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Tracing pollution in estuarine benthic organisms and its impacts on food webs of the Vitoria Bay estuary
Estuarine, Coastal and Shelf Science 229 (2019) 106410
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Estuarine, Coastal and Shelf Science journal homepage: http://www.elsevier.com/locate/ecss
Tracing pollution in estuarine benthic organisms and its impacts on food webs of the Vitoria Bay estuary Cíntia S. Varzim a, Heliatrice L. Hadlich a, Ryan Andrades b, Ana Carolina de A. Mazzuco a, Angelo F. Bernardino a, * a b
Grupo de Ecologia B^entica, Departamento de Oceanografia, Universidade Federal do Espírito Santo, Vit� oria, ES, 29075-910, Brazil Laborat� orio de Ictiologia, Departamento de Oceanografia, Universidade Federal do Espírito Santo, Vit� oria, ES, 29075-910, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Sewage Coprostanol Stable isotopes Isotopic niche Macrofauna
Estuaries in South America commonly receive untreated effluents from nearby metropolitan areas demanding ecosystem-based management solutions to access pollutant impacts. In this study we investigated how organic enrichment in Vit� oria Bay changes benthic macrofaunal isotopic signatures (δ13C and δ15N) and if highly contaminated areas would exhibit lower food web diversity. Geochemical markers of sewage input (coprostanol) revealed low to high levels of pollution in the bay area. The δ15N isotope signatures of benthic deposit-feeders and omnivores were lower in contaminated sites indicating preferential intake of raw sewage and that the ef fect size of sewage input changes among benthic feeding modes. Sites with high sediment coprostanol concen trations (>0.5 μg g 1) were associated with a lower abundance of basal resources and niche shrinking of the benthic food web, suggesting that eutrophication leads to a funtional loss in the sediment-water interface in this estuary. Our study revealed that pollution in the Vit� oria Bay estuary has functional impacts to the estuarine food webs and support previous ascertions that stable isotopes may be a useful and low-cost method to indicate ecosystem health monitoring in estuarine ecosystems.
1. Introduction Estuaries are transitional ecosystems with ecological and economic relevance that are continually impacted by organic pollution. Pollution typically deteriorates water and sediment quality, with consequences for estuarine biodiversity and productivity (Lotze et al., 2006). To effi ciently track those impacts, a common practice is to look for multiple ecological indices of habitat quality that can integrate ecosystem man agement strategies in estuaries (Elliot and Quintino, 2007; Borja et al., 2009). Many ecological and biogeochemical indices can be used in estuarine management programs with accurate, reliable and direct in formation of ecosystem status (Borja et al., 2003). Macrobenthic assemblages are long used as tools to determine sediment ecological status to assess anthropogenic disturbances in estuarine ecosystems (Borja et al., 2003; Muniz et al., 2005; Bernardino et al., 2018a). Benthic indices may rely on assemblage richness, diversity and functional attributes (Borja et al., 2000; Brauko et al., 2016), which offer direct assessment of ecosystem health based on predictive re sponses of populations and assemblages to eutrophication (Pearson and
Rosenberg, 1978). Chemical markers such as sterols are used in parallel to biological indices in order to determine correspondence between sewage contamination and ecological impacts. Sterols including copro stanol (5β-cholestan-3β-ol) and epicoprostanol (5β-cholestan-3α-ol) are not natural in marine sediments, but are present in human fecal material and thus can indicate anthropogenic pollution (Martins et al., 2010). The ratio between sediment epicoprostanol and coprostanol concen trations can additionally provide information on sewage treatment before it is released into estuarine and marine ecosystems (Mudge and Duce, 2005). Multiple organic biomarkers of sediment quality have suggested that �ria Bay, a tropical estuary located in the Eastern Brazil Marine Vito Ecoregion, is predominantly eutrophic or hypertrophic given a high input of untreated sewage in the estuary (Hadlich et al., 2018). The estuary receives nearly 16,000 m 3day 1 of untreated sewage from nearby metropolitan areas through several small river tributaries, and organic and trace metal pollution have been impacting the bay for several decades (Grilo et al., 2013; Costa et al., 2015; Hadlich et al., 2018). The coprostanol concentrations in sediments of Vitoria Bay are
* Corresponding author. E-mail address: [email protected] (A.F. Bernardino). https://doi.org/10.1016/j.ecss.2019.106410 Received 27 March 2019; Received in revised form 13 September 2019; Accepted 8 October 2019 Available online 10 October 2019 0272-7714/© 2019 Elsevier Ltd. All rights reserved.
