Marine Pollution Bulletin xxx (xxxx) xxx–xxx

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Can we assess the ecological status of estuaries based on larval fish

assemblages?
Régis Vinícius Souza Santosa,⁎, Sandra Ramosb,c, Ana Cristina Teixeira Boneckera
a
Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Laboratório Integrado de Zooplâncton e Ictioplâncton, Av. Carlos Chagas Filho,
373, Prédio do CCS, Bloco A, 21.941-902 Rio de Janeiro, RJ, Brazil
b
CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, ECOBIOTEC, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal
c
Institute of Estuarine and Coastal Studies, University of Hull, Hull HU6 7RX, UK

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

Keywords: Fish larvae of four SE Brazilian estuaries were investigated to assess if the larval fish assemblages reflect the
Fish larvae ecological status of estuaries. All samples were collected in the same water mass to guarantee similar natural
Human pressures water parameters, assuring that major differences among estuaries were related to anthropogenic pressures.
Ecological quality assessment Water temperature, oxygen, pH, chlorophyll a, faecal coliforms, nutrient load and total particulate matter were
Estuaries
obtained at each sampling area. A pressure index was used to assess the overall anthropogenic pressures acting in
Brazil
each estuary. Results showed that fish larvae were sensitive to water contamination, reducing the diversity and
especially exhibiting a high dominance of few species. Furthermore, this study reinforced the idea that the high
sensitivity of fish larvae can increase the accuracy of the environmental assessments when tackling short-time
events of hydrological controls (physical barriers and control of the freshwater input), representing an advance
in the water ecological quality assessments.

1. Introduction communities (Whitfield and Elliott, 2002; Harrison and Whitfield,

Estuaries are essential fish habitats, compiling several functions
such as nursery grounds, migration routes and refuge areas for a variety
of fish species (Cattrijsse and Hampel, 2006). Nevertheless, numerous
anthropogenic perturbations affect estuarine environments (Elliott and
Whitfield, 2011), contributing to habitat alteration and changes in the
structure and dynamics of biotic communities (Kennish, 2002), com-
promising the long-term ecological function of these ecotones (e.g.
Whitfield and Elliott, 2002; Gilliers et al., 2006; Coates et al., 2007; Le
Pape et al., 2007). In response to the increasing degradation of marine
ecosystems observed worldwide (Halpern et al., 2007, 2008), several
studies have assessed the ecological status of aquatic ecosystems and
the impacts of anthropogenic activities (e.g. Borja et al., 2013; Ruaro
and Gubiani, 2013; Martinez-Haro et al., 2015), in order to trace ef-
fective management plans and mitigation actions in these ecosystems.
In estuarine environments, several groups of organisms have been
used as quality indicators of ecosystem health, mainly macro-
invertebrates and fishes (e.g. Harrison et al., 2000; USEPA, 2000;
Alvarez et al., 2013; Nebra et al., 2014; Alves et al., 2015). Ecological
indicators based on fish communities have been recognized as useful
tools to assess anthropogenic impacts affecting estuarine fish
2006; Borja and Dauer, 2008; Fonseca et al., 2013). Typically, a fish
community facing increased anthropogenic induced stress tends to
show a decreased of number and abundance of species with high ha-
bitat and environmental specificities, i.e. sensitive and low tolerance
fish species, while generalist species tend to increased dominance, re-
sulting in a simplified community structure (e.g. Odum, 1983; Karr and
Chu, 1999; Fonseca et al., 2013). Nevertheless, the use of fishes as in-
dicators of environmental quality can also have limitations because of
their high mobility, temporal variability (seasonality) and specific
sampling requirements (Whitfield and Elliott, 2002; Harrison and
Whitfield, 2004).
Given these limitations, early larval stages of fishes can be con-
sidered good indicators of ecosystem integrity, since they have reduced
sampling costs and sampling bias, low mobility (planktonic phase) and
sensitivity to reflect anthropogenic pressures acting on a water body
(Ramos et al., 2015). Indeed, fish larvae show a high vulnerability to
environmental changes (Ramos et al., 2006a; Eick and Thiel, 2014;
Strydom, 2015; Santos et al., 2017), highlighting their importance as
pollution indicators (Longwell et al., 1992; Gordina et al., 2001;
Westernhagen et al., 2001). Ramos et al. (2012) demonstrated that
estuaries with different levels of anthropogenic pressures exhibit

⁎
Corresponding author.
E-mail address: regisvinicius@gmail.com (R.V.S. Santos).

