 2017). In
response to this concern, Harris et al. (2014) published 12 “Principles of
Sound Ecotoxicology”. In a follow-up review, Harris and Sumpter (2015)
used the most objective three of these principles to evaluate 173 eco­
toxicological articles from three reputable journals. They found that,
while most of the studies used more than one treatment concentration
and reported measured concentrations, the large majority described
results from a single experiment, which raises some concern around the
reproducibility (and therefore the reliability) of the results. In addition,
while many peer-reviewed ecotoxicological studies provide novel per­
spectives on the environmental risk of toxicants, this is often achieved at
the cost of deviating from standard experimental protocols such as those
provided by the Organisation for Economic Co-operation and Develop­
ment (OECD) guidelines (Ågerstrand et al., 2011). This inconsistency
raises some doubt regarding the reliability of such “non-standard” ex­
periments, which are therefore often overlooked by regulatory bodies
(Küster et al., 2009; Ågerstrand et al., 2011). Lack of transparency on the
reporting of the experimental methodology, poor study design, or both
may also play a role in the perceived quality issues in peer-reviewed
ecotoxicological studies (Ågerstrand et al., 2014). Given all this, when
resources are limited and repeatability is unfeasible, it is therefore
paramount to not just ensure transparency in reporting at the writing
stage but also that the study design of the experiment is sound and
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Environmental Advances 7 (2022) 100164
robust.
1.3. The three focal pharmaceuticals
In the European Union, approval and release of new pharmaceuticals
require both human safety and environmental quality standards and
incorporate acute and chronic tests on rodent and non-rodent models
(Smith, 2015). However, these rules only apply to pharmaceuticals
developed since 2006, precluding legacy pharmaceuticals from the
scrutiny of chronic toxicity testing (Gunnarsson et al., 2019). Legacy
pharmaceuticals extensively studied by ecotoxicological research
include antibiotics, those targeting endocrine functions, non-steroidal
anti-inflammatory drugs (NSAIDs), antiepileptics, and anxiolytics
(Saggioro et al., 2018). Carbamazepine, diclofenac, and ibuprofen are
among the most frequently studied pharmaceuticals, arising from their
widespread and extensive use (Daughton, 2016). Table 1 presents the
range of reported concentrations of these three pharmaceuticals in
global surface waters (i.e. rivers, streams, lakes and estuaries), sedi­
ments and a selection of aquatic organisms.
Carbamazepine was first synthesised in 1960 and is widely used for
treating epilepsy, pain, and some mental disorders (McCleane, 2009). It
binds to voltage-gated sodium channels and, to a certain extent, calcium
channels, thereby inhibiting action potential and decreasing synaptic
transmission (Gambeta et al., 2020). Little is known about the mecha­
nisms of how carbamazepine may affect aquatic organisms, but other
sodium-channel blockers such as the neurotoxic pesticides indoxacarb
and pyrethroids have well-documented adverse environmental effects
(Dong, 2007). These pesticides are heavily used in commercial agri­
culture and aquaculture, respectively, and are known to be highly toxic
to arthropods (Arena et al., 2018; Major and Brander, 2020). Carba­
mazepine is a persistent contaminant in the environment due to its
resistance to degradation, even in modern multistage sewage treatment
processes (Saggioro et al., 2018).
Diclofenac and ibuprofen are NSAIDs used as analgesia and were first
synthesised in the 1970s and 1960s, respectively (Halford et al., 2012;
Altman et al., 2015). They inhibit two cyclooxygenase enzymes involved
in the synthesis of prostaglandins, which lead to inflammation, pain, and
fever (Bushra and Aslam, 2010; Gan, 2010). Prostaglandins are known
to be involved in invertebrate functioning, including ion flux regulation,
temperature control, reproduction, cell aggregation and host-parasite
interactions (Stanley-Samuelson, 1987). Thus, non-target effects could
occur in invertebrates via these biological pathways. Both diclofenac
and ibuprofen are biodegradable but not fully removed in modern
sewage treatment processes (Costa et al., 2019). They are considered to
be ‘pseudo-persistent’ as their constant use results in their continuous
replenishment within receiving environments (Daughton and Ternes,
1999).
1.4. Research objectives
Because of the vast number of pharmaceutical compounds in the
environment, we focused this review on carbamazepine, diclofenac and
ibuprofen as they are widely prescribed and used as medicines,
commonly detected in surface waters globally, and therefore frequently
studied to assess environmental impacts of pharmaceuticals. Our critical
review consists of both a qualitative and a quantitative component to (i)
evaluate the effects of short-term and long-term exposures of these three
pharmaceuticals on aquatic organisms, particularly at environmentally
relevant concentrations, (ii) examine and critique the experimental de­
signs of the reviewed studies, and (iii) compare the measured toxicities
of the three drugs.
N.S.P. Batucan et al. Environmental Advances 7 (2022) 100164

Table 1 2. Methods
Concentrations of the emerging organic contaminants (EOCs) carbamazepine,
diclofenac, and ibuprofen detected in aquatic surface waters, sediments, and 2.1. Article collation, inclusion and exclusion criteria
biota. d.w. = dry weight, w.w. = wet weight, n.d. = not detected.
EOC Concentration We reviewed experimental studies published since 2010 that
Water Sediment Biota References involved bioindicator species from running and standing freshwaters or
Carbamazepine 0.2 ng 0.14 – 1.6 ng g− 1 Ternes (1998); estuarine ecosystems and included carbamazepine, diclofenac, and/or
L− 1 237.1 ng (fish liver w.w.) Heberer (2002); ibuprofen in their exposure treatments, which may also comprise other
11.6 μg g− 1 0.88 ng g− 1 Metcalfe et al. natural and anthropogenic stressors. An earlier critical review by Santos
L− 1 (fish muscle w. (2003);
et al. (2010) considered experimental studies from the period
w.) Rabiet et al.
n.d. – 1.9 ng g− 1 (2006); Kim et al. 1996-2009 and catalogued acute and chronic studies on freshwater
(bivalve spp. w. (2007); Loos species involving multiple pharmaceutical groups. Our review serves as
w.) et al. (2009); a follow-up of Santos et al. (2010), but with a focus on more recent
Zhou et al.
assessments of the toxicity of the three focal pharmaceuticals.
(2011); Ferguson
et al. (2013); We exclusively searched the Web of Science Core Collection over the
Du et al. (2014); decade encompassing January 2010 to June 2020. Much effort was
Zhou and made to ensure the search process was as inclusive as possible. We used
Broodbank the search terms ‘pharmaceutical* AND (freshwater OR aquatic) AND
(2014); Matongo
effect*’ as it returned a good number of hits while providing a reason­
et al. (2015);
Bernot et al. able estimate of the real number of relevant studies reported over the
(2016); target period. The initial search yielded 2758 articles, out of which 113
Koba et al. articles met our criteria for inclusion. We excluded studies that did not
(2018); Rojo
directly manipulate the stressors in an experimental set-up. In vitro de­
et al. (2019);
Golovko et al.
signs were also omitted as we aimed to evaluate stress effects on whole
(2020); organisms or communities. The annual publication rate was calculated
K’oreje et al. by dividing the total number of articles published in a year by 12. For
(2020) 2020, this number was divided by six as our review only covered the
period up to June 2020.
