Ecotoxicology (2021) 30:130–141

https://doi.org/10.1007/s10646-020-02304-2

Occurrence of organic micropollutants in an urbanized sub-basin

and ecological risk assessment
Juliana Azevedo Sabino1 André Luís de Sá Salomão
●
1 ●
Priscila Maria de Oliveira Muniz Cunha1 ●

Rodrigo Coutinho1 Marcia Marques1

●

Accepted: 26 October 2020 / Published online: 11 November 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
The João Mendes River - an important contributor to the Piratininga/Itaipu lagoon system in Rio de Janeiro State, Brazil -
receives untreated sewage from the population occupying the drainage basin with no proper sanitation infrastructure. The
present study assessed the ecological risk resulting from the presence of five organic micropollutants (17α-ethynylestradiol,
ibuprofen, trimethoprim, sulfamethoxazole, bisphenol A) based on four monitoring campaigns which included three
sampling points and one reference area. Chronic ecotoxicity assays were conducted with the bioindicators R. subcapitata,
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C. dubia and O. niloticus. Estrogenicity was assessed with genetically modified S. cerevisiae based on YES protocol. The
Ecological Risk Assessment was conducted based on the Chemical and the Ecotoxicological Lines of Evidence (LoE). In
order to analyse the results from different sampling points, principal component analysis (PCA) was performed using a
correlation matrix. Micropollutants below limit of detection or in very low concentrations were detected in the reference
area; no significant differences were observed when samples from the reference area were compared to the negative controls
for the ecotoxicity assays. A PCA including selected variables revealed the latent relationships among the three sampling
points (not verified for the reference area), which confirmed the analytical results. An extreme ecological risk index was
estimated for all sampling points in all campaigns. The extreme ecological risk index was mostly associated to the high
concentrations of 17α-ethynylestradiol and the antibiotic sulfamethoxazole.
Keywords Urbanized basin Micropollutants Ecological Risk Assessment Aquatic toxicology Endocrine disruptors
● ● ● ●

Introduction endocrine system of different organisms, with the ability of

Wastewater from untreated or insufficiently treated sewage
is an important source of emerging contaminants (i.e., such
as antibiotics, synthetic hormones, drugs of abuse) once
discharged into the aquatic environment (Cunha et al.
2019). Worldwide, several of these emerging micro-
pollutants have been proved to act as endocrine disrupting
chemicals (EDC) or endocrine modulators that disrupt the
Supplementary information The online version of this article (https://
doi.org/10.1007/s10646-020-02304-2) contains supplementary
material, which is available to authorized users.
* André Luís de Sá Salomão
andre@andresalomao.com
1 diclofenac) (Boillot et al. 2015). Moreover, the EU has
Department of Sanitary and Environmental Engineering, Rio de
Janeiro State University – UERJ, Rua São Francisco Xavier, 524,
5024E, Maracanã, CEP: 20550-900 Rio de Janeiro, RJ, Brazil
working as agonists or antagonists to endogenous hormones
(Yu and Wu 2012). The heterogeneous group of natural or
synthetic (xenobiotic) EDC can modify the levels and
functions of hormone receptors, induce or inhibit signaling
pathways, and mimic steroid hormones (estrogens and
androgens) (Souza et al. 2013). These EDC can bind to
hormone receptors and interfere in the synthesis, secretion,
storage, release or block, transport, metabolism, and action
of the hormones in the bloodstream (Rawat et al. 2011).
Because of the risks associated with the presence of such
compounds in the environment, the European Union
(Decision 2015/495/EU) (European Parliament 2015) and
other countries (e.g. USA) have developed a priority list of
substances widely used to monitor surface water quality.
This list includes pesticides, several antibiotics, natural
hormones and pharmaceuticals (e.g., 17α-ethynylestradiol,
established a risk assessment protocol for pharmaceuticals
(17α-ethynylestradiol and diclofenac, a non-steroidal
Occurrence of organic micropollutants in an urbanized sub-basin and ecological risk assessment
inflammatory drug), herbicides, several antibiotics, some
natural hormones and sunscreen products based on the
Predicted No-Effect Concentration (PNEC) of chemicals on
aquatic organisms, representing different trophic levels
(e.g., algae, crustacean and fish) (European Medicines
Agency 2018). However, due to the large variety of com-
pounds produced, many micropollutants have not yet been
fully examined for their adverse environmental effects on
aquatic biota and, ultimately, on humans.
In this context, the Ecological Risk Assessment (ERA) is
an important tool for predicting risks from exposure to
contaminants and managing contaminated sites. The ERA
aims to evaluate the ecological changes caused by the
various anthropogenic activities in a given environment
(Kolluru et al. 1998). By using ERA as a diagnosis tool, it is
possible to identify potential adverse effects of con-
taminants on the environment, through chemical and bio-
logical analyses (Jensen and Mesman 2006) at different
levels of biological organization (Chapman 1990).
In Brazil there is a lack of standard protocol to be applied
to ecological risk assessment of contaminated sites and
therefore, the current risk analysis performed in the country
are based on well-established international methodologies.
In the aquatic environment, for example, the Sediment
Quality Triad (SQT) developed by Long and Chapman
(Long and Chapman 1985) is the most used one. This
method was further adapted to the terrestrial environment,
known as the Dutch Triad, by Jensen and Mesman (Jensen
and Mesman 2006). The Dutch Triad was successfully
applied in Brazil to evaluate the risk of contaminated soil
(Niemeyer et al. 2015) and surface waters (Mendes et al.
2017). The Triad uses three Lines of Evidence (LoE) to
assess the risk: chemical, ecotoxicological and ecological.
The chemical LoE consists of detecting the concentration of
the chemical substances of interest (CSI) in environmental
matrices and comparing them with the reference values
established in the national or international legislation. The
ecotoxicological LoE assesses the toxic effects on the biota
of the existing mixture of chemical compounds (evaluated
or not in the chemical LoE) present in environmental
matrices. To do that, bioassays are conducted with standard
test organisms of different trophic levels. Ecological LoE
assesses the effects of environmental pollution on local or
in situ ecological receptors. This effect is evaluated
according to specific indexes and analyses of community
structure (wealth, abundance, diversity and species density)
(Sanchez 2012).
The aim of this study was to conduct an ecological risk
assessments for the João Mendes river (Rio de Janeiro State,
Brazil), based on the presence and concentration of five
common organic micropollutants (i.e., 17α-ethynylestra-
diol, Bisphenol A, Ibuprofen, Trimethoprim, Sulfamethox-
azole) resulting from the continuous release of untreated or
131
insufficiently treated sewage from the irregular urban
occupation. The study’s goal is to identify potential risks to
the aquatic ecosystem, which is considered of great ecolo-
gical, cultural, and economic importance for the state of Rio
de Janeiro.
Materials and methods
Study area
The João Mendes river (Fig. 1), the main tributary to the
Piratininga/Itaipu lagoon system in the oceanic region of
Niteroi Municipality, Rio de Janeiro State, Brazil has been
suffering from a continuous discharge of untreated sewage
(Monteiro-Neto et al. 2008; Prefeitura de Niterói and
AGRAR 2014). The hydrographic subbasin of João Mendes
river has an area of approximately 17 km² and the main
river course has about 7 km long and 15 tributaries before
reaching the Piratininga-Itaipu Lagoon. According to the
Environment State Institute (Instituto Estadual do Ambiente
- INEA), in 2015 the João Mendes river presented poor to
very poor environmental quality index, the worst among the
rivers forming the Piratininga-Itaipu lagoon system.
The Piratininga-Itaipu lagoon system is formed by sev-
eral lagoons connected by channels to their respective
beaches, which are part of the Itaipu Extractive Conserva-
tion Unit (RESEX). The RESEX aims to preserve the
region’s marine biodiversity, and to support artisanal fish-
ing. Besides the discharge of untreated urban sewage along
the drainage basin, solid waste and debris reach the beaches
coming from the tributary rivers (Monteiro-Neto et al. 2008;
Silva et al. 2015, 2016).
The lagoon system and the connection channel with the
coastal region covers different habitats. This complex also
includes a group of islands (Pai, Mãe and Menina) that
protect the bay from the waves. Thus, ensuring a region
suitable for small-scale fishing and the development of
different fish species, dolphins and turtles are often
observed by bathers. Among the fish species found, several
of them have commercial value, being a source of income
for a traditional small fishing community that occupies the
Itaipu beach. The products of fishing is sold in the local
markets but also at the Municipal Market São Pedro, one of
the most famous fish markets of Rio de Janeiro State (State
Government of Rio de Janeiro 2013).
Sampling points
Four sampling campaigns including three surface water
sampling points (SP1 - 22°56'2.31” S, 43° 0'31.51” W; SP2
- 22°56'19.68” S, 43° 1'19.9” W and SP3 - 22°56'47” S,
43°2'5” W) distributed along the river were conducted with
132
Fig. 1 João Mendes river basin and sampling points SPref (reference area), SP01, SP02 and SP03. Niteroi, Rio de Janeiro State, Brazil
intervals of two months (Fig. 1). An additional sampling
point considered not affected by the anthropogenic pollu-
tion (i.e., sewage discharge) at the source of the João
Mendes river was included as the reference area (SP0 - 22°
54'45” S, 42°59'42” W). Each water sample (collected five
centimeters below the water surface) was divided into
several subsamples and appropriately stored and preserved
for chemical analysis (1.2 μm glass fiber membrane filtra-
tion and SPE extraction) and ecotoxicity assays (frozen at
−20 °C for up to 2 months), according to the recommended
national guidelines (ANA/CETESB 2011). The physical-
chemical parameters (Table S1) were analyzed following
the standard methods described in the American Public
Health Association (APHA 2012).
Chemical risk index (ChemRI)
Five chemical substances of interest (CSI) were selected for
the Chemical Line of Evidence: 17α-ethynylestradiol (EE2,
CAS: 57-63-6), a synthetic hormone used in contraceptives
and hormone replacement therapy; Bisphenol A (BPA,
CAS: 80-05-7), a plasticizer widely used in packaging and
J. A. Sabino et al.
plastic bottles banned since 2011 from baby bottles, paci-
fiers and toys manufactured in Brazil, through the Resolu-
tion RDC 41/2011 (ANVISA 2011); Ibuprofen (IBU, CAS:
15687-27-1), the non-steroidal anti-inflammatory widely
consumed in Brazil; Trimethoprim (TMP, CAS: 738-70-5)
and Sulfamethoxazole (SMZ, CAS: 723-46-6), two anti-
biotics among the most commonly used in Brazil.
When the concentration of the CSI were below the Limit
of Quantification (LQ) of the method, as long as the CSI
had been quantified in other samples or campaigns, the
concentrations in the samples were estimated by using the
Eq. 1 (Gilbert 1987):
X ¼ LD þ ððLQ  LDÞ=2Þ ð1Þ
When the concentration of a CSI in a sample were found
below the Limit of Detection (LD) of the method, as long as
the CSI had been at least detected in other samples or
campaigns, the concentration was estimated applying the
Eq. 2 as follows:
X ¼ LD=2 ð2Þ
where: X = Estimated concentration.
Occurrence of organic micropollutants in an urbanized sub-basin and ecological risk assessment
The concentrations of the CSI found in each sampling
point were compared to the PNEC included in the Watch
List of Decision 2015/495/EU (European Parliament
2015) and Environment Canada, Health Canada (Envir-
onment Canada and Health Canada 2008), which are also
used by the Environmental Protection Agency (USEPA
2010). The threshold values selected were based on
international directives or legislation of several countries
and helped to establish the maximum permissible levels of
these compounds, according to international environ-
mental policies, which can be more or less restrictive
depending on the local water uses, environmental vul-
nerabilities and risks to human health (Buch et al. 2020).
The adopted threshold values were: 0.035 ng L−1 for EE2;
0.175 μg L−1 for BPA; 20.0 μg L−1 for IBU; 16.0 μg L−1
for TMP and 0.118 μg L−1 for SMZ.
The concentration of each CSI found in each water
sample was then subtracted by the background concentra-
tion found in the reference area (P0) (Dagnino et al. 2008).
The Chemical Risk due to the presence of the CSI was
calculated according to Jensen & Mesman (2006) modified
by Mendes et al. (2017) (Eqs. 3–5). Finally, for each sam-
pling point, data obtained in the previous step were summed
up to derive the toxic pressure coefficient (TPC), assuming
an additive effect of chemicals found in water samples.
Then, a ChemRI ranging from 0 to 1 was calculated
(Dagnino et al. 2008), being 0.00–0.25 very low risk;
0.25–0.50 moderate risk; 0.50–0.75 high risk; and >0.75
extreme risk.
R3 ¼ 1=ð1 þ expððlog R2  log R1ÞÞ=β
R4 ¼ ðR3  R3ref Þ=ð1  R3ref Þ