http://dx.doi.org/10.1016/j.marpolbul.2017.07.043
Received 1 February 2017; Received in revised form 3 July 2017; Accepted 17 July 2017
0025-326X/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Santos, R.V.S., Marine Pollution Bulletin (2017), http://dx.doi.org/10.1016/j.marpolbul.2017.07.043
R.V.S. Santos et al.
distinct larval fish assemblages, and also emphasized the potential role
of larval stages of fishes as indicators of hydrological pressures that are
difficult to measure (Ramos et al., 2015). Moreover, the use of fish
larvae to assess ecological conditions of estuaries integrates valuable
information about spawning and nurseries areas, and about con-
nectivity pathways between marine/riverine habitats.
Estuarine fish larvae assemblages continually vary in time and
space, according to reproductive seasons of the species and due to en-
vironmental fluctuations (Barletta-Bergan et al., 2002; Ramos et al.,
2006a; Montoya-Maya and Strydom, 2009; Strydom, 2015). Conse-
quently, the environmental conditions may interfere with ecological
quality assessment when fish larvae are used as quality indicators, as
well as temporal variability patterns associated with natural fluctua-
tions of species-specific reproductive seasons, considered as a major
driver of the estuarine larval fish assemblages (Rakocinski et al., 1996).
The Estuarine Quality Paradox (Elliott and Quintino, 2007), which
suggests that anthropogenic stress is difficult to detect in estuaries be-
cause their existing communities also show the natural variability, has
been reflected in many studies of ecological quality assessments (e.g.
Courrat et al., 2009; Dauvin and Ruellet, 2009; Ramos et al., 2015;
Tweedley et al., 2015). This is a particular problem in estuaries since
their natural environmental conditions vary both spatially and tempo-
rally (Sheaves, 2016). Thus, the constancy of environmental char-
acteristics is fundamental to the spatial comparisons of the category and
level of anthropogenic pressures, by removing the potentially con-
founding effects of natural variability on fish assemblages (Elliott and
Quintino, 2007). In this context, we tested the hypothesis that estuaries
with similar environmental characteristics, but distinct anthropogenic
pressures, carry distinctly different larval fish assemblages, by in-
vestigating the larval fish assemblages of four similar tropical estuaries
with different types and levels of human pressures.
2. Material and methods
2.1. Methodological approach
To test the hypothesis that estuaries with similar environmental
characteristics suffering from specific anthropogenic pressures com-
prise distinct larval fish assemblages, we selected four estuaries located
in the same biogeographic area of SE Brazil, in order to remove as-
semblage differences due to geographic location of the estuaries. In
addition, all samples were collected in the same water mass (salinity
15–25) to guarantee similar natural water parameters allowing inter-
2
Marine Pollution Bulletin xxx (xxxx) xxx–xxx
Fig. 1. Location of the four estuaries of the study. (1)
Macaé, (2) São João, (3) Bracuí and (4) Perequê-Açu, in Rio
de Janeiro State, SE Brazil.
estuarine comparisons. Considering that estuarine larval fish assem-
blages are highly seasonal (Ramos et al., 2006b; Arévalo-Frías and
Mendoza-Carranza, 2015; Strydom, 2015), we analyzed rainy/dry
seasons separately, ensuring that possible assemblages differences were
not related to seasonal influences. Hence, we restricted inter-estuarine
variability in terms of natural environmental conditions, assuring that
major differences among estuaries were related to type and level of
anthropogenic pressures. The anthropogenic pressures acting in the
estuaries were assessed based on contamination indicators, as nutrient
load (nitrate, nitrite, ammonia and phosphate) and sewage con-
tamination determined by microbial faecal coliforms contamination.
Also, a pressure index was calculated for each estuary based on human
activities and hydro-morphological pressures.
2.2. Estuarine models
Four estuaries of SE Brazil, Macaé, São João, Bracuí and Perequê-
Açu were investigated (Fig. 1). Macaé (22° 22′ S and 41° 46′ W) and São
João (22° 35′ S and 41° 59′ W) are larger systems directly connected to
the ocean with 130 km and 120 km in length, respectively; and total
area of 1765 km2 and 2160 km2, respectively. Bracuí (22° 57′ S and 44°
23′ W) and Perequê-Açu (23° 13′ S and 44° 42′ W) are smaller systems
with 32 km in length and a total area of 190 km2, and 22 km and area of
110 km2, respectively; connected to a coastal bay (Ilha Grande Bay).
The four estuaries are shallow (mean depth of 2–4 m), channel type,
microtidal and partially mixed, with semidiurnal regime.
The levels of anthropogenic pressures vary among the estuaries. In
Macaé and Perequê-Açu estuaries, the unplanned urbanization process
promoted the removal of mangroves in the estuarine region and dis-
charge large loads of organic matter and other compounds of human
waste without treatment directly into the water (INEA, 2014, 2015).
Both estuaries are used for navigation, mainly tour boats in Perequê-
Açu Estuary, which relies on constant dredging to improve navigability.
Macaé Estuary is used as navigation route for support vessels to oil and
gas drilling platforms. The freshwater input of Macaé River is highly
controlled by water abstraction for human consumption, industrial use
and to cool the power-producing equipment in thermoelectric plants
(INEA, 2014). Additionally, there are several dams, agricultural activ-
ities and commercial sand extraction along the Macaé River. São João
Estuary still holds mangrove areas in the middle and lower sections,
although there are agricultural and fishing activities that might re-
present a potential pressure. Also, the main river is obstructed by a
dam, which controls the river flow reaching the São João Estuary.
R.V.S. Santos et al.
Bracuí Estuary still has natural banks with mangrove areas without
hydro-morphological pressures, and human activities are limited to
some aquaculture and agricultural activities.
2.3. Sampling and sample processing
Sampling surveys were conducted every two months between May
2013 and March 2015. All samples were taken in the same salinity
range (15–25) during nightly ebb tides. Triplicates of surface trawls
were performed in each estuary with a Bongo net (500 μm) and the
volume of the water filtered was determined by a flowmeter (General
Oceanics®). At each sampling area, subsurface (0.5–1 m depth) water
temperature and pH were obtained by a multiparameter probe (Hach
HQ40d) and water samples were collected for further analyses.
Chlorophyll a and inorganic nutrients (nitrate, nitrite, ammonia and
phosphate) were determined following previously described methods