Diclofenac 1.7 ng <2.8 – 14 ng g− 1 Buser et al.
L− 1 300 ng (fish liver ww) (1999); Heberer
31.3μg g− 1 19.0 ng g− 1 (2002); Heberer 2.2. Information extraction
L− 1 (fish muscle w. et al. (2002);
w.) Kolpin et al.
All studies were categorised into carbamazepine, diclofenac and
1.4–68.0 ng g− 1 (2002);
(fish liver d.w.) Metcalfe et al. ibuprofen. A ‘mixed’ category was included for multiple-stressor studies
(2003); which involved at least one of the three focal pharmaceuticals. The
Rabiet et al. durations of acute and chronic exposures varied with the bioindicators
(2006); used and their life history. For example, three days’ exposure can be
Kim et al. (2007);
regarded as a ‘chronic’ growth inhibition test for single-celled algae due
Loos et al. (2009);
Ferguson et al. to their high turnover rate (OECD, 2006), but not for fish. There were
(2013); semantic grey areas, such as classifying a nine-day exposure duration as
Liu and Wong ‘sub-chronic’ (e.g. Nassef et al., 2010). To address these ambiguities, we
(2013);
classified exposure durations as either ‘short-term’ or ‘long-term’.
Du et al. (2014);
Cantwell et al. Short-term (acute) exposure included all durations lasting <15 days,
(2016); whereas long-term (chronic) exposure was ≥15 days (Peake et al.,
Koba et al. 2016). Some studies incorporated both short-term and long-term com­
(2018); Xie et al. ponents, so these were included in both categories. From each study, the
(2019); K’oreje
information extracted comprised (i) taxa used as indicator species, and
et al. (2020)
developmental stage (except for algae and community indicators); (ii)
Ibuprofen 150 ng 5–900 29 ng g− 1 Ternes (1998); stressors involved, exposure compartment, nominal concentrations or
L− 1 – 10 ng/g− 1 (phytoplankton Ashton et al. degree of exposure, and duration of exposure; (iii) endpoints used; (iv)
μg L− 1 d.w.) (2004); Loos
experimental set-up. For species whose developmental stage was un­
10.48 ng g− 1 et al. (2009);
(zoobenthos d. Lahti and Oikari clear, their reported size was recorded.
w.) (2011);
n.d.–49.0 ng g− 1 López-Serna et al. 2.3. Qualitative analysis: research outcome evaluation
(fish liver d.w.) (2012);
Zhou and
Broodbank
Endpoints used in the studies were categorised into ‘Community’,
(2014); Matongo ‘Structure’, ‘Behaviour’, ‘Physiology’, and ‘Biomarker’. These categories
et al. (2015); relate to the levels of biological organisation. ‘Community’ included
Gumbi et al. endpoints that measured community-level elements (e.g. diversity and
(2017); Xie et al.
community structure), while individual-level endpoints were encapsu­
(2019); K’oreje
et al. (2020) lated in ‘Structure’ (e.g. morphological changes), ‘Behaviour’ (e.g.
mobility, feeding, reproductive behaviour), ‘Physiology’ (e.g. growth
and reproduction), and ‘Biomarker’ (i.e. cellular and subcellular com­
ponents underpinning physiological functions). For cellular species (i.e.
algae and cyanobacteria), we modified the categorisation so that end­
points such as mortality, growth and reproduction (which are usually
measured using cellular-level markers, e.g. Chl α) were categorised

3
N.S.P. Batucan et al.
under ‘Physiology’ instead of ‘Biomarker’. This was done to maintain
consistency of categorisation across taxa.
The patterns of response were evaluated based on whether a ‘clear
effect’ was determined in a study. We defined ‘clear effect’ as: (i) when
exposure to the pharmaceuticals elicited an overall spatio-temporal
response that was significantly different from the control; (ii) the
response showed an unambiguous direction (either positive or nega­
tive); (iii) for individual-level endpoints, the response pattern was suf­
ficiently unambiguous to allow tentative inferences of the effects of the
contaminant on higher levels of biological organisation. The first qual­
ifier disambiguated findings based on repeated measures, where the
detection of significant effects varied across time. Here, we took the
latest measurement as the overall effect, accounting for the occurrence
of an apparent adaptive process or a delayed response. The second
qualifier disambiguated significant effects that were not dose-
dependent. ‘Negative’ denoted that the contaminant appeared to cause
harm; ‘positive’ denoted an apparent enhanced fitness (at least within
the context and scope of the experiment). Where multiple endpoints
were examined, we judged whether the overall pattern of response was
either ‘positive’ or ‘negative’ by prioritising effects that were significant.
The third qualifier disambiguated responses where at least two end­
points were used in tandem to evaluate an overlying biological function.
For example, acetylcholinesterase activity (a biomarker for neurotox­
icity) was compared with mobility to establish consistency; if not, these
responses were classed as inconclusive. We focused on extracting in­
formation from the Results section of each research article to maintain
objectivity and to avoid speculative interpretations that might occur in
the Discussion sections. To determine the most sensitive indicator spe­
cies, we took the five most frequently studied species and compared
these to the top five species for which clear effects were identified.
We noted the interaction types of responses in multiple-stressor
studies. Only a limited number of studies described interaction types
using mathematical models such as Concentration Addition (Beren­
baum, 1985), Independent Action (Bliss, 1939), Combination Index
(Chou, 2006), and Combination Index-Isobologram (Chou, 2006). We
classified these interactions as ‘additive’ when the overall effect was
equal to the sum of the individual effects of each component of a
mixture, ‘synergistic’ when the overall effect was greater than the sum of
individual effects, and ‘antagonistic’ when it was less than the sum of
individual effects (EPA United States Environmental Protection Agency,
2000). Interactions that were not mathematically modelled were cat­
egorised into ‘amplifying’ or ‘dampening’ effects, following the broad
definitions in (Orr et al., 2020). Accordingly, an amplifying effect had a
net effect that was greater than the individual effects (without dis­
tinguishing between additive or synergistic), whereas a dampening
response occurred when the net effect was less than the sum of the
combined effects of the individual components.
2.4. Qualitative analysis: study design evaluation
Research validity refers to the accuracy of measuring the phenome­
non of interest (Sechrest, 2005). To evaluate validity of the reviewed
studies, we noted whether nominal pharmaceutical concentrations were
confirmed by analytical measurement because this affects the validity of
the exposure tests (Moermond et al., 2016). An algorithm adapted from
the CRED project (Criteria for Reporting and Evaluating Ecotoxicity
Data, Moermond et al., 2016) was used to classify the studies into
‘Reliable’, ‘Reliable with restrictions’, ‘Requires further inquiry’, and
‘Not reliable’ (see Supplementary Material, Fig. S1 for definitions).