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within the range of other coastal metropolitan bays under heavy to moderate organic input such as Guanabara Bay (Rio de Janeiro), Cap ibaribe estuary (Pernambuco), and Barbitonga Bay (Santa Catarina) �ria (Hadlich et al., 2018). The metropolitan occupation along the Vito Bay is likely the main cause of the low environmental health of its estuarine ecosystems, which is a common reality of coastal bays and estuaries of South America (Lana and Bernardino, 2018; Barletta et al., 2019). Organic and metal pollutants are of concern as these typically accumulate in estuarine sediments and affect benthic organisms with observed changes in assemblage structure (Venturini et al., 2004; Souza et al., 2013). Although there is strong evidence for geochemical and ecological impacts of pollution on estuaries, the effects of anthropogenic pollution on benthic food webs have been rarely assessed. Stable isotopes have also been used as indicators of pollution impacts for decades (Sweeney et al., 1980). Changes in nitrogen stable isotopic signatures can determine anthropogenic organic inputs as nitrogen isotopes have end-members that vary by 9‰ between natural or sewage inputs; and yet the δ15N of raw effluents are similar to its refractory deposited fraction (Sweeney et al., 1980). Therefore, stable isotope signatures may indicate sewage input over spatial and temporal scales and additionally trace these inputs into coastal food webs (Connolly et al., 2013; Souza et al., 2018). The pollution impacts on benthic food webs are less clear and usually limited to identifying changes in organism’s isotopic signatures. Previ ous work has focused on detecting spatial-temporal changes in stable isotope signatures of selected taxa across pollution gradients (Sampaio et al., 2010; Connolly et al., 2013; Gorman et al., 2017). However, changes in faunal isotope signatures do not provide an integrated overview of food web diversity in coastal ecosystems, which is a func tional indice of interest. Otherwise, Bayesian stable isotope estimates from the SIBER model (Stable Isotope Bayesian Ellipses in R; Jackson et al., 2011), provide robust metrics allowing to test niche shrinking and trophic diversity loss in aquatic food webs under pollution and other
anthropogenic impacts (Bernardino et al., 2018a; Carvalho et al., 2019; Price et al., 2019). In this study, stable isotope signatures of benthic macrofaunal as semblages were assessed and applied in food web models to understand the long-term (scales of weeks to months) pollution impacts on benthic food webs in a tropical estuary. Benthic isotopic signatures spanning a range of feeding modes including suspensivores, deposit-feeders, car nivores and omnivores were used to estimate benthic food web trophic width and amplitude along sewage contamination gradients. The large�ria Bay were studied in detail scale gradients of organic pollution in Vito by Hadlich et al. (2018) and indicated spatially distinct contamination levels, which were used as a contemporary baseline for the food web assessment in this study. We asked if the δ13C and δ15N isotope signa tures of benthic organisms and the benthic food web diversity would be �ria spatially distinct across levels of sedimentary pollution in the Vito Bay estuary. So here we tested if the isotopic signatures of benthic or ganisms would vary spatially with respect to i) sediment coprostanol concentrations and ii) benthic feeding modes. 2. Materials and methods 2.1. Study area and sampling �ria Bay (20� 180 S, 40� 200 W; Fig. 1) is an urbanized estuary on the Vito eastern coast of Brazil with historical high levels of sewage contami nation (Bernardino et al., 2015; Grilo et al., 2013; Bissoli and Bernar �ria Bay has an area of 18.2 km2 and a catchment area of dino, 2018). Vito 1728 km2 with an average freshwater discharge of 18.7 m3 s 1 (Lessa et al., 2019). The bay is connected to the coastal ocean through two channels; the southern port channel is wider (~160 m wide) and deeper (5–24 m) than the northern passagen channel that is on average 35 m wide and 1–8 m deep. The channel bed morphology and an abundant mangrove vegetation in Vitoria Bay results in a strong tidal
Fig. 1. Sampling sites along the Vit� oria bay metropolitan estuary. River tributaries (names in yellow) and small channels with input of raw sewage to the bay are indicated by arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2
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potential preservation artifacts by adding 1‰ to δ13C macrofaunal sig natures (Sarakinos et al., 2002; Demopoulos et al., 2007).