(Grasshoff et al., 1999). Oxygen saturation was calculated as the per-
centage of dissolved oxygen concentration relative to the temperature
and salinity of the water, and the dissolved oxygen concentration was
determined by the Winkler method (Grasshoff et al., 1999). Total par-
ticulate matter was determined following APHA (2005) method. Faecal
coliforms were determined by the fermentation method in multiple
tubes, which determines the Most Probable Number (MPN) of faecal
coliforms per 100 mL water sample (APHA, 2005). Monthly precipita-
tion data were obtained from the National Agency of Water (ANA,
2015). Fish larvae were sorted from 144 ichthyoplankton samples and
identified to the highest possible taxonomic separation.
2.4. Data analysis
Months were aggregated into two seasonal groups, namely: dry
comprising May, July, September and November; and rainy comprising
January and March. All analyses were performed for each season se-
parately. Differences in water parameters (temperature, pH, total par-
ticulate matter, chlorophyll a and oxygen saturation) and contamina-
tion indicators (nutrients and faecal coliforms) among the four estuaries
were analyzed by one-way MANOVA (Multivariate Analysis of
Variance) with estuary as fixed factor (Sokal and Rohlf, 1995; Zar,
1996). One-way ANOVA was used to investigate differences in the
community descriptors (abundance, number of species and guilds, di-
versity and equitability) among estuaries (Zar, 1996). All variables,
excepting pH, were log transformed in order to stabilize the variance
and to fit data to a normal distribution, fulfilling one of the ANOVA and
MANOVA assumptions. Homogeneity of variance was tested by a Co-
chran test, and whenever variance was still heterogeneous, conclusions
from ANOVA and MANOVA results were only accepted for those cases
where significance levels were < 0.01. Furthermore, in the event of
significance, a posteriori LSD (Least Significant Difference) test was
used to determine which means were significantly different at a 0.05
level of significance.
A Pressure Index (PI), adapted from Aubry and Elliott (2006), as-
sessed anthropogenic pressures acting in each estuary. PI included 13
descriptors relevant for larval stages of fishes, namely: habitat loss,
Table 1
Mean values and standard deviation of water parameters and precipitation in Macaé, São João, Bracuí and Perequê-Açu estuaries.
Dry
Macaé São João
Temperature (°C) 23.2 ± 2.1 23.0 ± 1.2
Oxygen saturation (%) 72.0 ± 6.7 77.0 ± 8.7
pH 8.2 ± 0.2 8.2 ± 0.3
Chlorophyll a (μg L− 1) 3.1 ± 1.7 3.7 ± 3.1
Total particulate matter (mg L− 1) 22.0 ± 10.6 14.8 ± 4.7
Precipitation (mm) 95.2 ± 42.8 128.0 ± 62.6
Marine Pollution Bulletin xxx (xxxx) xxx–xxx
dredging, percentage of land for industrial and agricultural use, popu-
lation density, presence of marinas and ports, tourism and recreation,
physical barriers (dams), aquaculture, fisheries, freshwater input
(amount of freshwater that naturally reaches the estuary) and sewage
pollution (discharge of loads of organic matter and other compounds of
human waste without treatment). These descriptors may have appar-
ently included an element of double counting or redundancy, which can
lead to the over-weighting of particular pressures. However, we con-
sider that a single number of indicators surely cannot capture the
complexity involved in environmental state (Purvis and Hector, 2000;
Derous et al., 2007; Borja et al., 2014), and that the ecosystem-based
approach implies that overall environmental status can only be ap-
propriately described by combining the different aspects of the en-
vironment into a holistic view (Berg et al., 2015). The 13 selected de-
scriptors were classified as: no change (0), very low (1), low (2),
moderate (3), high (4) and very high change (5), and the PI resulted
from the average value of the individual pressures scores. Simple
average of all descriptors is not necessarily the best solution in every
circumstance, considering that different descriptors meet various
screening criteria differently (Shin et al., 2010; Borja et al., 2014,
2016). However, an adequate basis for weighting descriptors is not
always available and in such cases equal weighting is recommended by
Ojaveer and Eero (2011).
Differences on the larval fish assemblages were investigated at the
structural (abundance, diversity and equitability) and functional (eco-
logical guilds) levels. Abundance data were standardized into number
of larvae per 100 m3 of water filtered. Diversity of the larval fish as-
semblage was expressed by Shannon-Wiener index (H′) based on nat-
ural log (Shannon and Weaver, 1963), and equitability was measured
by Pielou's evenness index (J′) (Pielou, 1966). Fish species were as-
signed to an ecological guild according to their estuarine use patterns,
following Elliott et al. (2007): amphidromous species (AM), anadro-
mous species (AN), catadromous species (C), estuarines (ES), freshwater
species (F), marine migrants (MM; spawn at sea and regularly enter
estuaries in large numbers, including marine species using estuaries as
nursery grounds) and marine stragglers (MS; spawn at sea and enter
estuaries accidentally in low numbers).
Inter-estuarine differences in terms of larval fish assemblage were
investigated by one-way analysis of similarity (ANOSIM) (Clarke and
Warwick, 1994), at a significance level of P < 0.05 and considering R
statistic > 0.5 (Clarke and Warwick, 1994). The tests were based on a
Bray-Curtis rank similarity matrix, calculated using log transformed
[log 10(x + 1)] fish larval data. Non-metric multidimensional scaling
(nMDS) was used to investigate similarities among estuarine samples,
based on the previously described Bray–Curtis similarity matrix (Bray
and Curtis, 1957). Similarity percentages (SIMPER) (Clarke, 1993) were
used to identify the percentage contribution of each taxon to the
average dissimilarity between samples of the various estuaries pair
combinations. Multivariate analyses were performed with the software
package PRIMER 6 (Plymouth Routines Multivariate Ecological Re-
search) (Clarke and Warwick, 1994).
To evaluate the influence of environmental variables on the larval
fish assemblages of each estuary a canonical correspondence analysis
Rainy
Bracuí Perequê-Açu Macaé São João Bracuí Perequê-Açu
23.2 ± 2.8 25.1 ± 2.1 25.9 ± 1.1 27.4 ± 1.0 28.7 ± 2.6 28.5 ± 2.5
81.2 ± 6.3 80.8 ± 9.3 78.6 ± 14.6 77.6 ± 4.5 88.2 ± 4.3 86.2 ± 14.7
8.2 ± 0.2 8.3 ± 0.1 8.1 ± 0.2 8.0 ± 0.2 8.1 ± 0.3 7.9 ± 0.4
2.2 ± 0.7 4.6 ± 2.5 9.5 ± 6.2 5.6 ± 2.8 1.9 ± 1.2 3.6 ± 3.1
8.8 ± 3.6 11.7 ± 6.1 19.0 ± 3.5 11.4 ± 3.5 9.7 ± 4.5 11.1 ± 9.0
124.1 ± 45.1 119.4 ± 24.6 132.0 ± 56.8 159.8 ± 50.2 211.6 ± 84.3 208.6 ± 69.8
3
R.V.S. Santos et al. Marine Pollution Bulletin xxx (xxxx) xxx–xxx