Scientific reliability (as defined by Moermond et al., 2016) is the extent
to which consistent results are achieved with repetition. Among other
factors, reliability is affected by statistical power – the probability that a
true effect is detected in a statistical test if it exists in the data (Quinn and
Keough, 2002). To evaluate reliability of each study, we noted the levels
of replication of each treatment level and whether standardized effect
sizes were reported (as recommended by Nakagawa and Cuthill, 2007
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Environmental Advances 7 (2022) 100164
and in numerous recent studies). Generalisability is the extent to which
the findings can be extrapolated to a larger ecological context (Myers
et al., 2010). We evaluated research generalisability by examining the
level of biological organisation of endpoints used, determining whether
effects were detected at environmentally relevant concentrations, and
noting whether the studies were laboratory- versus mesocosm-based.
2.5. Quantitative analysis
For all single-exposure studies that detected ‘clear effects’ at envi­
ronmentally relevant concentrations (as defined by the concentration
ranges measured and reported in the field surveys summarised in
Table 1), we extracted the lowest observed effect concentrations
(LOECs) of exposures to carbamazepine, diclofenac, and ibuprofen
regardless of model species and endpoints used. We compared these
LOECs to determine if the three pharmaceuticals differed significantly in
their overall toxicity. We also compared short-term vs. long-term ex­
posures by pooling the LOECs of the individual pharmaceutical expo­
sures in each exposure duration. For the comparison between
pharmaceuticals, we used the statistical software R (version 3.6.2) to
perform separate one-way ANOVAs each for short-term and long-term
exposures with Type III sums of squares, due to unequal sample sizes
between the pharmaceuticals. The same method was applied for the
comparisons between short-term vs. long-term LOECs. These ANOVAs
were supplemented by standardised effect sizes calculated as partial-eta
squared values.
3. Results
3.1. Publication rates and exposure durations
Over the last 10 years, ecotoxicological studies on the exposure to
carbamazepine, diclofenac, ibuprofen, and multiple stressors containing
at least one of these pharmaceuticals were dominated by short-term
exposure durations (78 short-term vs. 51 long-term studies, Fig. 1).
(Sixteen studies were counted twice here as they comprised both short-
term and long-term components; thus, the total of 129 is greater than the
113 reviewed studies.) In 2019, there was a notable increase in publi­
cation rates of both short- and long-term studies, with the former in­
crease being steeper.
The average duration of short-term studies was 6.4 days, with a
minimum of 25 min. One short-term study (González-Naranjo and
Boltes, 2014) did not explicitly indicate the exposure duration but cited
the OECD (2008) methodology. The average long-term exposure dura­
tion was 43.1 days, with a maximum of 365 days. However, the 365-day
exposure was an outlier, as this experiment (Yan et al., 2016) was
designed to detect effects on a wetland plant. Removing this outlier
produced an average of 37.8 exposure days, based on studies conducted
using algae, cyanobacteria, smaller macrophytes (i.e. Lemna spp.), and
animal indicator species. One long-term study (Heye et al., 2019) did not
explicitly indicate the exposure duration but the experiment lasted up to
six generations of Daphnia magna. A similar study (Grzesiuk et al., 2020)
stated that this was equivalent to approximately three months; there­
fore, we used this estimate when calculating the average long-term
duration.
3.2. Short-term studies
Twenty-six studies examined the effects of short-term exposure to
carbamazepine. Sixteen (62%) of these included environmentally rele­
vant concentrations, and eight (31%) found clear effects (Supplemen­
tary Material, Table S1.1). Thirty-two studies examined diclofenac,
sixteen (50%) were environmentally relevant, and three (9%) detected
clear effects (Table S1.2). Twenty-two studies examined ibuprofen,
twelve (54%) were environmentally relevant, and five (23%) detected
clear effects (Table S1.3). There were 21 multiple-stressor studies, of
N.S.P. Batucan et al.
Fig. 1. Monthly publication rates from January 2010 to June 2020 of short-term and long-term ecotoxicological studies examining the effects of carbamazepine,
diclofenac, ibuprofen, and mixed stressors containing them.
which 13 (62%) were environmentally relevant, and seven (33%) found
clear effects (Table S1.4). Most studies that detected clear effects indi­
cated that the responses were negative, and only two (Pietrini et al.,
2015, Table S1.3; Tlili et al., 2017, Table S1.4) suggested that phar­
maceutical exposure enhanced physiological functioning. (See
Tables S3.1–S3.4 for a summary of response patterns of short-term
studies.)
Of the 21 multiple-stressor studies, only three studies categorized
interaction types using mathematical models (see Methods 2.3 for model
types). Five studies found a combination of enhancing and dampening
effects, and this was determined by the endpoint examined and the
exposure concentration. For example, in the fish study by Shi et al.
(2019), 1 μg L− 1 cadmium combined with 1 μg L− 1 of carbamazepine
resulted in vitellogenin levels close to that produced by 1 μg L− 1 of
cadmium alone, but 10 μg L− 1 carbamazepine seemed to dampen the
effect of 10 μg L− 1 cadmium on vitellogenin. Further, 1 μg L− 1 cadmium
appeared to enhance the effect of 1 μg L− 1 carbamazepine on lactase
dehydrogenase, but when higher concentrations were combined (at 10
μg L− 1 each) cadmium dampened the carbamazepine effect. Four studies
found solely enhancing effects, while five detected only dampening ef­
fects. The remaining four studies did not investigate interaction types
because their study designs did not allow this.
3.3. Long-term studies
Eighteen studies examined the long-term effects of carbamazepine;
twelve (66%) of these involved environmentally relevant exposure
concentrations and seven (39%) showed clear effects (Supplementary
Material, Table S2.1). For diclofenac, we identified 19 studies, ten of
which (53%) were environmentally relevant and two detected clear
effects (19%) (Table S2.2). Ten studies examined ibuprofen, seven
(70%) involved environmentally relevant concentrations, and four
(40%) found clear effects (Table S2.3). There were 13 multiple-stressor
studies, ten (77%) were environmentally relevant and five (39%)
detected clear effects (Table S2.4). Only a single study (Grzesiuk et al.,
2020, Table S2.1) found that pharmaceutical exposure produced a
positive effect, while all others identified a negative effect. (See
Tables S4.1–S4.4 for a summary of response patterns of long-term
studies.)
Of the 12 multiple-stressor studies, three detected a combination of
enhancing and dampening effects, depending on the exposure concen­
tration and endpoint assessed. Another three found only dampening
effects while the remaining six (50%) did not investigate interaction
types because their study designs did not allow this.