amplification inside the estuary, with stronger ebb flows and higher sediment bed load transport out of the bay (Rigo, 2004; Lessa et al., 2019). On areas near the coast both channels are highy impacted by urban development that resulted in loss of intertial mangrove forests for �ria Bay also the construction of an industrial port and local marinas. Vito receives a significant amount of treated and untreated sewage from nearby cities through its river tributaries, resulting in a historical accumulation of organic and trace metal pollutants in estuarine sedi ments (Grilo et al., 2013; Costa et al., 2015; Hadlich et al., 2018). �ria Bay was sampled at 11 sites in November 2014 along a The Vito salinity gradient from the inner estuary towards the coastal ocean which are all under influence of multiple pollution discharges into the bay (Fig. 1). Based on vertical CTD casts during sampling, average salinity within the bay ranged between 23.1 and 36.2 PSU and the average temperature ranged between 22.1 and 27 � C. The sampling sites 02, 06, 07, 09 and 15 exhibited polyhaline conditions and the other sites 17, 19, 21, 24, 30 and 34 were euhaline at the time of sampling. In each site, two sediment samples (i.e., replicates) were collected with a Day Grab (0.1 m2), and the top 3 cm of undisturbed sediment from each independent replicates were mixed into one composite sam ple for sterol biomarker analyses. Samples for biomarkers were imme diately stored in pre-cleaned aluminum containers and remained frozen ( 20 � C) until laboratory analysis. Benthic macrofaunal organisms (>0.5 mm) were sampled from the remaining sediments from each in dependent replicate along the 11 stations. All faunal samples were sieved live through a 0.5 mm mesh and fixed in buffered 10% formal dehyde and transferred to 70% Ethanol until laboratory analysis.
2.3. Statistical analysis Spatial differences of macrofaunal isotopic signatures and food web niche width were compared between contaminated and noncontaminated sites using all macrofaunal taxa and by grouping taxa into feeding modes. Analysis of Variance (ANOVA) tests were used to test for spatial variation of isotopic signatures across contamination levels and benthic trophic modes. Pairwise multiple comparisons were tested by Tukey’s HSD and adjusted Bonferroni-Dunn tests with applied corrections (Quintana et al., 2015). The coprostanol concentration in each site were used to determine sewage contamination levels adapted from Hadlich et al. (2018). Sites were classified as “non contaminated” if average coprostanol concentrations were below <0.5 μg g 1; whereas “contaminated” sites had sediment coprostanol concentrations above 0.5 μg g 1. Differences in isotopic signatures among sites and the correspon dence between benthic isotopic signatures and coprostanol concentra tions were analyzed using a canonical analysis of principal coordinates (CAP) complemented by multidimensional scaling based on the Euclidean distance between sites (Anderson and Willis, 2003). Addi tionally, canonical discriminant function analyses DFA were performed in order to test the classification of samples in trophic groups and levels of contamination (Venables and Ripley, 2002; Mazzuco et al., 2019). The results from the DFAs were interpreted based on the linear discriminant coefficients, which described the relationship of the iso topic signatures and coprostanol concentrations. Jackknife re-samplings were included in the analyses to test the accuracy of the classifications by DFAs (Tukey, 1958; Ripley, 1996). All analyses were performed in R (R Core Team, 2016). Isotopic niche metrics were evaluated through the method of Stable Isotope Bayesian Ellipses in R (SIBER package; Jackson et al., 2011) based on the Layman’s niche metrics δ15N range, δ13C range, distance to centroid (CD), mean nearest neighbour distance (MNND) and standard deviation of the nearest neighbour distance (SDNND) (Layman et al., 2007). Total Area (TA) is a metric that indicates the trophic niche width or space and is highly sensitive to variations in δ13C and δ15N signatures (Brind’Amour and Dubois, 2013; Andrades et al., 2019). Niche metrics were calculated considering all macrofaunal δ13C and δ15N signatures in the studied area (i.e., all feeding modes) in order to have enough sample size and to avoid underrepresentation of feeding groups along contam ination gradients.