Table 2
Mean values and standard deviation of contamination indicators in Macaé, São João, Bracuí and Perequê-Açu estuaries.

Dry Rainy

Macaé São João Bracuí Perequê-Açu Macaé São João Bracuí Perequê-Açu

Faecal coliforms (MPN 100 mL− 1) 101.2 ± 221.5 8.7 ± 8.3 18.1 ± 27.8 56.0 ± 171.1 60.0 ± 91.0 19.8 ± 13.7 15.5 ± 20.5 356.8 ± 629.1
Nitrate (μM) 5.5 ± 2.7 2.7 ± 2.3 1.8 ± 1.1 6.4 ± 2.9 5.4 ± 0.9 2.9 ± 2.4 2.5 ± 0.7 6.5 ± 2.7
Nitrite (μM) 0.4 ± 0.2 0.2 ± 0.1 0.2 ± 0.2 0.2 ± 0.1 0.5 ± 0.3 0.2 ± 0.1 0.1 ± 0.1 0.2 ± 0.1
Ammonia (μM) 18.4 ± 11.7 5.5 ± 5.6 2.4 ± 2.2 6.1 ± 4.6 18.7 ± 4.8 3.4 ± 1.0 2.5 ± 1.1 7.1 ± 3.8
Phosphate (μM) 1.0 ± 0.5 0.3 ± 0.2 0.3 ± 0.1 0.7 ± 0.2 0.8 ± 0.3 0.2 ± 0.1 0.2 ± 0.1 0.5 ± 0.2