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Environmental Advances 7 (2022) 100164
3.4. Indicator species, effect detection rates, and endpoints
In short- and long-term studies, the most common model species
were (in decreasing order) fish, algae, microcrustaceans, and anurans
(Supplementary Material, Fig. S2A, C). These major taxon groups
generally showed clear effects at environmentally relevant concentra­
tions, except for algae in short-term studies, which generally used
exposure concentrations considerably higher than those found in the
environment. Species at early life stages (i.e. embryo, neonate, larva,
and juvenile) comprised 54% and 53% of taxa tested in short-term and
long-term studies, respectively. Adult species comprised 25% (short-
term) and 24% (long-term). For the remaining species the develop­
mental stage was not indicated. In these cases, either body size was
substituted for age (for one annelid, platyhelminth, ciliate, and macro­
phyte), or developmental stage was indistinguishable (i.e. phyto­
plankton and community indicators).
The zebrafish Danio rerio and the cladoceran Daphnia magna were the
most frequently used indicator species in short-term and long-term
studies, respectively (Supplementary Materia, Fig. S2B, D). The five
most frequently studied species detected clear effects in less than half
the experiments they were used in. The only species exhibiting a 100%
detection rate (5 out of 5) was the freshwater clam Corbicula fluminea.
Biomarkers were the most prevalent endpoint used in short-term
studies, followed narrowly by physiological endpoints, then by behav­
ioural, structural, and community-level endpoints (Fig. 2A). Several of
the short-term studies employing physiological endpoints (e.g. mortal­
ity, growth inhibition) primarily aimed to establish the median effect
concentration (EC50) which informed subsequent long-term exposure
tests. If these studies were not counted, biomarkers would be the most
frequently used endpoint by a much larger margin. Physiological end­
points that detected clear effects at environmentally relevant exposure
levels included moulting (from micro- and macro-crustaceans) and
photosynthetic response variables (from natural stream periphyton).
Only a small proportion of short-term studies (regardless of endpoint)
detected clear effects (Fig. 2A). There was no obvious superiority of one
endpoint type over all others, although physiology and community
endpoints were surpassed by biomarkers and behaviour in the relative
frequency of detecting clear effects.
In long-term studies, physiology was the most prevalent endpoint,
followed by biomarkers, structural, and community-level endpoints
(Fig. 2B). Overall, detection frequency of clear effects was similarly low
as for short-term studies. In absolute terms, biomarkers detected the
highest number of clear effects; however, given their low total fre­
quency, community-level endpoints detected comparably more clear
effects relative to the others. These endpoints included community di­
versity, evenness, and environmental nutrient uptake (Lawrence et al.,
N.S.P. Batucan et al.
Fig. 2. Frequency of endpoints that detected clear effects at environmentally relevant concentrations in short-term (A) and long-term (B) studies.
2012; Jarvis et al., 2014b; Pomati et al., 2017; Baho et al., 2019).
3.5. Experimental design features
Treatment replication ranged between one and 20. In both short-
term and long-term studies, triplicates were the most commonly used
level of replication for each exposure concentration (Fig. 3A, B). Thir­
teen studies incorporated experiments that were pseudoreplicated (see
Hurlbert, 1984), indicated by a single replicate in Fig. 3. We observed
that as replication increased, the number of test organisms within rep­
licates generally decreased. Some examples of this pattern were: one
short-term study (Van den Brandhof and Montforts, 2010) had 20 rep­
licates containing one fish embryo each, another (Han et al., 2010) had
10 replicates with a single fish neonate each, and a long-term study
(Ribas et al., 2016) had 10 replicates with a single fish each. Eight
studies were classed as ‘unclear’ as the degree of replication was not
explicitly indicated.
Only 20% of all studies provided effect sizes for their findings
(Supplementary Material, Table S5 and S6). Most of these studies
Fig. 3. Replication levels in short-term (A) and long-term (B) studies.
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reported correlation coefficients of regression analyses. A single study
(Galus et al., 2014) stated that they had performed a statistical power
analysis and linked this analysis to the limitations of their findings.
Of all the studies included in this review, 38% verified their exposure
concentrations and were reliable (according to the algorithm described
in Section 2.4), 5% were reliable with restrictions, and <1% required
further inquiry or were not reliable (Fig. 4). The remaining studies
(55%) did not verify their exposure concentrations. Most of these were
not reliable and the rest required further inquiry.
Most studies were conducted under laboratory conditions and only
five were mesocosm studies, all with different experimental set-ups. One
study involved 180-L containers in a simulated wetland within a
controlled greenhouse (Yan et al., 2016), another used 75-L containers
as outdoor mesocosms (Jarvis et al., 2014b), and a third exposed 3-L
beakers within two ponds (Bácsi et al., 2016). The final two studies
used floating outdoor mesocosm systems that consisted of dialysis bags
containing local lake phytoplankton; these bags measured 25 cm length
x 2 cm diameter in Pomati et al. (2017) and 2.5 m length x 3 cm width in
Baho et al. (2019).
N.S.P. Batucan et al.
Fig. 4. Percentages of studies that verified or did not verify their nominal exposure concentrations.
Most of the studies used waterbourne exposures. A single study
(Gilroy et al., 2012) utilised sediment treated with carbamazepine.
Three studies (Vernouillet et al., 2010; Guiloski et al., 2015, Ribas et al.,
2016) used biotic vehicles (i.e. treated prey) for the delivery of carba­
mazepine and diclofenac. Finally, one study (Quinn et al., 2011) directly
injected treatments of diclofenac into the muscle of the bivalve Dreissena
polymorpha.
3.6. Pharmaceutical toxicity comparison
Few single-exposure studies detected ‘clear effects’ at environmen­
tally relevant concentrations (16 short-term and 13 long-term studies for
all three pharmaceuticals combined) and were thus suitable for inclu­
sion in the quantitative toxicity comparison. There were no statistically
significant differences between LOECs calculated for the three phar­
maceuticals in both short-term (F2,13 = 1.799, p = 0.204, η2 = 0.217)
and long-term studies (F2,10 = 0.140 p = 0.871, η2 = 0.027), and clear
effects were detected at ≤10 µg L− 1 in both settings (Supplementary
Material, Fig. S3). Further, the pooled LOECs were similar across short-
term and long-term exposures (F1, 27 = 0.371, p = 0.547, η2 = 0.014;
Fig. S4).
The overall lowest LOEC determined from short-term studies was
0.05 µg L− 1 (carbamazepine and ibuprofen); the highest was 9 µg L− 1
(ibuprofen). Two short-term studies of carbamazepine and ibuprofen
were based on pseudoreplicated designs. However, their removal did not
alter the range of concentrations within which clear effects were
detected, so these data points were retained. The lowest overall LOEC
determined from long-term studies was 0.1 µg L− 1 (carbamazepine); the
highest was 10 µg L− 1 (carbamazepine and ibuprofen).
4. Discussion
4.1. Brief summary of the existing published evidence
Our qualitative review evaluated the evidence on the potential
environmental risk of carbamazepine, diclofenac, ibuprofen, and
multiple-stressor exposures containing at least one of these pharma­
ceuticals by examining the scientific literature published on this topic
since 2010. About half of the reviewed studies used environmentally
relevant exposure concentrations and, of these, 9-50% identified clear
effects, most of which were negative. As an overall average, ‘clear’
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negative effects (according to the criteria in 2.3.) were observed in 35%
of single or mixed pharmaceutical exposures of short-term (~6 d) or
long-term (~38 d) duration. This is just over a third of the 113 reviewed
studies. At first glance, this finding would be consistent with the current
consensus that these pharmaceuticals pose a relatively low risk at con­
centrations typically encountered in the environment. However, we
have several important concerns about the existing evidence in the
published literature, which we will discuss below.