2.2. Laboratory analysis All organic contaminant analysis (sterols) were performed on the top 3 cm of undisturbed sediment samples. Sterols were extracted from sediments with a Soxhlet apparatus for 8 h with 80 mL of n-hexane: dichloromethane (DCM) (1:1) after being spiked with surrogate stan dard (5α-androstanol, Wisnieski et al., 2016). The extracts were concentrated to 1 mL, purified and fractionated by silica and alumina liquid chromatographic column with an elution of 5 mL of ethanol/DCM (1:9, v/v), followed by 15 mL of ethanol. The purified extract fractions were dried, derivatized (BSTFA/TMCS (99:1) for 90 min at 70 � C), spiked with internal standard (5α -cholestane) before instrumental an alyses. Sterols were analyzed using an Agilent 7890A gas chromato graph equipped with a flame ionization detector (GC/FID) and an Agilent 19091J-015 capillary fused silica column coated with 5% phe nylmethylsiloxane (50 m, 0.32 mm ID and 0.17 μm film thickness). The oven temperature was programmed from 40 to 240 � C at 5 � C min 1, then to 250 � C at 0.25 � C min 1 (holding for 5 min), then to 280 � C at 5 � C min 1 and to 300 � C at 20 � C min 1 (holding for 10 min). Calibra tion was based on external standard mixtures of coprostanol at nine different concentrations between 0.25 and 15.0 ng μL 1; R2 > 0.995). Benthic organisms were counted and identified and the 12 dominant families, which contributed to over 90% of the total abundance present in the samples were classified by their feeding mode and selected for stable isotopic analysis (Arruda et al., 2003; Jumars et al., 2015). Stable isotopic signatures from sediment organic matter and macrofaunal samples were obtained after samples were dried at 60 � C for 24 h and reduced until fine powder using a mortar and pestle. Carbonate contents of samples were removed by adding 1M HCl to avoid isotopic signals from inorganic carbon. Macrofaunal individuals at each site were pooled according to taxonomic classification to ensure enough sample mass for isotopic analysis. All samples were analyzed using an isotope ratio with a mass spec trometer. Values are expressed in parts per thousand differences from the standard: δX ¼ [(Rsample/Rstandard)-1] x 103, where X is 13C, 15N. R is the corresponding ratio 13C/I2C, 15N/14N. The standard reference ma terials were Vienna Pee Dee Belemnite for carbon and air for nitrogen. Samples previously fixed with formaldehyde 4% were corrected for
3. Results Pollution along Vitoria Bay was evidenced by sediment coprostanol concentrations that ranged from 0.08 to 13.8 μg g 1 (Supplementary Table 1). The contaminated sites with coprostanol values higher than 0.5 μg g 1 included stations 2, 6, 15, 17, 19, 21 and 24 (Fig. 2). The stations with highest coprostanol concentrations (1.4–13.8 μg g 1) were located along the two oultlet channels of Vitoria Bay to the coastal ocean. The stations with lowest coprostanol concentrations and classi fied as non-contaminated were either located on the coastal areas (sta tions 30 and 34) or in the inner estuary (stations 07 and 09). The sediment isotopic signatures had limited variability along the bay indicating a mixture of organic matter sources at all sites. Sediment δ15N values ranged from 5.1‰ to 6.8‰ and were on average similar between contaminated (5.91‰ � 0.09) and non-contaminated sites (5.6‰ � 0.07; ANOVA F ¼ 0.718, p ¼ 0.577). Sediment δ13C signatures were also not directly related to contamination levels. Sediments along the contaminated sites had similar δ13C signatures ( 25.7‰ � 0.08) �ria Bay when compared to other non-contaminated sites in Vito ( 25.6‰ � 0.16; Supplementary Fig. S1). Benthic assemblages were dominated by annelid polychaetes 3
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Estuarine, Coastal and Shelf Science 229 (2019) 106410
Fig. 2. Sediment coprostanol concentrations (μg.g
1
) and contamination levels at the study sites. Values > 0.5 μg g
1
suggest sewage contamination.