(CCA) (Ter Braak, 1986) was performed using CANOCO software and Bracuí estuaries were the least impacted systems, classified as low
(version 4.5, Microcomputer Power, Ithaca, NY). Monthly precipitation, and very low impacted, respectively (Table 3). Macaé Estuary suffers
water temperature, oxygen saturation, pH, TPM, chlorophyll a, nitrate, from several human pressures, mainly alteration of the amount of
nitrite, ammonia, phosphate, faecal coliforms and PI were standardized freshwater that naturally reaches the estuary, large-scale habitat loss, a
and used as explanatory environmental variables. All 59 taxa were used high percentage of land use for agriculture, sewage pollution and
in the CCA in order to have a comprehensive picture of larval fish dredging activities. In Perequê-Açu Estuary, habitat loss, tourism and
distributions. Larval abundances were square root transformed to nor- recreation, sewage pollution and dredging were the most severe pres-
malize the data and downweighting of rare species was selected. Sta- sures observed. The most important pressures detected in São João
tistical significance of the correlations between explanatory and species Estuary were the percentage of land for agricultural use and the
variables extracted from the CCA was determined by the Monte–Carlo freshwater input, which is controlled by a large dam located upstream.
test, based on 499 permutations. Environmental variables were con- Bracuí Estuary exhibited few pressures, associated with some aqua-
sidered to be biologically important when inter-set correlation coeffi- culture and land use for agriculture.
cients were |≥0.4| (Hair et al., 1984; Rakocinski et al., 1996).
3.4. Larval fish assemblages
3. Results
The larval fish assemblages varied significantly among the four es-
3.1. Environmental characteristics tuaries during both seasons, not only in terms of abundance (dry:
F = 13.07, P < 0.001; rainy: F = 8.58, P < 0.001), but also the
In general, high precipitation values were observed during the rainy number of species (dry: F = 18.63, P < 0.001; rainy: F = 2.86,
season in the four estuaries (Table 1), although mean seasonal pre- P = 0.05), diversity (dry: F = 23.74, P < 0.001; rainy: F = 17.08,
cipitation varied among estuaries. During the rainy period, Bracuí and P < 0.001) and equitability (dry: F = 15.36, P < 0.001; rainy:
Perequê-Açu estuaries registered the highest precipitation values F = 17.19, P < 0.001). Perequê-Açu Estuary showed significantly
(Table 1). Macaé Estuary registered the lowest mean precipitation higher abundances (P < 0.001) during both seasons, with a mean of
during both rainy and dry seasons. MANOVA results showed some 291 ± 306 larvae 100 m− 3 during the dry season, and 2791 ± 3901
significant differences in particular water parameters, namely tem- larvae 100 m− 3 in the rainy period (Table 4). Such high abundances
perature during the dry season and TPM, oxygen and chlorophyll a were associated with significantly (P < 0.001) lower diversity (dry:
during the rainy season (Table 1). During the dry season, Perequê-Açu H′ = 0.8 ± 0.4, rainy: H′ = 0.5 ± 0.4) and equitability values (dry:
Estuary reached the highest water temperature (Wilks' lambda = 0.40, J′ = 0.5 ± 0.3, rainy: J′ = 0.3 ± 0.3). During the dry season, Macaé
P < 0.05) and Macaé Estuary registered significantly lower dissolved larval fish assemblages were significantly (P < 0.01) more abundant
oxygen (Wilks' lambda = 0.40, P < 0.05). During the rainy season,
Macaé Estuary exhibited significantly lower water temperature (Wilks' Table 3
lambda = 0.08, P < 0.05) and highest chlorophyll a concentration Pressure descriptors used to calculate the Pressure Index (PI) for Macaé, São João, Bracuí
and Perequê-Açu estuaries. PI varied from 0 (no change), 1 (very low), 2 (low), 3
(Wilks' lambda = 0.08, P < 0.05). In addition, Macaé Estuary pre-
(medium), 4 (high) and 5 (very high). INEA: State Environmental Institute (www.inea.rj.
sented significantly high TPM values (Wilks' lambda = 0.08, gov.br), GE: Google Earth, EJ: expert judgment, IBGE: Brazilian Institute of Geography
P < 0.05) during both seasons. The remaining water characteristics and Statistics (www.ibge.gov.br).
did not vary among estuaries.
Pressure indicators Data Macaé São João Bracuí Perequê-Açu
source
3.2. Contamination indicators
Habitat loss INEA, GE 5 2 1 5
Contamination indicators differed significantly among the four es- Dredging INEA, EJ 5 1 0 5
tuaries during both seasons (dry: Wilks' lambda = 0.25, P < 0.001; % industrial land INEA, GE 4 0 1 2
% agricultural land INEA, GE 5 4 3 3
rainy: Wilks' lambda = 0.05, P < 0.01), with Macaé and Perequê-Açu
Presence of marinas INEA, GE 4 2 0 4
estuaries exhibiting lower water quality than Bracuí and São João es- Presence of ports INEA, GE 4 0 0 0
tuaries. Overall, Macaé and Perequê-Açu estuaries were more con- Physical barriers INEA, GE 3 4 0 0
taminated by faecal coliforms and carried a higher nutrient load, par- Aquaculture INEA, GE, 1 2 3 1
EJ
ticularly higher concentrations of nitrate, ammonia and phosphate
Fisheries INEA, EJ 2 3 1 1
(Table 2). Sewage pollution INEA, EJ 5 2 1 5
Population density INEA, 4 3 2 4
3.3. Pressure index IBGE
Tourism and INEA 3 3 0 5
Recreation
According to the pressure index (PI) the four estuaries differed in
Freshwater input EJ 5 4 0 0
terms of anthropogenic pressures, with Macaé Estuary classified as Pressure index 4 2 1 3
highly impacted and the most impacted estuary, followed by Perequê- High Low Very low Medium
Açu Estuary, classified as medium impacted estuary (Table 3). São João