Short-term outnumbered long-term studies. This illustrates the
persistence of the bias for experiments of shorter duration, even though
the paucity of long-term studies has been repeatedly highlighted as a
continuing knowledge gap in ecotoxicological studies of pharmaceuti­
cals (Calisto and Esteves, 2009; Santos et al., 2010; Gunnarsson et al.,
2019).
4.2. Endpoints
Most of the endpoints used in short-term studies were biomarkers
and physiological indicators. Biomarkers showed the highest effect
detection rate, confirming that biomarkers can provide early warning
signals of harm to higher levels of biological organisation (Adams et al.,
2001, Roméo and Giamberini, 2012). However, biomarker responses in
the short-term studies were not always linear or their effect direction as
expected, which made interpreting such findings difficult. For example,
acute exposure to 1 μg L− 1 diclofenac led to increased acetylcholines­
terase levels in zebrafish and coincided with an observed reduction in
fish swimming activity (Sun et al., 2020). Acetylcholinesterase is a
biomarker for neurotoxicity which is usually inhibited as a response to
neurotoxic exposure, such as seen for organophosphate toxicity to fish
(Roméo and Giamberini, 2012; Barboza et al., 2018; Sandoval-Herrera
et al., 2019). That said, abnormal acetylcholinesterase activity can lead
to abnormalities in locomotion that could lead to hypo- or hyperactivity,
depending on the degree of inhibition (Kristoff et al., 2006; Azevedo-­
Pereira et al., 2011). Therefore, complementing biomarkers with other
lines of evidence, such as behaviour, aided in disambiguation in this
case. In fact, we found that behaviour was a sensitive marker even for
short-term exposures averaging ~6 days.
In several cases, interpreting biomarker responses also became
ambiguous when several were investigated in parallel. Take for example
lipid peroxidation and lysosomal membrane stability – biomarkers of
oxidative cell damage, and catalase, superoxide dismutase, glutathione
N.S.P. Batucan et al.
peroxidase, and ethoxyresorufin O-deethylase – biomarkers for oxida­
tive stress defence (Roméo and Giamberini, 2012). Acute exposure of the
fish Cyprinus carpio to 17.6 mg L− 1 ibuprofen led to no changes in he­
patic lipid peroxidation yet coincided with increased catalase activity
(but no changes in superoxide dismutase and glutathione peroxidase),
suggesting that catalase may have counteracted oxidative cell damage
(Islas-Flores et al., 2017). On the other hand, the biomarker profile in
gill tissue was different, with lipid peroxidation being elevated despite
the increased activities of catalase and superoxide dismutase (Islas-­
Flores et al., 2017). The question, then, is how these compartmentalised
biomarker changes affected the overall performance of the fish. Taking
this idea further, chronic exposure of the bivalve Corbicula fluminea to
0.1-1 μg L− 1 carbamazepine resulted in reduced lysosomal membrane
stability (measured using neutral red retention time [NRRT]), despite
increased ethoxyresorufin O-deethylase and glutathione peroxidase ac­
tivities (Aguirre-Martínez et al., 2015). The bivalves exposed to carba­
mazepine at these concentrations were considered to be “stressed but
compensating” (Aguirre-Martínez et al., 2015). This interpretation was
supported by Martínez-Gómez et al.’s (2008) NRRT criteria, which had
been developed by studying lysosomal membrane stability trends in the
haemolymph of the marine bivalve Mytilus galloprovincialis in pop­
ulations along a gradient of chemical pollution on the Iberian Mediter­
ranean coast. As illustrated by these two examples, biomarker responses
remain somewhat speculative and open to interpretation unless they are
calibrated against endpoints from a higher level of biological organisa­
tion (Gagné, 2014).
In cases with strong correlations with higher organisational levels of
the ecosystem, biomarkers can be valuable tools for environmental risk
assessment. Their use is endorsed in the ‘adverse outcome pathway
paradigm’, which advances the concept that causal relationships exist
between different levels of biological organisation so that it is possible to
predict higher-level responses using lower-level endpoints (Ankley
et al., 2011). The two-pronged approach of using molecular- and
community-level methods offer a legally defensible framework for
linking stressors as the causal agent to observed qualitative changes in
ecosystem state (Adams and Tremblay, 2003), which is indispensable to
environmental policymaking and management. ‘Omics’ technologies (i.
e. genomics, transcriptomics, proteomics, and metabolomics) are
increasingly recognised as having great utility in identifying the un­
derlying mechanisms of qualitative changes in ecosystems (Zhang et al.,
2018). For example, in a mesocosm study by Deng et al. (2020), the
authors demonstrated that antibiotic exposure at trace concentrations
caused temporal changes in the antibiotic resistant gene (ARG) profile in
water and sediments, and these changes were correlated with changes in
bacterial community structure. A shift in dominance of tolerant bacterial
species was observed, and these were speculated to be the compatible
hosts of the ARGs. Deng et al. (2020) also found that certain ARGs
persisted in the water and sediments even after removal of the antibi­
otics, indicating the persistence in compatible host bacteria. Here, the
shift in ARG profile helped to directly link the change in bacterial
community to the presence of antibiotics.
Physiology appeared to be a poorly sensitive endpoint at detecting
effects at environmentally relevant exposures. This may not be unex­
pected as many physiological endpoints measured mortality, growth,
development, and reproduction. At such low concentrations it may be
less likely to detect effects even at long-term exposure timeframes ana­
lysed herein, and this highlights the advantage of using biomarkers in
assessing sublethal physiological conditions (Saggioro et al., 2018).
Behaviour was surprisingly not a very sensitive endpoint, which is
contrary to the findings of Melvin and Wilson’s (2013) meta-analysis on
the sensitivity of behavioural responses to contaminants. However, in
their analysis pharmaceutical contaminants comprise a relatively small
proportion of reviewed contaminants relative to pesticides and metals,
thus their analysis was inherently biased for the latter two. It is also
possible that, while the mode of action for the three focal drugs in many
aquatic species is unknown, these drugs may act on biological pathways
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not readily detected by behaviour changes unlike certain pharmaceu­
tical groups that are designed to produce psycho-behavioural effects,
such as anti-depressants and benzodiazepines (Stanley et al., 2007,
Brodin et al. 2014).
Among short-term studies of the three pharmaceuticals, only expo­
sure to carbamazepine resulted in significant morphological effects.