(Aristizabal-Ciro et al. 2017; Márta et al. 2018). The mass
R5 ¼ 1  ð1  R4Þ1 ð1  R4Þ2 ð1  R4Þ3 ¼ ¼ ¼ ð1  R4Þ n
Where: β = Default value established via ecotoxicological
assays is 0.4; R1 = Compound concentration in each
sampling point; R2 = Environmental Quality Standard
adopted; R3 = Toxic pressure per compound; R4 = Correc-
tion for background concentration value; R5 = Integrated
Chemical risk index.
Chromatographic analyses
Samples were filtered through glass fiber membranes
(1.2 μm) followed by solid phase extraction (SPE) and fil-
tration on polytetrafluoroethylene membrane (0.22 μm) for
subsequent UPLC-MS/MS injection.
The solid phase extraction of surface water samples
(500 mL, pH 3.0) was carried out using Bond Elut® C18
cartridge (500 mg, 3 mL, flow rate 3 mL min−1). The
133
cartridge was conditioned with 6 mL of hexane, 2 mL of
acetone, 6 mL of methanol and 10 mL of ultrapure water at
pH 3.0. Ten minutes after extraction, samples were kept in
the vacuum, until the cartridge was completely dried. Elu-
tion was then performed with 4 mL of methanol.
The analytes were identified and quantified using ultra-
performance liquid chromatography equipped with a Xevo
TQD MS/MS mass spectrometer (UPLC-MS/MS, Waters
ACQUITY®). UPLC separation was performed by injecting
5 μL of sample on a Waters Acquity UPLC BEH C18
column (2.1 × 50 mm, 1.7 μm particle size) at a temperature
of 50 °C. Chromatographic separation was achieved by
gradient elution at an flow rate of 0.4 mL min−1, starting
with 98% of phase A up to 4 min, being then reduced to 1%
of phase A up to 5 min, and finally returning and remaining
in the initial condition until the end of the run. Mobile
phases used during analysis were 0.01% ammonium
hydroxide in Milli-Q water (A) and in methanol (B). Total
run time was 8 min.
Electrospray ionization mass spectrometry (ESI-MS)
was performed on a Waters tandem quadrupole detector
(Xevo TQD) in positive and negative ionization multiple
reaction monitoring mode (MRM). Capillary voltage was
3.2 kV. Source temperature was 150 °C and the desolva-
tion temperature was 600 °C. The desolvation gas (nitro-
gen) and cone gas (nitrogen) were set to 1100 and 150 L h
−1
, respectively. Collision gas (argon) flow was 0.15 mL
min−1. Two MRM transitions (confirmation and quantifi-
cation) were monitored for the analytes of interest,
according to a cone voltage and collision energy optimized
ð3Þ for maximum intensity. Except for ibuprofen, which due to
the poor fragmentation under the conditions applied, only
ð4Þ one transition was available 205.1 m/z to 161.1 m/z
spectrometer method is summarized in Table 1. Data
ð5Þ acquisition and processing were performed using Mass
Lynx v 4.1 (Waters®).
The analytical parameters evaluated in the SPE-UPLC-
MS/MS method included calibration, recovery, repeat-
ability, limit of detection (LOD) and limit of quantification
(LOQ). In order to compensate for the matrix effects and to
reduce the signal suppression/enrichment phenomena
caused by the complexity of the matrix, the matrix-matched
standard method was used. This method of calibration is
also laborious and generally less expensive compared to the
internal standard (IS) (Ngumba et al. 2016). In addition, the
standard matrix matching method has a high sample
throughput that can be performed efficiently (Ngumba et al.
2016). In this way, the calibration curve and the evaluation
of the matrix effect were performed using the matrix-
matched standard method and using the reference area
matrix as a liquid medium (spiking surface water). The
linearity of the analytical method for the quantification of
134 J. A. Sabino et al.