4
R.V.S. Santos et al.
Table 4
Mean values and standard deviation of abundance, number of species, diversity (H′) and equitability (J′) indices in Macaé, São João, Bracuí and Perequê-Açu estuaries.
Dry
Macaé São João Bracuí
−3
Abundance (larvae 100 m ) 111 ± 94 49 ± 88 39 ± 40
Number of species 13 ± 4 7 ± 2 7 ± 3
Diversity (H′) 1.6 ± 0.3 1.2 ± 0.4 1.4 ± 0.3
Equitability (J′) 0.6 ± 0.1 0.7 ± 0.2 0.8 ± 0.1
than those of São João and Bracuí, with an average of 111 ± 94 larvae
100 m− 3 (Table 4), and comprised the significantly (P < 0.001)
highest number of species of all study (Table 4).
The nMDS grouped Macaé and Perequê-Açu samples on opposite
sides of the plots, while Bracuí and São João samples were dispersed in
both plots (Fig. 2A–B). According to ANOSIM results, the structure of
the larval fish assemblages differed among the four estuaries, during the
dry (R = 0.6, P < 0.01) and rainy seasons (R = 0.5, P < 0.01).
During both seasons, Perequê-Açu samples were highly dominated by
Anchoviella lepidentostole (Supplementary data), which was responsible
for at least 20% of the average dissimilarity between Perequê-Açu Es-
tuary and the other estuaries. During the dry period, Macaé assem-
blages were characterized by high abundances of Micropogonias furnieri
that reached a mean of 40 larvae 100 m− 3, while in the other estuaries
did not surpass 2 larvae 100 m− 3 (Supplementary data). On the other
hand, São João and Bracuí samples were not clearly separated from
each other (R < 0.5, P < 0.01), presenting similar larval fish as-
semblages, characterized by high abundances of Anchoviella spp. in
both seasons (Supplementary data).
The larval fish assemblages of the four estuaries studied comprised
seven functional guilds (AN, ES, MM, MS, AM, C, F) (Supplementary
data). The majority of the species collected in the four estuaries were
AN, ES, MM MS (Fig. 3). In dry surveys, AN was the main group in São
João, Bracuí and Perequê-Açu estuaries, which reaching 47%, 54% and
79% of the total larvae captured in each estuary, respectively (Fig. 3A).
On the other hand, Macaé larval fish assemblage was dominated by MM
that represented 53% of the total larvae (Fig. 3A). In rainy surveys, AN
was the most abundant group in the four estuaries, reaching 36%, 68%,
80% and 98% of the total catch in Macaé, São João, Bracuí and Per-
equê-Açu estuaries, respectively (Fig. 3B). The number of ecological
guilds was significantly higher in Macaé Estuary during the dry
(F = 3.52, P < 0.05), that comprised two further ecological guilds
(AM and F), but did not differ during the rainy period (F = 1.30,
P = 0.28). According to ANOSIM results, the guild structure of the four
estuaries did not vary significantly during the dry season (R = 0.2,
P = 0.001) or the rainy season (R = 0.3, P = 0.001). Also, estuarine
samples of the four estuaries were not separated by the nMDS of the dry
(Fig. 4A) and rainy seasons (Fig. 4B).
In order to allow inter-season comparisons, CCA analyses of the dry
and rainy seasons were performed separately. During the dry season
Estuary
A Macaé
B Macaé
São João
Bracuí
Perequê-Açu
5
Marine Pollution Bulletin xxx (xxxx) xxx–xxx
Rainy
Perequê-Açu Macaé São João Bracuí Perequê-Açu
291 ± 306 45 ± 39 96 ± 97 97 ± 143 2791 ± 3901
7 ± 3 9 ± 3 8 ± 4 6 ± 3 7 ± 2
0.8 ± 0.4 1.7 ± 0.2 1.2 ± 0.6 1.0 ± 0.4 0.5 ± 0.4
0.5 ± 0.3 0.8 ± 0.1 0.7 ± 0.2 0.7 ± 0.2 0.3 ± 0.3
(Fig. 5A), the first CCA axis (CCA1) was negatively correlated with
temperature and positively correlated with TPM, ammonia and PI
(Table 5). And the second CCA axis (CCA2) was negatively correlated
with nitrate and PI (Table 5). Samples tended to cluster according to
their estuarine origin, with the four estuaries discriminated along the
two CCA axes. Bracuí and Perequê-Açu samples clustered on the left
side of CCA1 associated with high water temperature and lower nu-
trient load, and were also separated along CCA2, with Perequê-Açu
samples positively correlated with nitrate and PI (Fig. 5A). São João
and Macaé samples were positively correlated with TPM and ammonia
(clustering on the right side of CCA1) and were discriminated by CCA2,
since Macaé samples were positively correlated with nitrate and PI
(Fig. 5A).
CCA analysis of the rainy season (Fig. 5B) showed that the first CCA
axis (CCA1) was negatively correlated with temperature and oxygen,
while the second CCA axis (CCA2) was positively correlated with nu-
trient load (nitrate, nitrite, ammonia and phosphate) and PI (Table 5).
Perequê-Açu samples were isolated on the left side of CCA1 associated
with high water temperature and dissolved oxygen (Fig. 5B). Bracuí,
São João and Macaé samples were negatively correlated with tem-
perature (clustering on the right side of CCA1) and were mainly dis-
criminated by CCA2, with Macaé samples positively correlated with
nutrient load and PI and separated from the less contaminated São João
and Bracuí samples (Fig. 5B).
4. Discussion
One of the most critical phases in fish life cycle is the larval stage
(Miller and Kendall, 2009), which is particularly sensitive to environ-
mental conditions (Ramos et al., 2006a; Eick and Thiel, 2014; Strydom,
2015), including hydrological alterations of estuarine environment
(Ramos et al., 2012, 2015) and contamination (Longwell et al., 1992;
Gordina et al., 2001; Westernhagen et al., 2001). The level of con-
tamination impact on the estuarine environments may vary according
to estuarine geomorphology and hydrodynamics, specifically the degree
of connectivity with the marine ecosystem. This characteristic directly
influences the water residence time and dictates the time over which
the fish are exposed to any stressors (Elliott and Whitfield, 2011).
However, in the present study, the descriptors associated with the larval
fish assemblages were affected more by the anthropogenic pressures on
Fig. 2. Non-metric multi-dimensional scaling (nMDS) con-
Estuary figuration of species similarity matrix for Macaé, São João,
Bracuí and Perequê-Açu larval fish assemblages during the
São João
Bracuí
dry (A) and rainy (B) seasons.
Perequê-Açu
R.V.S. Santos et al. Marine Pollution Bulletin xxx (xxxx) xxx–xxx

Fig. 3. Percentage of anadromous (AN), estuarine (ES),

A B marine migrants (MM) and marine stragglers (MS) species
100 100
observed in the larval fish assemblages of Macaé, São João,
90 90
Bracuí and Perequê-Açu estuaries during the dry (A) and
80 80
rainy (B) season.
70 70
60 60

%
%

50 50
40 40
30 30
20 20
10 10
0 0
Macaé São João Bracuí Perequê-Açu Macaé São João Bracuí Perequê-Açu
AN ES MM MS AM F C AN ES MM MS