These studies were done on larval zebrafish (D. rerio) and the cnidarian
Hydra circumcincta. For D. rerio, a minimum 1 μg/L carbamazepine
exposure up to 96 hours caused greater body length, increased swim
bladder appearance and accelerated yolk sac absorption (Qiang et al.,
2016). For H. circumcincta, increased deviations in morphology relative
to controls were detected after 14 days exposure to 900 ng/L carba­
mazepine alone and in combination with two of its common metabolites
(Desbiolles et al., 2020). A six-week exposure to 10 μg/L carbamazepine
caused male D. rerio to produce abnormal sperm (Galus et al., 2014) and,
when combined with three other drugs of different classes, an increased
incidence of abnormal embryo development occurred (Galus et al.,
2013). Similarly, an increased incidence of deformed embryos in
D. magna was observed after exposure to 4 μg/L ibuprofen over three
generations (Grzesiuk et al., 2020). Exposure of the rainbow trout
Oncorhynchus mykiss to 5 μg/L diclofenac for 21 days led to intestinal
villi hyperplasia and increased fusion (Mehinto et al., 2010). In general,
more long-term than short-term studies detected morphological
changes, although this result may be an artefact of the greater absolute
number of long-term studies that used morphological endpoints.
Compared to other endpoints, morphology had the lowest proportion of
detected effects relative to the total number of studies that used it as an
endpoint. Nonetheless, because quite a few morphological effects were
detected when summed across all long-term exposures, morphology
remains a promising endpoint in future chronic ecotoxicological tests.
In long-term studies, community-level endpoints performed best at
detecting clear stressor effects at environmentally relevant concentra­
tions. These endpoints included community diversity, evenness, and
nutrient-cycling markers, all of which are considered to have greater
ecological relevance compared to biomarkers (Adams and Tremblay,
2003). For example, a single pulse of a mixture of the three focal drugs
and nine other pharmaceutical and personal care products caused a
significant change in phytoplankton community structure in two
Swedish lakes 20 days later, where larger-sized species (>5 μm) domi­
nated (Baho et al., 2019). Further, in Lawrence et al.’s (2010) study on
natural river biofilms, carbon utilisation tended to decrease with eight
weeks exposure to 5 μg/L diclofenac. The paucity of long-term, eco­
system-level studies has been highlighted in previous reviews (Santos
et al., 2010; Tiedeken et al., 2017), but their continued rarity may reflect
the logistical challenges and resource-intensity attached to them
(Bradbury et al., 2004). Thus, it was not unexpected to see the pre­
dominance of laboratory-based experiments in the research literature
we assessed. Even among community-level mesocosm studies, a sole
study (Jarvis et al., 2014b) employed a simulated, multi-trophic inver­
tebrate assemblage that also included microscopic primary and sec­
ondary producers. The remaining experiments used only microscopic
species (i.e. phytoplankton and periphyton communities), presumably
because their logistics and resource-intensity were easier to manage.
Short-term and long-term multiple-stressor studies identified com­
plex interactions, with both ‘amplifying’ and ‘dampening’ effects
occurring depending on the endpoint and concentration of exposure,
with dampening effects being slightly more common than amplifying
ones (18 vs. 16, respectively). In Jackson et al.’s (2016) meta-analysis on
the effects of binary stressors on aquatic ecosystems, the net effect of
dual stressors at least at an organismal level tended to be antagonistic.
By contrast, when we examined binary stressor combinations (for 21
short-term studies and 1 long-term study), we found a marginal preva­
lence of amplifying over dampening effects (10 vs. 8, respectively).
However, when >2 stressor combinations were examined, dampening
effects were more frequent than amplifying effects (10 vs. 6). All these
studies used single-species models. In contrast to Jackson et al.’s (2016)
N.S.P. Batucan et al.
observation, a systematic review by Cedergreen (2014) determined that
additive multiple-stressor effects were the most common interaction
type in binary exposures of pesticides, metal ions and antifouling agents,
followed by antagonisms and synergisms. Matthaei and Lange’s (2016)
review of multiple-stressor effects on freshwater fish found a similar
pattern and they examined major stressors such as climate change,
habitat modification, and nutrient enrichment. That said, it is often
difficult to predict interaction type as its variability can be affected by
the type of stressor, interspecies response variability, the levels of bio­
logical organisation, and the characteristics of the ecosystem in question
(Jackson et al., 2016).
The small sample size in our study makes it difficult to generate
conclusions regarding which interaction types involving pharmaceutical
contaminants are important. The effects of pharmaceuticals on the
environment can be subtle (Brodin et al. 2014), which makes predicting
their individual effects challenging much less modelling their interactive
effects with other stressors (Piggott et al., 2015). Thus, from the
perspective of environmental management, focusing on pervasive
stressors (e.g. habitat degradation, disturbance of flow regime, climate
change) may be more practical and informative, as these already pose an
immediate threat with drastic consequences that are already
well-established (Matthaei and Lange, 2016). While micropollutants are
also among the major threats to global freshwater ecosystems (Tiedeken
et al., 2017), future multiple-stressor studies will need to establish how
pharmaceutical contaminants may modulate the disruptive effects of
known pervasive stressors.
4.3. Indicator species
The use of species in their early life stages was common, which is an
advantage as early life stages are generally more vulnerable to con­
taminants than adults due to the former’s greater surface-to-volume area
and sensitive developmental processes (Luckenbach et al., 2001).
Therefore, the risks being estimated were realistic (Matthaei and Lange,
2016). The four taxon groups most frequently studied were fish,
microcrustaceans, anurans, and algae. Fish was the major group inves­
tigated in both short-term and long-term studies. Fish can be sensitive to
pharmacological compounds designed for humans, perhaps due to our
close evolutionary history resulting in many homologous physiological
and anatomical features (Garcia et al., 2016). For example, the zebrafish
Danio rerio shares more pharmaceutical target receptors with humans
than other common model species, such as the cladoceran Daphnia pulex
and the green alga Chlamydomonas reinhardtii (Gunnarsson et al., 2019).
As with the case of the Gyps vultures, fish feminisation from synthetic
oestrogens is among the most well-known adverse effects of micro­
pollutants in aquatic environments, underpinning the continued risk of
pharmaceuticals to aquatic ecosystems (Kidd et al., 2007; Kümmerer,
2009; Toma and Crişan, 2018).
D. rerio and D. magna were the most-studied species in both short-
term and long-term tests in our review, in keeping with their well-
established and prevalent use in aquatic ecotoxicology (Martins et al.,
2007; Matsumoto et al., 2009; Chatterjee et al., 2018). However, the
frequency of detecting clear effects at environmentally relevant con­
centrations with these two organisms was relatively low, suggesting
they are not very sensitive to the three focal pharmaceuticals. This result
is surprising for D. rerio because it is generally known to be a sensitive
model for pharmaceutical testing (Gunnarsson et al., 2019). Perhaps
exposure via the water compartment, the prevalent mode of exposure in
our reviewed articles, underestimated the three drugs’ effects on
zebrafish and other fish models, highlighting the need for incorporating
a more varied approach to exposure delivery (Brodin et al. 2014). By
contrast, the insensitivity of D. magna has been observed for various
other contaminants. For example, D. magna has been gradually recog­
nised as being extremely tolerant to neonicotinoid insecticides, but their
continued use as a test model initially led to a serious underestimation of
the environmental toxicity of neonicotinoids (Sánchez-Bayo et al.,
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2016).