Table 1 Optimized MS/MS parameters for the chemical substances of interest (CSI)
CSI Mode of ionization (ESI) Precursor (m/z) Quantitation Confirmation
Product (m/z) CE (V) Cone (V) Product (m/z) CE (V) Cone (V)

17α-ethynylestradiol − 295.0 145.0 43 60 159.0 33 60

Bisphenol A − 227.0 212.0 18 45 133.0 25 45
Ibuprofen − 205.1 161.1 6 20 – – –
Trimethoprim + 291.3 231.3 25 50 261.3 25 50
Sulfamethoxazole + 254.0 92.00 30 40 156.0 15 40
ESI electrospray ionization, CE collision energy

Table 2 UPLC‐MS/MS analytical quality control parameters

Analyte Linearity (μg L−1) R2 Standard Error RSD1 (%) RSD2 (%) LOD (μg L−1) LOQ (μg L−1) REC (%)

17α-ethynylestradiol 2.5–50.0 0.9923 0.0077 10.78 10.53 1.14 2.5 63.72

Bisphenol A 1.0–50.0 0.9918 0.0082 12.14 1,65 0.40 1.0 83.46
Ibuprofen 1.0–50.0 0.9939 0.0061 9.32 9.20 0.12 1.0 48.91
Trimethoprim 0.5–50.0 0.9984 0.0016 4.08 1.89 0.02 0.5 53.82
Sulfamethoxazole 0.1–50.0 0.9993 0.0007 10.19 6.04 0.02 0.1 2.37
R²: coefficient of determination; RSD1 relative standard deviation of repeatability, RSD2 relative standard deviation of intra-day, LOD limit of
detection, LOQ limit of quantitation, REC recovery