Fig. 4. Non-metric multi-dimensional scaling (nMDS) con-

figuration of guild similarity matrix for Macaé, São João,
A Estuary
Macaé
B Estuary
Macaé Bracuí and Perequê-Açu larval fish assemblages during the
São João São João
Bracuí Bracuí
dry (A) and rainy (B) seasons.
Perequê-Açu Perequê-Açu

the systems than by their degree of connectivity with the ocean (i.e. if
they flow directly into the open sea or into large coastal bays). Differ-
ences in the structure and functioning of assemblages associated with
the estuarine impact level may be identified by the different patterns of
fish species occurrence and abundance in the four estuaries.
In Perequê-Açu Estuary, the reduced ecosystem health (high nu-
trient load and faecal contamination) negatively influenced the larval
fish assemblage, which showed some environmental stress symptoms,
namely reduced number of species, low diversity and especially a high
dominance of few species (Odum, 1983; Fausch et al., 1990). Macaé
Estuary was highly contaminated by sewage, which might have po-
tentially increased respiration processes and consequently lead to an
oxygen decrease of water (Thurman, 1985). This scenario might have
been the reason for the lower oxygen detected in Macaé dry samples,
since high faecal coliforms coincided with high nutrient load and TPM
values. Ramos et al. (2012) observed a similar effect in a small urban
estuary where the high contamination levels of faecal indicators and
high concentrations of dissolved organic carbon were associated to an
oxygen decreased of the water. However, Macaé larval assemblage
structure did not reflect the effect of high sewage contamination and
nutrient load observed in this study. In this case, the local pressure level
probably was not sufficiently high to cause significant impacts in the
structure of larval fish assemblages. This ability of the estuary to absorb
an amount of a contaminant added to the system is regarded as the
“assimilative capacity”. It can vary according to the part of the estuary
that receives the inputs, as well as being related to environmental
factors, such as water circulation (McLusky and Elliott, 2004). Ac-
cording to Elliott and Jonge (2002), some estuarine areas have high
assimilative capacity often caused by increased dilution or flushing.
Considering this, the large water abstraction and flow changes observed
in Macaé Estuary may have increased the seawater intrusion and,
consequently, the assimilative capacity of this estuary by dilution. On

Fig. 5. Ordination of sampling estuaries along the first two

axes of the canonical correspondence analysis (CCA) re-
lating species with environmental variables (arrows)
during the dry (A) and rainy (B) season. Pp: precipitation;
T: water temperature; O2: oxygen saturation; TPM: total
particulate matter; Chla: chlorophyll a; NO3: nitrate; NO2:
nitrite; NH3: ammonia; PO4: phosphate; FC: faecal coli-
forms; PI: pressure index.

6
R.V.S. Santos et al. Marine Pollution Bulletin xxx (xxxx) xxx–xxx

Table 5
Results of canonical correspondence analysis (CCA) performed on larval fish abundance, relating species with environmental variables of Macaé, São João, Bracuí and Perequê-Açu
estuaries during the dry and rainy seasons. Underscores values are the highest correlation values for each variable. Inter-set correlations ≥ |0·4| were highlighted and correspond to
biologically important variables.

Dry Rainy

Axes Axes

1 2 3 4 1 2 3 4

Eigenvalues 0.39 0.23 0.12 0.08 0.51 0.36 0.18 0.14

Species-environment correlations 0.91 0.82 0.74 0.73 0.96 0.97 0.92 0.89
Cumulative percentage variance
Of species data 12.3 19.5 23.2 25.8 17.8 30.3 36.7 41.6
Of species-environment relation 36.0 56.9 67.8 75.3 33.9 57.5 69.7 78.9
Sum of all unconstrained eigenvalues (total inertia) 3.17 2.84
Sum of all canonical eigenvalues 1.08 1.49
Summary of Monte Carlo test
Test of significance of all canonical axis
Trace 1.08 1.49
F-ratio 3.60 3.24
P-value 0.002 0.002
Temperature − 0.67 −0.17 0.05 − 0.09 − 0.65 − 0.26 − 0.11 − 0.01
Oxygen saturation − 0.38 0.17 −0.08 − 0.03 − 0.65 − 0.05 − 0.09 0.04
pH − 0.07 0.10 −0.30 − 0.42 − 0.18 − 0.01 − 0.16 − 0.17
Chlorophyll a − 0.36 −0.26 −0.12 0.05 0.01 0.27 0.37 − 0.09
Total particulate matter 0.47 −0.33 −0.17 0.03 − 0.15 0.30 − 0.07 − 0.16
Precipitation − 0.16 0.00 0.15 0.38 − 0.35 − 0.20 0.35 − 0.02
Faecal coliforms 0.06 0.02 0.29 − 0.17 − 0.38 0.07 − 0.06 − 0.18
Nitrate 0.01 −0.44 0.38 0.01 − 0.32 0.43 0.04 0.17
Nitrite 0.26 −0.15 0.52 − 0.30 0.22 0.43 0.31 − 0.05
Ammonia 0.42 −0.16 0.41 − 0.11 0.31 0.65 0.12 0.13
Phosphate 0.25 −0.38 0.40 − 0.30 0.23 0.45 0.31 0.24
Pressure Index 0.57 −0.55 −0.02 − 0.14 0.30 0.78 0.14 − 0.23