(58.3%) including Capitellidae, Spionidae, Cirratulidae, Orbiniidae, Goniadidae, Onuphidae and Nereididae, with a predominance of deposit-feeding groups (Supplementary Table 2). Molluscs were also abundant (41.7% of total abundance) and included mostly suspension feeding (Mytilidae, Solecurtidae and Veneridae) and deposit feeding taxa (Hydrobiidae, Tellinidae). Carnivores were only represented by annelid Goniadidae polychaetes and omnivores were represented by annelid Onuphidae and Nereididae polychaetes. Average δ13C signa tures of benthic macrofauna were similar in non-contaminated �ria bay ( 19.7 � 0.09) and contaminated ( 20.1 � 0.05) sites in Vito (ANOVA F ¼ 0.44, p ¼ 0.516). In general, there was no distinction in δ13C signatures between feeding modes across pollution gradients in Vitoria Bay (Table 1). Average δ15N signatures of benthic macrofauna were significantly lower in contaminated sites (6.4 � 0.05; ANOVA F ¼ 17.26, p ¼ 0.0002; Table 1). Deposit feeders and omnivores had lower δ15N signatures in contaminated sites if compared to noncontaminated sites (Fig. 3; ANOVA F ¼ 18.43, p < 0.01 and ANOVA F ¼ 9.02, p ¼ 0.01, for deposit feeders and omnivores, respectively). The food web δ13C range at non-contaminated sites (2.9062) sug gests that more types of basal resources were available, compared to contaminated sites (0.9207). In a similar pattern, the δ15N range, CD, MNND and SDNND indicated a higher trophic length and diversity at Table 1 Average δ13C and δ15N signatures of deposit-feeders (DF), omnivores (Omni), carnivores (Ca) and suspension feeders (SF) in contaminated and noncontaminated sites of Vitoria Bay. Values averaged within feeding groups. Standard errors in brackets. Non-contaminated δ DF SF Omni Ca All
13
C
19.0 (0.24) 21.6 (0.1) 18.9 (0.48) 20.4 (0.27) 19.7 (0.09)
Fig. 3. Box plots (mean, 5%, 95% CI and outliers as circles) of δ15N signatures of benthic macrofaunal organisms grouped by trophic groups in noncontaminated (Non-C) and contaminated (Cont) sites in Vit� oria Bay.