the compounds was evaluated through the construction of
an adjusted analytical curve. This curve was based on the
evaluation of the relative standard deviation of the data
(RSD ≤ 20%) and the determination of the linear correlation
coefficient (r ≥ 0.99) (ANVISA 2003). The recovery (REC
%) of the extraction was verified by water enriched in the
concentration range of the compounds of 20 μg L−1, in
500 mL, according to the analytical extraction method of
the present study. The accuracy of the method, in terms of
interday repeatability, met the recommendations for relative
standard deviation (RSD ≤ 20%, n = 10), as well as the
interday (n = 3) performed on three alternate days (Table 2).
The LOD was obtained from the calibration curve, using the
equation LOD = 3.3*σ/S, where σ is the standard deviation
of the residuals and S is the slope of the calibration curve.
LOQ was defined as the lowest concentration that can be
measured by the analytical curve (ICH 1995; IMETRO
2007). Acceptance criteria were that all concentrations
points comprising the linearity should have a coefficient of
variation (CV) less than 20%. The accuracy for each con-
centration level tested should be within 80.0–120.0%.
Ecotoxicological risk index (EtoxRI)
The Ecotoxicological Line of Evidence (Etox LoE) was
based on three chronic ecotoxicity assays with three dif-
ferent trophic level organisms (algae, microcrustacean and
fish) to obtain an Ecotoxicological Risk Index (EtoxRI).
An additional assay was conducted to evaluate the sam-
ples estrogenic activity. The chronic toxicity assay with
the microalgae Raphidocelis subcapitata followed the ISO
8692 (Baścik-Remisiewicz et al. 2011) and the national
ABNT-NBR 12.648 (ABNT 2018). The growth inhibitory
effect was evaluated with 96 h of exposure time, under
static condition nonrenewal technique. Four days before
conducting the assays, an initial algae culture was pre-
pared to ensure the exponential growth phase in the
beginning of the assays. The tests were carried out in
125 mL Erlenmeyer containing 75 mL of sample (n = 3)
and known density algal suspension (final density of 105
cells mL−1). The assays were carried out under continuous
cool white light 6000 lux, and 25 ± 2 °C. The cells
counting was performed under an optical microscope with
400x magnification, using the Neubauer chamber. The
growth inhibitory effect of algal biomass was statistically
evaluated by comparing the results of the exposed algae
with the negative controls. The chronic toxicity assay with
the microcrustacean Ceriodaphnia dubia followed the
ISO 20665 (2008) and ABNT NBR: 13.373 (ABNT
2017). The effect on reproduction rate was evaluated after
168 h (7 days) of exposure (n = 10) with no dilution. In
each vial of 20 mL were added 15 mL environmental
sample (test solution) or culture solution (negative con-
trol) and a female with 6 to 24 h old. The vials were
cleaned, and the neonates were counted daily. The assay
was conducted in a light: dark cycle of 16: 8 h photoperiod
incubator (cold light) at 25 °C, and organisms were fed
every two days with algal cultures. The assay was vali-
dated according to the criterion in the ISO 20665 (2008)
and ABNT NBR: 13373 (2017).
Occurrence of organic micropollutants in an urbanized sub-basin and ecological risk assessment
The chronic toxicity assay with the fish species Oreo-
chromis niloticus (Nile tilapia) followed the experimental
protocol described in our previous publications (Salomão
et al. 2014, 2015). The genotoxicity effect was evaluated
after 168 h (7 days) of exposure at 24 °C in pH 6.8; with
dissolved oxygen (DO) 6 mg L−1; and 14:10 h (light: dark)
photoperiod in 2 L aquariums. The initial length (7.4 ±
0.6 cm) and weight (14.2 ± 3.7 g) of the fish exposed had no
statistically significant differences (p < 0.05). After 168 h,
the blood samples were collected from the caudal vein using
syringes (BD Ultra-Fine™ 50 U 12.7 mm × 0.33 mm nee-
dle) rinsed with heparin. The slides were prepared by
smearing one drop of blood (10 μL) and after drying it was
submitted to a 15-minute bath in methanol (100%). After
being completely dried, the slides were stained for 10 min in
5% Giemsa. A total of 2000 erythrocytes were examined for
each specimen (n = 5) under oil immersion at ×1000
magnification with a microscope (Carrasco et al. 1990). The
frequencies of micronuclei (MN) were expressed per 1000
cells (‰). The slides were coded, randomized, and scored
by a single observer using a blind review. The MN observed
were classified (Carrasco et al. 1990; Fenech et al. 2003) as:
non-refractory particles, small nuclei with the same col-
oration as the nucleus cell and with a round or ovoid shape.
The determination of estrogenic activity was performed
with a recombinant yeast strain Saccharomyces cerevisiae
used in the Yeast Estrogen Screen (YES) assay (Routledge
and Sumpter 1996). Assays were performed in a laminar
flow hood. Pre-inoculum: prepared with 10 mL of culture
medium and 2 mL of precipitate of the thawed strain in a T-
tube (orbital shaker, 150 rpm, 48 h, 28 °C). Inoculum: pre-
pared with 10 mL of culture medium and 100 μL of pre-
inoculum in a new sterile T-tube (orbital shaker, 150 rpm,
24 h, 28 °C). Tube 1 (50 mL sterile Falcon tube): 4 mL of
culture medium and 3 mL of yeast inoculum added to obtain
approximately 4 ×107 cells (in a spectrophotometer at
620 nm; ideal absorbance ranging from 0.8 to 1.0). Tube 2
(50 mL sterile Falcon tube): 25 mL of the culture medium,
250 μL of CPRG (Chlorophenol Red-β-D-galactopyrano-
side, CAS Number: 99792-79-7 Sigma-Aldrich) solution
(10 mg mL−1) and 25 μL of the content of tube 1.
The analyses were performed in two sterile 96-well
microplates, flat bottomed with a lid and in duplicate. Plate
1 (dilutions plate): 100 μL of methanol (HPLC grade) was
added to the wells of the sample dilution row, with the
exception of the first well, in which, 200 μL of the extract of
the sample was added. In the second well, 100 μL of the first
well was added and so on. The standard curve of
17β-estradiol (E2, 54.48 μg L−1, >98%, Sigma-Aldrich® in
methanol; HPLC grade, Tedia®), positive control, was used
as a reference in the assay, by repeating the same procedure
as the sample. Plate 2 (assay plate): 10 μL were transferred
from each well of Plate 1, in duplicate and the total
135
evaporation was awaited to proceed with the assay. Nega-
tive control was made by adding 10 μL of methanol and
waiting for complete evaporation. After that, 200 μL of the
analysis medium (tube 2) were added to each well. The
plates were shaken for 2 min and incubated (72 h, 30 °C).
After 72 h, each well was read for absorbance at 575 and
620 nm (SoftMax, SpectraMax M3).
Bioassay results from each sample point were compared
with those obtained from exposure to samples obtained at
the reference area (SP0). When the difference between the
sample and SP0 was statistically not significant (p < 0.05),
risk values were set equal to zero. When the difference was
statistically significant, results of each bioassay were cal-
culated using the Eqs. 6–10 (Jensen and Mesman 2006), to
obtain an Ecotoxicological Risk Index (EtoxRI) ranging
from 0 to 1, being 0.00–0.25 very low risk; 0.25–0.50
moderate risk; 0.50–0.75 high risk; and> 0.75 extreme risk.
R1 ¼ X=100 ð6Þ
R2 ¼ ðR1  Ref Þ=ð1  Ref Þ ð7Þ
R3 ¼ logð1  R2Þ ð8Þ
R4 ¼ ðR31 þ ¼ R3n Þ=n ð9Þ
R5 ¼ R1  ð10^ R4Þ ð10Þ
Where: X = Ecotoxicity results; Ref. = Values obtained in
the reference site; R1 = Division of percentual results by
100; R2 = Risk values; R3 = Transformation of the values;
R4 = Combined risk of ecotoxicity assays; R5 = Integrated
Ecotoxicological risk index.
Integrated environmental risk index (EnvRI)
Results from the Chemical LoE and Ecotoxicological LoE
were integrated to estimate the Integrated Risk Index
(EnvRI) (Eqs. 11–13). The integration procedure did not
include any weighting factor (wf) to the indexes and
therefore, the EnvRI was calculated as the mean value of the
two indexes (Jensen and Mesman 2006).
R1 ¼ ð1  log XÞ ð11Þ
R2 ¼ ððR1ChemicalLoE  AÞ þ ðR1EtoxLoE  BÞÞ=A þ B
ð12Þ
R3 ¼ 1  ð10^ R2Þ ð13Þ
Where: R1 = Transformation of the Risk values of each
LoE; X = Risk value between 0 and 1 associated with each
LoE; R2 = Weighted average value of each R1 of the two
136 J. A. Sabino et al.

LoE; A = Chemical LoE weighting factor; B = Ecotoxico-
logical LoE weighting factor; R3 = Final Environmental
Risk Index.
Statistical analyses
Chronic toxicity assays with R. subcapitata and C. dubia
were evaluated by comparing the collected samples with the
control, using the GraphPad Prism 5 (v. 5.02 for Windows,
San Diego, USA) with one-way analysis of Variance fol-
lowed by Dunnett’s Multiple Comparison Test (p < 0.05). The
EC50 results and the equivalent estrogen (EQ-E2) from the
Yeast Estrogen Screen (YES) assays were calculated using
Origin v. 8.0 (do Nascimento et al. 2018; Saggioro et al.
2019). For genotoxicity assays (micronucleus frequency) with
O. niloticus, the statistical comparison between the control
group and collected samples was performed using the
GraphPad Prism 5 (v. 5.02 for Windows, San Diego, USA)
with non-parametric Mann–Whitney test (p < 0.05).
Correlation analysis and hierarchical cluster analysis
were performed to test relationships between study vari-
ables (integrated risk, chemical substances of interest and
test organisms). To interpret the dataset by reducing their
dimensionality in an interpretable way, such that most of the
information in the data is preserved (Jollife and Cadima
2016), a principal component analysis (PCA) - descriptive
tool which does not need any distributional assumption -
was applied to the results obtained using a correlation
matrix. Loading of each variable contributes to the original
data for the major components and allows grouping of data
with similar behaviors. Both statistical analyses were per-
formed using R software (version 3.0.1).
Results and discussion
Chemical risk index (ChemRI)
Most of the samples showed high concentrations for the CSI
during the four campaigns (Table 3) when compared to the
adopted PNEC values from Canada and Europe directives.
Except for the reference area, EE2 was found at a very high
concentration in all samples (from 16.1 μg L−1 to 32.2 μg L
−1
) compared to the global environmental concentration for
surface waters reported in the literature (usually less than
0.001 up to 0.3 μg L−1) (Laurenson et al. 2014; Reyhanian
Caspillo et al. 2014; Dias et al. 2015; Valdés et al. 2015;
Gárriz et al. 2017).
In Brazil the concentration of EE2 in treated effluents has
been found in the range of <0.02–5.2 μg L−1 (Aquino et al.
2013). Raw sewage (domestic sewage from condominiums)
in Minas Gerais state, for instance, has shown EE2 con-
centrations up to 175 μg L−1, with an average of 86 μg L−1
(Quaresma et al. 2018). Therefore, the high concentrations
of EE2 in João Mendes river are probably related to the
discharge of untreated domestic sewage generated by the
population living in the drainage basin.
Among the CSI, EE2 was the main compound to con-
tribute to a high toxic pressure of chemicals (TPC) (>0.99,
extreme risk) in all campaigns and sampling points; fol-
lowed by SMZ (0.25 to 0.96) posing extreme risk to most
sampling points during the 4 campaigns and the BPA (0.12
to 0.83) posing high risk to most sampling points (Fig. 2).
The IBU and TRI presented low risk (<0.25) to most
sampling points.
High concentrations of EE2 (5.3–12.1 μg.L−1) were
also found at the Guanhe river, the main source of
drinking water for residents of northern Jiangsu Province
in China (Han et al. 2019). Similarly, to the present study,
EE2 was one of the main compounds contributing to the
high ecological risk (equivalent to the extreme risk in the
present study). Recently, the concentration of EE2 in the
Paranoá Lake (an artificial reservoir located in the Brazi-
lian Federal District) was detected ranging from 3.7 to
5.6 ng L−1 and the risk quotients indicated the potential
for adverse effects to human health and the environment
(Sodré and Sampaio 2020).
Sulfamethoxazole was found in the surface water of
Atibaia river, in São Paulo, Brazil at the concentration of
0.1 μg L−1 (Montagner and Jardim 2011). In Europe,