Interestingly, in our review the freshwater clam Corbicula fluminea
detected clear effects to carbamazepine and ibuprofen in all five studies
which used this test organism, making it a promising candidate for
future related research. This observation is especially pertinent to the
ongoing efforts to minimise using vertebrate models in toxicity testing
for ethical concerns. Freshwater bivalves are sentinels to environmental
perturbations with global populations of various species in serious
decline in recent years and pollution is found to be particularly
impacting (Cope et al., 2008; Lopes-Lima et al., 2018). They have also
been shown to demonstrate higher bioaccumulation of different classes
of pharmaceutical contaminants compared to other model species such
as algae and fish (Du et al., 2014; Burket et al., 2019). This perhaps
suggests the readiness in the bioavailability of pharmaceuticals to bi­
valves and therefore are more likely to show effects, furthering the
support of their use as an alternative to vertebrate species, but with a
focus on utilising non-threatened species.
Aquatic algae appeared non-sensitive to environmentally relevant
concentrations of the three pharmaceuticals, despite their known
sensitivity to many other contaminants owing to their rapid genera­
tional turnover rates (Gökçe, 2016). However, most of the reviewed
algae studies used exposure concentrations orders of magnitude higher
than those found in the environment, limiting the use of the data for
field-based risk assessment. Our review methods overlooked the occur­
rence of hormesis, a pattern sometimes seen in organisms characterised
by a stimulating effect at low-dose stress and an inhibiting effect at high
doses (Mattson, 2008). This biphasic response unavoidably contradicted
our second qualifier for identifying a ‘clear effect’ (see 2.3), which
rendered this response pattern ‘inconclusive’. That said, of the four
environmentally relevant studies involving algae, only one study (Haase
et al., 2015), on Parachlorella keissler and Neochloris pseudoalveolaris,
showed a biphasic response of growth and chlorophyll pigments to acute
exposure to carbamazepine. Further, some algal species appear to be
highly tolerant to the three pharmaceuticals. For instance, Chlorella
pyrenoidosa, Chlorella vulgaris and Pseudokirchneriella subcapitata all had
median inhibition concentrations ≥49.4 mg L− 1 when exposed to car­
bamazepine or a mixture of pharmaceuticals (Vannini et al., 2011;
Zhang et al., 2012; Geiger et al., 2016). These concentrations far exceed
environmentally relevant concentrations. Perhaps the use of freshwater
algae is most informative when used as part of a test community in
mesocosms, given that clear effects of the three pharmaceuticals were
detected in most community-level studies (which included assemblages
of phytoplankton and/or periphyton) in long-term exposures.
4.4. Study design evaluation
The majority of reviewed studies were laboratory-based and had low
replication of each treatment level, triplicates being the most common.
Perhaps this low degree of replication (which results in low statistical
power) primarily reflects restrictions caused by limited resources. To
some extent, it could also be an artefact of existing standard protocols.
For instance, while the OECD guidelines generally specify rigorous
quality assurance instructions for toxicity tests, protocols for acute
toxicity and juvenile growth tests on fish allow do not require replication
at the tank level and permit the use of individual tanks and the perfor­
mance of statistical analysis at the level of individual fish (OECD, 2000,
2019). However, these recommendations only apply to fish for these
specific tests, and protocols for other tests and/or other species recom­
mend a minimum of 4 replicates (e.g. OECD, 2010, 2012, 2015).
Further, there may be an element of conservatism based on ethical
grounds, particularly when using vertebrate models. While all these
factors may affect the ability of researchers to increase replication, it still
remains paramount to aim for high replication. If increasing replication
is possible after considering all restricting factors including ethical as­
pects, it would be a false economy to stop at the minimum number
recommended by guidelines because the occurrence of Type II errors
N.S.P. Batucan et al.
(false negatives) can be highly likely due to the low statistical power
inherent in low-replicated designs.
Adding to the high frequency of low replication, the few higher-
replication studies also shared an important design weakness. As the
number of replicate experimental units per treatment increased, the
number of test animals within these replicates decreased, to the point
that there was just a single individual per experimental unit. Because
there is no guarantee that each individual will survive through the
experimental duration, fewer replicates will be left if some of these in­
dividuals die, most likely also resulting in an unbalanced study design.
This pattern of spreading as few test individuals as possible across as
many replicate units as possible could suggest a certain level of ‘data
dredging’ (Nakagawa and Parker, 2015), the act of designing a study
with the aim of maximising the chances of finding significant p-values.
On the other hand, this pattern could also be an artefact of some OECD
guidelines. For example, the reproduction test protocol for daphnids in
semi-static systems recommends having a minimum of 10 individuals,
each one individually held and representing one replicate (OECD, 2012).
The problems of low replication and associated low statistical power
could have been alleviated by the reporting of standardised effect sizes
(Nakagawa and Cuthill, 2007), but unfortunately this was done by very
few studies in our review. The influence of pharmaceutical contami­
nants on the environment could then be assessed by comparing effect
sizes across multiple studies in a meta-analysis. However, even the ev­
idence generated by such a meta-analysis can only be as sound as its
individual components; with low-replication studies being common, the
evidence body is already of limited quality. Another problem is that
fewer than half of the studies verified their nominal exposures concen­
trations through direct analytical measurement; thus, most studies were
unreliable in that regard. Exposure solutions tend to vary over time due
to sorption processes and natural degradation; therefore, validating the
true exposure concentrations increases the reliability of linking obser­
vations to the intended exposure concentrations (Moermond et al.,
2016).
A review by Boxall et al. (2012) examined salient questions around
pharmaceutical and personal care products in the environment and
indicated the identification of exposure pathways as an important topic.
In our review, most studies used waterbourne exposures. This is another
limitation of the existing evidence since contaminants distribute within
different compartments in the environment; thus, focusing on a single
pathway may risk underestimating actual exposure levels occurring in
the field. It is worth investigating if simultaneously operating multiple
exposure pathways change the likelihood of detecting meaningful ef­
fects of pharmaceutical and personal care products, for example aqueous
exposure combined with exposure to contaminated food sources. As
some contaminants are known to bioaccumulate (Memmert et al., 2013;
Bickley et al., 2017; Nkoom et al., 2019), exposure could be higher
through biotic vehicles versus the aqueous compartment. Moreover,
different routes of absorption (e.g. absorption through gills vs. inges­
tion) may vary in their pharmacokinetics, which could influence the
level of potency of exposure (Toutain et al., 2010).