Contaminated δ
15
N
8.5 (0.14) 5.3 (0.18) 10.7 (0.29) 11.2 (0.43) 8.7 (0.1)
δ
13
C
20.2 (0.09) 22.0 (0.48) 19.0 (0.11) 18.5 (0.54) 20.1 (0.05)
δ
15
non-contaminated sites, if compared to contaminated sites (Fig. 4), with more niche diversification at unpolluted sites. The macrofaunal δ15N and δ13C isotopic signatures followed sedimentary patterns with higher niche width at non-contaminated sites (TA ¼ 0.3841), compared to contaminated sites (TA ¼ 0.3033). Isotopic niche width suggest that non-contaminated sites had more resources being exploited by the
N
6.1 (0.07) 4.3 (0.17) 7.7 (0.2) 9.5 (0.26) 6.4 (0.05)
4
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probabilities of groups (pg) and levels of accuracy (ac) of the classifi cation varied between groups, and were higher for deposit-feeders (pg ¼ 0.53, ac ¼ 72%) and lower for carnivores (pg ¼ 0.09, ac ¼ 8%). Similarly, samples were separated according to the contamination levels using only the isotopic signatures with an accuracy of 51%. Lower levels of contamination had higher probability and accuracy of classification (Table 3). 4. Discussion Integrated approaches are valuable tools to assess ecosystem health and our study supported previous assertions that nitrogen stable iso topes are greatly useful to identify and track pollution in estuaries. Based on sediment coprostanol concentrations that are specific biomarkers indicative of sewage contamination, we showed that increased pollution decreased δ15N isotope signatures of benthic macrofaunal deposit feeders and omnivores in Vitoria Bay. More importantly, benthic food web niche width and mean isotopic signatures of dominant benthic trophic groups were spatially associated with geochemical markers of sediment contamination. Our results showed a strong contrast between contaminated and non-contamined sites based on these metrics, sup porting that stable isotope data can generate useful indices for moni toring estuarine ecosystems (Connolly et al., 2013; Gorman et al., 2017; Souza et al., 2018). �ria Bay receives significant inputs of sewage and other pollutants Vito from several river tributaries, local marinas and other anthropogenic sources, which lead to chronic contamination in some areas (Hadlich et al., 2018). High coprostanol sediment concentrations and other geochemical indices support a strong input from untreated sewage into Vitoria Bay, as observed in other major metropolitan areas in Brazil (Carreira et al., 2002; Hadlich et al., 2018). Untreated sewage is mostly composed of ammonium and has low δ15N signatures (<3‰; Sweeney et al., 1980), which is in contrast to the high nitrogen isotope signatures of treated effluents typically discharged into coastal regions (Gorman et al., 2017). Although receiving high volumes of untreated sewage daily, the sediments of Vitoria Bay did not exhibit strong differences in carbon or nitrogen isotope signatures between contaminated and non-contaminated sites, suggesting a strong mixing with estuarine organic matter sources and supporting the use of biomarkers to identify sewage contamination. �ria Bay indicated The sedimentary and faunal δ13C signatures in Vito strong mixing of freshwater, estuarine and marine organic matter sources. Carbon isotopes did not clearly indicate sediment contamina �ria Bay, which supports previous observations about un tion in Vito specific δ13C signatures in coastal ecosystems and partially rejects our hypothesis. Sites with marked contrast in coprostanol concentrations had similar sedimentary δ13C, suggesting a strong input of natural organic matter sources and effective mixing processes in this bay, which was previously observed in other estuaries of the region (Bernardino et al., 2018a). This complex mixing of organic sources limits the use of sediment carbon isotopic signatures to trace pollution sources into estuarine ecosystems. However, in coastal areas that are very close to point sources of effluents, carbon signatures seem to be additionally useful to detect antropogenic organic matter into food webs (Waldron et al., 2001; Sampaio et al., 2010; Gorman et al., 2017; Souza et al., 2018). Benthic organisms can ingest a range of food particles deposited in estuarine sediments and their isotopic signatures will reflect the incor poration of organic carbon and nitrogen in the area. Our results partially supported our hypothesis of lower δ15N in benthic macrofaunal organ isms within contaminated sites and suggests the incorporation of raw sewage with predominantly light nitrogen. This is in contrast to previous work indicating incorporation of heavier nitrogen from treated effluent outfalls (Connolly et al., 2013; Mazumder et al., 2015; Gorman et al., 2017), and to our knowledge this is the first evidence of incorporation of light nitrogen into benthic organisms from untreated effluents. In