Table 3 17α-ethynylestradiol (EE2), Bisphenol A (BPA), Ibuprofen (IBU), Trimethoprim (TMP) and Sulfamethoxazole (SMZ) found in João
Mendes river surface water (μg L−1), in three sampling points (SP1, SP2 and SP3) and reference area (SPref) in four campaigns (n = 4)
CSI Campaign 1 Campaign 2 Campaign 3 Campaign 4
SPref SP1 SP2 SP3 SPref SP1 SP2 SP3 SPref SP1 SP2 SP3 SPref SP1 SP2 SP3

EE2 ND 20.6 27.9 32.2 ND 25.6 17.0 20.6 ND 19.9 17.3 21.4 ND 30.3 17.4 16.1
BPA 0.10 0.77 1.16 0.80 0.03 0.19 0.22 0.33 0.17 0.21 0.28 0.26 0.03 0.28 0.46 0.34
IBU < LOQ 4.6 4.2 3.0 <LOQ 3.9 2.7 1.3 <LOQ 1.8 2.4 2.1 <LOQ 10.7 3.9 2.1
TRI < LOQ 0.04 <LOQ 0.05 <LOQ 0.03 0.06 0.01 <LOQ 0.06 0.05 0.02 <LOQ 0.06 0.04 0.03
SMZ < LOQ 1.75 0.04 0.29 <LOQ 0.14 0.81 0.18 <LOQ 1.00 0.60 0.89 <LOQ 1.59 1.98 2.42

ND: not detected; <LOQ: under Limit of Quantification of the method (Table 2). Values in bold exceed the PNEC values (Environment Canada
and Health Canada 2008; European Parliament 2015)
Occurrence of organic micropollutants in an urbanized sub-basin and ecological risk assessment 137

Fig. 2 Chemical, Ecotoxicological and Integrated Risk Assessment for João Mendes river. Three sampling points (SP1- SP3) and four campaigns
(C1 – C4)

Table 4 Ecotoxicological Risk Index (0.0–1.0), based on the Ecotoxicological LoE and the Equivalent Estrogen (EQ-E2 in μg L−1) of surface
water from three sampling points (SP1, SP2, SP3) and reference area (SPref) and four campaigns

Campaign 1 Campaign 2 Campaign 3 Campaign 4

SPref SP1 SP2 SP3 SPref SP1 SP2 SP3 SPref SP1 SP2 SP3 SPref SP1 SP2 SP3
R. subcapitata 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.44 0.55 0.50 0.0 0.64 0.68 0.56
C. dubia - - - - 0.0 0.99 0.99 0.99 0.0 0.99 0.99 0.99 0.0 1.0 1.0 1.0
O. niloticus 0.0 0.78 0.75 0.71 0.0 0.0 0.0 0.2 0.0 0.67 0.67 0.80 0.0 0.7 0.7 0.8
YES 0.0 0.99 0.99 1.0 0.0 1.0 0.99 0.99 0.0 0.99 0.98 0.99 0.0 0.99 0.99 0.99
YES EQ-E2
0.02 0.53 0.70 0.45 0.04 0.41 0.43 0.30 0.02 0.38 0.86 0.32 0.09 0.39 1.07 0.83
(µg L-1)

In green: low risk; in yellow: moderate risk; in orange: high risk; in red: extreme risk