the other hand, the seawater intrusion may also have favored the mi-
gration of several marine species, which may have been the reason for
this estuary to be clearly more diverse, despite the high contamination
level. Recently, Teichert et al. (2016) demonstrated that the flow
changes are essential explaining components of fish distribution in es-
tuaries and, in addition with water pollution and oxygen depletion, are
stressors classified at the ranks of priority in the restoration of fish
ecological quality in estuaries.
It has been acknowledged that the pollution in estuaries has a re-
levant impact on fish species, not only in terms of the community
structure (reduced species diversity), but also at the function (reduced
abundance of migrant species and disturbance sensitive species) (e.g.
Harrison and Whitfield, 2004; Franco et al., 2006; Rochette et al., 2010;
Ramos et al., 2012; Fonseca et al., 2013). In this study, the results
showed that all estuaries exhibited similar number of ecological guilds,
but in different relative proportions of occurrence. In fact, the anthro-
pogenic pressures appeared to influence in different ways the estuarine
use patterns by fishes. River flow changes caused by water abstraction
and presence of barriers affected the migration routes of fish species,
influencing mainly the occurrence of the marine migrant M. furnieri,
and anadromous species A. lepidentostole and Anchoviella spp. The
croaker M. furnieri is a marine migrant species that systematically uses
estuarine environment along its ontogeny (Vieira, 2006; Costa et al.,
2013). Spawning occurs in the coastal zone (Macchi and Christiansen,
1996; Acha et al., 1999; Simionato et al., 2008; Braverman et al., 2009)
and eggs and larvae enter in the estuary associated with the intrusion of
salt water (Sinque and Muelbert, 1997; Elliott and Hemingway, 2002).
In this way, the river discharges may regulate croaker estuarine re-
cruitment by promoting high recruitment during low discharge periods
(Acha et al., 2012). Following this, the higher abundances of M. furnieri
in Macaé Estuary appears to be linked to the altered hydrological re-
gime caused mainly by water abstraction, which favored the estuarine
migration from the coastal area through the seawater intrusion.
Anadromous species, such as the anchovies A. lepidentostole and
Anchoviella spp., may also respond directly to hydro-morphological
pressures caused by dams. These species are characterized by migrating
upstream reaching the rivers where they spawn (Paiva-Filho et al.,
1986). Thus, the absence of physical barriers interfering with fish mi-
gratory routes, could have contributed to the higher abundances of A.
lepidentostole and Anchoviella spp. in Perequê-Açu and Bracuí estuaries.
Furthermore, the warmest water of Perequê-Açu Estuary might also
have contributed to impressive high densities of anchovies, since their
abundance, spawning and distribution have been already reported as
positively associated with temperature (Silva and Araújo, 2000; Ribeiro
et al., 2014; Santos et al., 2017). The high temperature in Perequê-Açu
Estuary during both seasons could have been a result of the influence of
a warmer and oligotrophic seawater mass as Tropical Water (TW),
which is predominant in this region as well as in the most of the Bra-
zilian continental margin (BCM) (Lopes et al., 2006). On the other
hand, this typical warming of Brazilian tropical marine ecosystems was
interestingly less pronounced in Macaé Estuary during the rainy, al-
though the seawater intrusion was high due to lower precipitation
observed in this system, associated with the large water abstraction.
The low water temperatures and also high chlorophyll a concentrations
observed in Macaé rainy samples could have been a result of the in-
fluence of a colder and nutrient-rich seawater mass as South Atlantic
Central Water – SACW (Acha et al., 2004; Braga and Niencheski, 2006).
The SACW intrusion over the shelf is more frequent during the rainy
season (spring-summer), when prevailing northeast winds cause Ekman
transport of surface waters offshore (Rodrigues and Lorenzzetti, 2001;
Castro et al., 2006a, 2006b; Calado et al., 2010; Campos et al., 2013;
Macedo-Soares et al., 2014), and its effect in the biota and seawater
properties has been observed along the Brazilian coast, mainly between
Cape São Tomé (22°S) and Cape Frio (23°S) (e.g. Macedo-Soares et al.,
2014; Moser et al., 2014; Suzuki et al., 2015). This is other example of
how the catchment events can contribute to processes and changes in
the environmental conditions within the estuary.
Macaé, São João, Bracuí and Perequê-Açu estuaries carried dis-
tinctly different larval fish assemblages as a reflection of different en-
vironmental status. Macaé and Perequê-Açu estuaries emerged as more

7
R.V.S. Santos et al.
impacted than Bracuí and São João estuaries. Faecal contamination and
nutrient load were the main anthropogenic factors influencing the
larval fish assemblages in Perequê-Açu Estuary, while in Macaé Estuary
the water contamination effect on community structure was apparently
diminished by seawater/marine species intrusion resulting from the
other anthropogenic factor: the hydrological manipulations. Finally,
this study showed the TW/SACW effect on the estuarine larval fish
assemblages and water properties, emphasizing the need to develop
more studies in order to more accurately define the limits of these
seawater masses influences in this region.
5. Conclusions
The present study showed that fish larvae were sensitive to water
contamination, reducing the diversity and especially exhibiting a high
dominance of few species. Also, hydro-morphological pressures caused
by physical barriers and control of the freshwater input interfered in
migratory fish routes. Thus, this study reinforced the idea that the high
sensitivity of fish larvae can increase the accuracy of the environmental
assessments, mainly when tackling short-time events of hydrological
manipulations or water pollution, representing an advance in the as-
sessment of water ecological quality assessments. Also, the present
study gives strategically important information for improved estuarine
management by integrating valuable information about the environ-
mental status of estuarine ecological functions, as nursery areas, and
the connectivity between marine and freshwater habitats, considered
vital to many fish species.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.marpolbul.2017.07.043.
Acknowledgments
This work was supported by the Coordination for the Improvement
of Higher Education Personnel (CAPES), through a PhD grant awarded
to Régis Santos (DS/1086381) and also by the Brazilian National
Council for Scientific and Technological Development (CNPq) (Split
Fellowship Program, ref. 204767/2014-8). Sandra Ramos was funded
by a FCT Post-doc fellowship (SFRH/BPD/102721/2014). The authors
thank all who participated in field surveys and laboratory processing.
We also thank to Dr. Rodolfo Paranhos, responsible for Hydrobiology
Laboratory, Department of Marine Biology of the Federal University of
Rio de Janeiro, for his support in water analysis.
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