4.5. Quantitative pharmaceutical toxicity comparison
The LOECs that caused clear effects at environmentally relevant
concentrations were similar across the focal pharmaceuticals in both
short- and long-term studies, suggesting the toxicity of the three phar­
maceuticals to freshwater test organisms may be similar. Similarly, the
comparison of LOECs between short- and long-term exposures showed
no significant difference. However, the latter result should be treated
with some caution because the small number of studies included in this
meta-analysis (2-8 studies for each pharmaceutical of either exposure
duration) resulted in low statistical power. For short-term exposures, the
effect size (η2 = 0.22) was still large enough to be biologically mean­
ingful (Nakagawa and Cuthill, 2007), whereas it was negligible (η2 =
0.03) for long-term exposures.
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When all three pharmaceuticals were considered together, their
LOECs derived from short-term (0.05-9.0 μg L− 1) and long-term studies
(0.1-10.0 μg L− 1) were comparable. Statistical analysis of the pooled
mean LOECs confirmed this similarity, which seems counterintuitive at
first glance. For example, a meta-analysis comprising data on over 150
chemicals obtained from the EnviroTox database (https://envirotoxdata
base.org/) (Hiki and Iwasaki, 2020) found that chronic exposures were
about 10 times more sensitive than acute exposures when detecting the
hazardous concentrations for 5% of test species including fish, algae,
invertebrates, and amphibians. Moreover, neonicotinoid insecticides
have acute-chronic ratios of median lethal concentrations (LC50) of up to
800:1 for insect test organisms (Sánchez-Bayo et al., 2016).
There are at least two potential explanations for the similarity of
pooled mean LOECs of short-term and long-term exposures. First, most
of the studies included in our quantitative analysis did not confirm their
actual exposure concentrations; thus, our comparison had to be based on
nominal concentrations. Had the measured concentrations been sup­
plied, potential differences in the LOECs could have been detected.
Indeed, further examination of the studies included in this meta-analysis
revealed that 63 % (10 of 16) of short-term and 62 % (eight of 13) of
long-term studies did not report measured concentrations. Alternatively,
the three focal pharmaceuticals, which were designed for therapeutic
purposes, might not elicit stronger adverse effects at environmentally
relevant concentrations during chronic compared to acute exposures, in
contrast to chemicals that are toxic by design, such as neonicotinoids.
This could be because individuals or populations of freshwater species
might be able to adapt to persistent low-dose exposure to pharmaceu­
ticals. For instance, Damásio et al. (2011) observed that the larvae of the
caddisfly Hydropsyche exocellata residing in industrially affected waters
of the Llobegat River had greater activities of detoxifying enzymes
compared to conspecifics transplanted into the same location for a
period of four days, and enhanced activity in detoxification enzymes
may indicate an adaptive response (Tenji et al., 2020). Further, sus­
tained exposure to anthropogenic stressors can lead to genetic adapta­
tion; for example, this evolutionary influence occurs widely in
populations of killifish from polluted estuaries in the US (Whitehead
et al., 2017).
4.6. Conclusion and recommendations
Based on the evidence in the published literature we reviewed, one
might initially conclude that the pharmaceuticals carbamazepine,
diclofenac and ibuprofen, and multi-stressor exposures containing them,
pose a low risk to the environment. However, our review highlighted
several important limitations of the existing evidence; therefore, we
judge that neither their perceived low risk nor a potentially higher risk is
supported by conclusive evidence. To improve this situation for the
three focal pharmaceuticals (and probably also for other pharmaceuti­
cals), we make the seven recommendations below for future research.
Notably, four of these recommendations had already been made in more
general terms by Harris et al. (2014). Our review shows clearly that
these issues remain pertinent:
(i) Change the framing of research questions from focusing on
lethality of selected test organisms to investigating the extent to
which these micropollutants modify their receiving ecosystems.
Instead of lethal doses, exposure concentrations should be based
on concentrations measured in the environment (see Harris et al.,
2014) as well as on projected future increases of the contaminants
in the environment. This shift in framing minimises experiments
which produce results with low field-realism, and steers research
into generating evidence with greater relevance for environ­
mental management and protection.
(ii) The paucity of long-term mesocosm based studies continues to
represent a significant limitation for assessing the risks of the
three focal pharmaceuticals in aquatic ecosystems, a limitation
N.S.P. Batucan et al.
that should be urgently addressed. When considering the per­
formance of short-term exposure tests, a strong rationale must be
in place that pivots on environmental relevance, so that the fre­
quency of short-term, laboratory-based studies contributing
marginally to the body of knowledge is minimised. Limited re­
sources can then be allocated more economically and efficiently.
(iii) As animals in the natural environment may be exposed to con­
taminants through more than one matrix, future experiments
need to incorporate more than one exposure compartment in
their study design to improve ecological realism (see also Harris
et al., 2014). Combinations of contaminated food supply (biota)
and at least one spatial matrix (water and/or sediment) are
particularly recommended.
(iv) Future studies should improve experimental rigour and quality
assurance by quantifying actual exposure concentrations (see also
Harris et al., 2014) and verifying that these are within acceptable
levels of +/- 20% of the nominal concentration (Moermond et al.,
2016). This approach ensures that any significant observations on
endpoints can be reliably attributed to the exposure treatments
and that more accurate effect concentrations (e.g. LOEC) can be
established. Further, future studies need to be more transparent
in reporting their methodologies, including better documentation
of experimental conditions, so that quality and rigour of the study
design can be accurately evaluated, and a more balanced weight
of evidence can be assigned (Ågerstrand et al., 2014).
(v) Future studies need to be more rigorous in their experimental
design (more true replicates with multiple test individuals per
unit) and statistical analysis, enabling a more accurate and robust
hypothesis testing and interpretation of findings (see also Harris
et al., 2014). Moreover, statistical results should always include
calculating standardized effect sizes to complement p-values.
Once enough such robust evidence has accumulated, another
quantitative meta-analysis should reassess the environmental
risks of the focal pharmaceuticals.
(vi) Future multiple-stressor experiments need to combine pharma­
ceutical pollutants with other known major stressors (e.g. climate
change, nitrogen and phosphorus pollution, and sedimentation),
to better understand how micropollutants may mediate the level
of ecosystem alteration caused by major stressors.
(vii) Future ecotoxicological studies can achieve a more accurate es­
timate of toxic effects by combining biomarker endpoints with
community-level endpoints. This two-pronged approach not only
helps resolve some ambiguities around interpreting biomarkers,
but also offers a potential causal mechanism for any changes
observed at the community or ecosystem level, thereby produc­
ing defensible evidence relevant to environmental policymaking
and management.
Author contributions
Niña Sarah P. Batucan: Conceptualization; Data curation; Formal
analysis; Methodology; Validation; Visualization; Writing - original
draft; Writing - review & editing
Louis A. Tremblay: Supervision; Writing - review & editing
Grant L. Northcott: Supervision; Writing - review & editing
Christoph D. Matthaei: Conceptualization; Formal analysis; Meth­
odology; Supervision; Writing - review & editing
Funding
This research was partially funded by the New Zealand Ministry of
Business, Innovation and Employment grant (CAWX1708).
Declaration of Competing Interest
No conflict of interest.
11
Environmental Advances 7 (2022) 100164
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.envadv.2021.100164.
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