sulfamethoxazole was detected and quantified within the
ranges: 0.01–2.0 μg L−1 in wastewater treatment plants
effluents and; 0.03–0.48 μg L−1 in environmental waters
(Hirsch et al. 1999). In the present study, sulfamethoxazole
was detected at higher levels in João Mendes River than
these mentioned values. Actually, the concentrations
reported in surface water in the present study were similar to
those detected in the WWTP effluents in Europe (Hirsch
et al. 1999).
BPA has been found in Brazilian surface waters at
concentrations ranging from 0.0012 to 64.2 μg L−1 (Ghiselli
2006; do Nascimento et al. 2018; Peteffi et al. 2019), while
in the present study, the range was 0.03–1.16 μg L−1. In
almost all samples, BPA was detected in higher levels than
the PNEC established in Canadian and European directives.
This means the João Mendes river ecosystem has been
exposed to concentrations which are expected to have
deleterious effects.
Trimethoprim and Ibuprofen were respectively detec-
ted and quantified in Brazilian water bodies at con-
centrations ranging from 0.0006 to 1.6 μg L−1 and 0.0005
to 4.15 μg L−1, respectively (do Nascimento et al. 2018).
In the present study, both compounds were detected and
quantified, respectively, raging from <0.02 (LOQ) to
0.06 μg L−1 and <0.12 (LOQ) to 10.7 μg L−1, values that
were lower than PNECs from Canada and Europe
regulations.
Ecotoxicological Risk Index (EtoxRI)
Chronic bioassays with the microalgae R. subcapitata
showed no inhibition effect for the first and second cam-
paigns. Photosynthetic algae growth stimulation was
observed in the samples, compared to the negative control
and reference area (SPref), probably due to the presence of
nutrients in the surface water (domestic wastewater con-
tamination). However, the third and fourth campaigns
registered growth inhibition, resulting in high risk
(0.5–0.75) for all sampling points (SP), excepting the
reference area (Table 4).
Chronic bioassay with the micro-crustacean C. dubia
was not performed for the first campaign. However, the
138
three following campaigns registered extreme risk (>0.99)
for all sampling points, excepting the reference area (Table
4). This extreme risk was based on the mortality of most
organisms exposed to the environmental samples. The
number of neonates in samples from the reference area was
statistically not different from those in the negative control.
Similar results are registered in the literature (Schroder et al.
2008) that evaluated the toxic effects on C. dubia and
D. magna exposed to effluents from MWWTP. In that
investigation, Schroder et al. (2008) observed 100% mor-
tality of C. dubia, while D. magna was less sensitive to the
effluent. C. dubia was more sensitive than D. magna and
more affected by the physical-chemical conditions of the
cultivation medium.
Genotoxic effect was observed in all samples (p <
0.05), excepted for the reference area (Table 4), and were
considered with high (0.5–0.75) and extreme (>0.75) risk.
Similarly, higher frequency of micronuclei has been
reported in O. niloticus exposed to surface water,
domestic and municipal wastewater, which could be also
explained by the presence of some endocrine disruptors
(Salomão et al. 2015). According to Du et al. (2017) the
continuous release of pharmaceuticals, such as antibiotics
for human and/or veterinary use in aquatic environments
has increased the possibility of bioaccumulation and
biomagnification in aquatic organisms. Multiple exposure
of these organisms to a mixture of contaminants, some-
times in concentrations below their individual LOEC, can
lead to additive, synergistic or antagonistic effects, which
increases the ecological risks for these organisms (Salo-
mão et al 2020).
The determination of estrogenic activity in the water
samples of João Mendes river using the recombinant yeast
strain S. cerevisiae in the Yeast Estrogen Screen (YES)
assay demonstrated no effect or estrogenic activity for the
reference area (SPref), when compared to the negative
control. However, the three sampling points (SP1, SP2,
SP3) showed high estrogenic activity in all campaigns.
Additionally, at higher concentrations (100%, and in some
cases, even 50%), the water samples were cytotoxic to the
tested yeast, which contributed to the extreme level of risk
(Table 4). Because EE2 and BPA are present at high
concentrations (Table 3) and both are well-known endo-
crine disruptors (Salomão et al. 2014; Li et al. 2015), it is
likely that these compounds together are responsible for
most of the positive estrogenic activity observed (Table 4).
In addition, there are several other natural and synthetic
hormones (e.g. progesterone, estrone, estradiol, levo-
norgestrel among others) that occur in domestic waste-
water, which were not analyzed in the present study could
contribute to the high estrogenic activity. Comtois-Marotte
et al. (2017) evaluated the EQ-E2 in two WWTP effluents
in Canada using the YES assay and the results ranged from
J. A. Sabino et al.
0.04 to 0.72 μg L−1 EQ-E2. The EQ-E2 values found in the
present study (Table 4) were similar or in some cases
higher than some found in the literature (Comtois-Marotte
et al. 2017), confirming that João Mendes river presents a
high degree of contamination by sewage.
It has been reported in our previous publications that
EE2 in concentrations above 0.016 μg L−1 is already cap-
able of causing endocrine disrupting effects in O. niloticus
males (Salomão et al. 2014), increasing the alkali-labile
phosphate (ALP) levels in plasma, which indicates
increasing synthesis of vitellogenin, a protein related to
reproduction in female fish. It is not implausible to assume
that continuous male fish exposure to high levels of endo-
crine disruptors might cause infertility and consequently,
extinction of more sensitive fish species. It is worth men-
tioning that in the present investigation the EE2 con-
centrations observed between 16 and 32 μg L−1 (sampling
point close to the lagoon) represent 1000 up to 2000 times
more than the minimum required to promote detectable
endocrine disruption in O. niloticus. This threat is posed to a
coastal area with important artisanal fishing activity that
supplies not only the local population, but also the largest
fish market in the Rio de Janeiro State.
Environmental Risk Index (EnvRI)
The integration of the two LoE (risk index) for all sam-
pling points and throughout the sampling period resulted
in extreme EnvRI (Fig. 2) for the surface waters of the
João Mendes river. This extreme risk reflects the lack of
sanitation and proper treatment of domestic sewage prior
to discharge into the water body, a prevalent problem
affecting many drainage basins. This level of pollution is
likely to pose high risks to the local population, tourists
and bathers and the population that consumes the products
of fishing.
Regarding the Correlation matrix (Fig. 3a) it was
observed that EE2 was strongly correlated to two assays: O.
niloticus genotoxicity assay and estrogenicity assay (YES).
Additionally, these three variables were strongly correlated
to the EnvRI.
It was also possible to confirm the degree of preservation
of the reference area (SPref, near the João Mendes river
source) in all samplings, by explaining the eigenvalues of
Component 2 (16.9% of the variance) represented by the
distance between groups I and II (Fig. 3b).
The PCA (correlation and hierarchical cluster analysis)
for the selected variables revealed the latent relationships
among all the sampling points (excepting the reference
area), confirming our analytical results. The points
affected by the Component 1 axis assessment (64.4% of
Variance) include the list of variables that contribute to
the EnvRI.
Occurrence of organic micropollutants in an urbanized sub-basin and ecological risk assessment 139

Fig. 3 João Mendes river,

Niteroi, RJ, Brazil: (a)
Correlation matrix and
hierarchical cluster analyses
with variables of the Ecological
risk assessment. Higher
correlation coefficients were
represented with elongated
shapes and dark colors; Lower
correlation coefficients were
represented with circular shapes
and light colors; Oval and
orange shapes represent
intermediate correlations (b)
PCA diagram for the study
variables at each sampling point.
The groupings identified by the
analysis are circled. SPref
reference area (blue circles);
SP1 sampling point 1 (orange
square); SP2 sampling point 2
(red triangle); SP3 sampling
point 3 (hollow squares). EE2:
17α ethynylestradiol; BPA:
Bisphenol A; IBU: Ibuprofen;
TMP: Trimethoprim and SMZ:
Sulfamethoxazole

Conclusion low concentrations of CSI with no significant differences

The ecological risk assessment carried out in the present
investigation demonstrated that João Mendes river located
in an Environmental Protection Area (APA) in Niteroi
municipality, part of an extractive reserve RESEX - created
to preserve marine biodiversity and sustain the local arti-
sanal fishing - is under extreme risk. Without basic sanita-
tion to promote adequate domestic sewage treatment, the
local ecosystem is threatened by emerging micropollutants
and endocrine disrupters, which in turn, might represent a
serious risk to the local population and consumers of the
fishing products, something yet to be investigated.
The integration of the chemical and ecotoxicological risk
index was essential to assess the extreme risk for this eco-
system affected by the sewage release, specially the high
concentration of EE2, BPA and SMZ. The comparison
between CSI concentrations and threshold values from
different Directives helped to verify the possible water
quality degradation and extreme risks to ecological health.
The surface water samples from the reference area presented
when compared to the controls of the ecotoxicology
bioassays and therefore, suitable to be used as a reference
for calculating risks.
Based on the type of micropollutants and the toxicity
assays carried out, the extreme ecological risk level is
related to the unplanned urban occupation of the region and
lack of adequate sanitation infrastructure. The results sug-
gest that urgent measures and remediation actions are
necessary to restore, as much as possible the ecosystem
integrity.
Funding We acknowledge the financial support by the Financing
Agency of Studies and Project (FINEP 01.14.0081.00), the National
Council for Scientific and Technological Development (CNPq
308.335/2017-1) and the Carlos Chagas Filho Research Support
Foundation (FAPERJ E-26/202.894/2018).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
140
Ethical approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed. This
project was approved by the Rio de Janeiro State University – UERJ
Ethics Committee for the Care and Use of Experimental Animals
(CEUA), under protocol 021/2018.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
ABNT-Associação Brasileira de Normas Técnicas (2018) Aquatic
ecotoxicology-Chronic toxicity-Test method with algae (Chlor-
ophyceae). NBR 12648 (original in Portuguese), Brazil
ABNT-Associação Brasileira de Normas Técnicas (2017) Aquatic
ecotoxicology-Chronic toxicity-Test method with Ceriodaphnia
spp (Cruatacea, Cladocera). NBR 13373 (original in Portu-
guese), Brazil
APHA-American Public Health Association (2012) Standard Methods
for Examination of Water and Wastewater, 22nd. Ed. American
Public Health Association, American Water Works Association,
Water Environment Federation
Aquino S, Brandt E, Chernicharo A (2013) Removal of pharmaceu-
ticals and endocrine disrupters in sewage treatment plants: lit-
erature review Remoção de fármacos e desreguladores endócrinos
em estações de tratamento de esgoto: revisão da literatura. Eng
Sanit Ambient 18:187–204
Aristizabal-Ciro C, Botero-Coy AM, López FJ, Peñuela GA (2017)
Monitoring pharmaceuticals and personal care products in
reservoir water used for drinking water supply. Environ Sci Pollut
Res 24:7335–7347. https://doi.org/10.1007/s11356-016-8253-1
Baścik-Remisiewicz A, Aksmann A, Żak A et al. (2011) Toxicity of
cadmium, anthracene, and their mixture to Desmodesmus sub-
spicatus estimated by algal growth-inhibition ISO standard test.
Arch Environ Contam Toxicol 60:610–617. https://doi.org/10.
1007/s00244-010-9585-3
Boillot C, Martinez Bueno MJ, Munaron D et al. (2015) In vivo
exposure of marine mussels to carbamazepine and 10-hydroxy-
10,11-dihydro-carbamazepine: bioconcentration and metaboliza-
tion. Sci Total Environ 532:564–570. https://doi.org/10.1016/j.
scitotenv.2015.05.067
Buch AC, Niemeyer JC, Marques ED, Silva-Filho EV (2020) Ecolo-
gical risk assessment of trace metals in soils affected by mine
tailings. J Hazard Mater in press: https://doi.org/10.1016/j.jhazma
t.2020.123852
Carrasco KR, Tilbury KL, Myers MS (1990) Assessment of the pis-
cine micronucleus test as an in situ biological indicator of che-
mical contaminant effects. Can J Fish Aquat Sci 47:2123–2136.
https://doi.org/10.1139/f90-237
Chapman PM (1990) The sediment quality triad approach to deter-
mining pollution-induced degradation. Sci Total Environ
98:815–825
Comtois-Marotte S, Chappuis T, Duy SV et al. (2017) Analysis of
emerging contaminants in water and solid samples using high
resolution mass spectrometry with a Q exactive orbital ion trap
and estrogenic activity with YES-assay. Chemosphere
166:400–411
Cunha DL, Mendes MP, Marques M (2019) Environmental risk
assessment of psychoactive drugs in the aquatic environment.
Environ Sci Pollut Res 26:78–90. https://doi.org/10.1007/s11356-
018-3556-z
Dagnino A, Sforzini S, Dondero F et al. (2008) A ‘“weight-of-evi-
dence”’ approach for the integration of environmental ‘“triad”’
data to assess ecological risk and biological vulnerability. Integr
Environ Assess Manag 4:314–326. https://doi.org/10.1897/IEAM
J. A. Sabino et al.
Dias ACV, Gomes FW, Bila DM et al. (2015) Analysis of estrogenic
activity in environmental waters in Rio de Janeiro state (Brazil)
using the yeast estrogen screen. Ecotoxicol Environ Saf
120:41–47. https://doi.org/10.1016/j.ecoenv.2015.05.013
do Nascimento MTL, Santos AD, de O, Felix LC et al. (2018)
Determination of water quality, toxicity and estrogenic activity in
a nearshore marine environment in Rio de Janeiro, Southeastern
Brazil. Ecotoxicol Environ Saf 149:197–202. https://doi.org/10.
1016/j.ecoenv.2017.11.045
Du J, Zhao H, Liu S et al. (2017) Antibiotics in the coastal water of the
South Yellow Sea in China: Occurrence, distribution and ecolo-
gical risks. Sci Total Environ 595:521–527. https://doi.org/10.
1016/j.scitotenv.2017.03.281
Environment Canada, Health Canada (2008) Screening assessment of
phenol, 4,4’ -(1-methylethylidene)bis-or bisphenol A
European Medicines Agency (2018) Guideline on the environmental
risk assessment of medicinal products for human use. United
Kingdom
European Parliament (2015) Watch list of substances for Union-wide
monitoring in the field of water policy (2015/495). European
Union
Fenech M, Chang WP, Kirsch-Volders M et al. (2003) HUMN project:
detailed description of the scoring criteria for the cytokinesis-
block micronucleus assay using isolated human lymphocyte
cultures. Mutat Res 534:65–75
Gárriz Á, del Fresno PS, Miranda LA (2017) Exposure to E2 and EE2
environmental concentrations affect different components of the
Brain-Pituitary-Gonadal axis in pejerrey fish (Odontesthes
bonariensis). Ecotoxicol Environ Saf 144:45–53. https://doi.org/
10.1016/j.ecoenv.2017.06.002
Ghiselli G (2006) Potable water quality in the Campinas region:
occurrence and determination of endocrine disruptors (EDs) and
pharmaceuticals and personal care products (PPCPs) (original in
Portuguese). Universidade Estadual de Campinas
Gilbert RO (1987) Statistical methods for environmental pollution
monitoring. Van Nostrand Reinhold Company, New York, NY
Han Z, Lin L, Fan Y, Zou C (2019) Environmental risk assessment of
the emerging EDCs contaminants in Guan river of Jiangsu in
China. Acad J Sci Res 7:363–369. https://doi.org/10.15413/ajsr.
2018.0474
Hirsch R, Ternes T, Haberer K, Kratz KL (1999) Occurrence of
antibiotics in the aquatic environment. Sci Total Environ
225:109–118. https://doi.org/10.1016/S0048-9697(98)00337-4
Instituto Nacional de Metrologia-IMETRO (2007) Guidance on vali-
dation of chemical test methods (DOQ-CGCRE-008) (original in
Portuguese). Brazil
International Conference on Harmonisation (ICH) (1995) Validation
of Analytical Procedures. Methodology, Q2 (CPMP/ICH/381/
95), London
International Organization for Standardization - ISO (2008) Interna-
tional Standard: Water quality—Determination of chronic toxi-
city to Ceriodaphnia dubia. Switzerland
Jensen J, Mesman M (2006) Ecological Risk Assessment of Con-
taminated Land-Decision support for site specific investigation.
Rijksinstituut voor Volksgezondheid en Milieu RIVM
Jollife IT, Cadima J (2016) Principal component analysis: A review
and recent developments. Philos Trans R Soc A Math Phys Eng
Sci 374: https://doi.org/10.1098/rsta.2015.0202
Kolluru R, Bartell S, Pitblado R, Stricoff S (1998) Manual de eva-
luación y administración de riesgos. McGraw-Hill, Méxic, D.F.
Laurenson JP, Bloom RA, Page S, Sadrieh N (2014) Ethinyl estradiol
and other human pharmaceutical estrogens in the aquatic envir-
onment: a review of recent risk assessment data. AAPS J
16:299–310. https://doi.org/10.1208/s12248-014-9561-3
Li L, Wang Q, Zhang Y et al. (2015) The molecular mechanism of
bisphenol A (BPA) as an endocrine disruptor by interacting with
Occurrence of organic micropollutants in an urbanized sub-basin and ecological risk assessment
nuclear receptors: insights from molecular dynamics (MD)
simulations. PLoS ONE 10:1–18. https://doi.org/10.1371/journal.
pone.0120330
Long ER, Chapman PM (1985) A sediment quality triad: measures of
sediment contamination, toxicity and infaunal community com-
position in Puget Sound. Mar Pollut Bull 16:405–415
Márta Z, Bobály B, Fekete J et al. (2018) Simultaneous determination
of ten nonsteroidal anti-inflammatory drugs from drinking water,
surface water and wastewater using micro UHPLC-MS/MS with
on-line SPE system. J Pharm Biomed Anal 160:99–108. https://
doi.org/10.1016/j.jpba.2018.07.016
Mendes MP, Salomão ALS, Niemeyer JC, Marques M (2017) Eco-
logical risk assessment in a tropical wetland contaminated with
gasoline: tier 1. Hum Ecol Risk Assess 23:992–1007. https://doi.
org/10.1080/10807039.2017.1294477
Montagner CC, Jardim WF (2011) Spatial and seasonal variations of
pharmaceuticals and endocrine disruptors in the Atibaia River,
São Paulo State (Brazil). J Braz Chem Soc 22:1452–1462. https://
doi.org/10.1590/S0103-50532011000800008
Monteiro-Neto C, Tubino RA, Moraes LES et al. (2008) Fish asso-
ciations in the coastal region of Itaipu, Niterói, RJ (original in
Portuguese). Iheringia Série Zool 98:50–59. https://doi.org/10.
1590/S0073-47212008000100007
National Health Surveillance Agency-ANVISA (2011) RESOLU-
TION - RDC No 41 (original in Portuguese). Brazil
National Health Surveillance Agency-ANVISA (2003) Guide for
validation of analytical and bioanalytical methods (original in
Portuguese). Brazil
Ngumba E, Kosunen P, Gachanja A, Tuhkanen T (2016) A multi-
residue analytical method for trace level determination of anti-
biotics and antiretroviral drugs in wastewater and surface water
using SPE-LC-MS/MS and matrix-matched standards. Anal
Methods 8:6720–6729. https://doi.org/10.1039/c6ay01695b
Niemeyer JC, Moreira-Santos M, Ribeiro R et al. (2015) Ecological
risk assessment of a metal-contaminated area in the tropics. Tier
II: Detailed assessment. PLoS ONE 10:1–25. https://doi.org/10.
1371/journal.pone.0141772
Peteffi GP, Fleck JD, Kael IM et al. (2019) Ecotoxicological risk
assessment due to the presence of bisphenol A and caffeine in
surface waters in the Sinos River Basin - Rio Grande do Sul -
Brazil. Brazilian J Biol 79:712–721. https://doi.org/10.1590/
1519-6984.189752
Prefeitura de Niterói, AGRAR (2014) Environmental Impact Report-
TransOceanic Road Corridor. Niterói (original in Portuguese -
Relatório de Impacto Ambiental – Corredor Viário TransOceâ-
nica), Niterói
Quaresma A, de V, Floripes TC, Souza CLde et al. (2018) Occur-
rence of drugs and endocrine disruptors in raw and treated
sewage in the city of Belo Horizonte (MG) [original in Portu-
guese - Ocorrência de fármacos e desreguladores endócrinos em
esgoto bruto e tratado na cidade de Belo Horizonte (MG)]. Eng
Sanit e Ambient 23:1199–1211. https://doi.org/10.1590/s1413-
41522018177703
Rawat I, Ranjith Kumar R, Mutanda T, Bux F (2011) Dual role of
microalgae: Phycoremediation of domestic wastewater and bio-
mass production for sustainable biofuels production. Appl Energy
88:3411–3424. https://doi.org/10.1016/j.apenergy.2010.11.025
Reyhanian Caspillo N, Volkova K, Hallgren S et al. (2014) Short-term
treatment of adult male zebrafish (Danio rerio) with 17α-ethinyl
estradiol affects the transcription of genes involved in develop-
ment and male sex differentiation. Comp Biochem Physiol Part -
C Toxicol Pharmacol 164:35–42. https://doi.org/10.1016/j.cbpc.
2014.04.003
141
Routledge EJ, Sumpter JP (1996) Estrogenic activity of surfactants and
some of their degradation products assessed using a recombinant
yeast screen. Environ Toxicol Chem 15:241–248
Saggioro EM, Chaves FP, Felix LC et al. (2019) Endocrine disruptor
degradation by UV/Chlorine and the impact of their removal on
estrogenic activity and toxicity. Int J Photoenergy 2019:1–9.
https://doi.org/10.1155/2019/7408763
Salomão AL de S, Hauser-Davis RA, Marques M (2020) Critical
knowledge gaps and relevant variables requiring consideration
when performing aquatic ecotoxicity assays. Ecotoxicol Environ
Saf 203: https://doi.org/10.1016/j.ecoenv.2020.110941
Salomão AL, de S, Marques M (2015) Estrogenicity and genotoxicity
detection in different contaminated waters. Hum Ecol Risk
Assess 21:1793–1809. https://doi.org/10.1080/10807039.2014.
987027
Salomão AL, de S, Marques M (2014) Quantification of alkali-labile
phosphate groups in the plasma of Oreochromis niloticus
exposed to intermittent discharges of estrogens: Effect of con-
centrations vs. loads. Int J Environ Anal Chem 94:1161–1172.
https://doi.org/10.1080/03067319.2014.930845
Sanchez LA (2012) Ecological risk assessment for the diagnosis of
environmental impacts in tropical freshwater ecosystems. Uni-
versidade Federal de São Carlos
Schroder GD, Ross‐Lewandowski S, Davis EM (2008) Evaluation of
the toxic effects of selected municipal wastewater effluents on
aquatic invertebrates. Environ Technol 12:757–768
da Silva ML, de Araújo FV, Castro RO, Sales AS (2015) Spatial-
temporal analysis of marine debris on beaches of Niterói, RJ,
Brazil: Itaipu and Itacoatiara. Mar Pollut Bull 92:233–236.
https://doi.org/10.1016/j.marpolbul.2014.12.036
Silva ML, da, Sales AS, Martins S et al. (2016) The influence of the
intensity of use, rainfall and location in the amount of marine
debris in four beaches in Niteroi, Brazil: Sossego, Camboinhas,
Charitas and Flechas. Mar Pollut Bull 113:36–39. https://doi.org/
10.1016/j.marpolbul.2016.10.061
Sodré FF, Sampaio TR (2020) Development and application of a
SPE-LC-QTOF method for the quantification of micropollutants
of emerging concern in drinking waters from the Brazilian
capital. Emerg Contam 6:72–81. https://doi.org/10.1016/j.
emcon.2020.01.001
Souza MS, Hallgren P, Balseiro E, Hansson L-A (2013) Low con-
centrations, potential ecological consequences: synthetic estro-
gens alter life-history and demographic structures of aquatic
invertebrates. Environ Pollut 178:237–243. https://doi.org/10.
1016/j.envpol.2013.03.038
State Government of Rio de Janeiro (2013) Decree no 44.417, Sep-
tember 30, 2013 - Creates the Itaipu Marine Extractive Reserve in
the city of Niterói (original in Portuguese - Decreto no 44.417 de
30 de setembro de 2013 - Cria a Reserva Extrativista Marinha de
Itaipu no Município de Niterói). Brazil
USEPA - U.S. Environmental Protection Agency (2010) Bisphenol A
Action Plan (CASRN 80-05-7) Accessed at: https://19janua
ry2017snapshot.epa.gov/sites/production/files/2015-09/
documents/bpa_action_plan.pdf
Valdés ME, Marino DJ, Wunderlin DAL et al. (2015) Screening
concentration of E1, E2 and EE2 in sewage effluents and surface
waters of the “Pampas” region and the “Río de la Plata” estuary
(Argentina). Bull Environ Contam Toxicol 94:29–33. https://doi.
org/10.1007/s00128-014-1417-0
Yu Y, Wu L (2012) Analysis of endocrine disrupting compounds,
pharmaceuticals and personal care products in sewage sludge by
gas chromatography-mass spectrometry. Talanta 89:258–63.
https://doi.org/10.1016/j.talanta.2011.12.023