Fig. 4. The six Layman metrics (NR; CR; TA; CD; MNND; SDNND) applied to all macrofaunal data from contaminated (A) and non-contaminated sites (B). Black dots represent their mean, and the red “x”, the corrected mean. Shaded boxes represent the 50, 75 and 95% confidence intervals from dark to light grey. NR ¼ δ15N range; CR ¼ δ13C range; TA ¼ total area of the convex hull; CD ¼ distance to centroid; MNND ¼ mean nearest neighbour distance; SDNND ¼ standard deviation of the nearest neighbour distance. (For interpre tation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
benthic macrofauna (Table 2; Supplemental Fig. S1). We detected a significant association between trophic group δ15N and δ13C isotopic signatures and coprostanol concentrations (CAP, F ¼ 7.26, p ¼ 0.004). The spatial ordination of the samples according to the CAP analysis suggests a separation of the stations along gradients of pollution and benthic trophic groups, explaining 20–60% of the vari ability (Fig. 5). Macrofaunal taxa sampled in contaminated areas had lower δ15N signatures in contrast to non-contaminated areas. The segregation of macrofaunal taxa was also partially driven by feeding modes, with deposit-feeders exhibiting lower δ15N isotopic signatures in sites with higher coprostanol concentrations (Fig. 5). It is possible to distinguish trophic groups by isotopic signatures and concentrations of coprostanol through linear functions, with an overall accuracy of 47%. LDs confirmed the negative relationship between the isotopic signatures and coprostanol in the samples (Table 3). The Table 2 Layman metrics results applied to δ13C and δ15N isotopic signatures of benthic macrofaunal taxa from contaminated and non-contaminated sites. TA ¼ total area of the convex hull; CD ¼ distance to centroid; MNND ¼ mean nearest neighbour distance; SDNND ¼ standard deviation of the nearest neighbour distance. δ13C range δ15N Range TA CD MNND SDNND
Contaminated
Non-contaminated
0.9207 1.4139 0.3033 0.7161 0.7755 0.6313
2.9062 2.8488 0.3841 1.4140 1.9863 0.1985
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Fig. 5. Canonical analyses of principal coordinates (CAP) of samples according to differences in isotopic signatures (δ15N e δ13C) and sediment coprostanol con centrations. Vector is based on Spearman correlation values higher than 0.5 (p < 0.05). Proportion of data explained by axis 1 and 2 are in parenthesis. Significance test result: F ¼ 7.26, p ¼ 0.004.
food web that has been rarely directly assessed. Isotopic niche analysis further captured functional impacts on benthic food webs from sewage input, supporting our hypothesis. Contaminated areas had a 2-times shorter food chain length (δ15N range), lower diversity of resources (based δ13C values) and higher trophic redundancy (CD and MNND values) among their benthic fauna, if compared to non-contaminated sites. These food web metrics support that pollution reduces feeding group niche diversity in estuarine sediments by excluding non-tolerant taxa (Pearson and Rosenberg, 1978). The high trophic redundance in benthic assemblages from contaminated sites revealed an impaired food web structure among benthic consumers that is in contrast to non-contaminated sites. In addition, niche analysis revealed that both δ13C and δ15N values were significantly associated (47% accuracy) with coprostanol levels, even in an ecosystem with a broad environmental variability. These results give additional support to far-reaching impacts that chronic raw sewage discharges have in the Vitoria Bay. In contaminated sites, benthic consumers also had lower δ15N signatures which could be traced to higher trophic levels (Connolly et al., 2013), and could have ecological implications in areas under raw or treated effluent discharges. Contaminated areas can therefore be traced by ni trogen stable isotopes in benthic organisms with accuracy, and more importantly, they could also trace food web integrity and represent functional changes as a result of eutrophication in estuaries and coastal bays. The food web niche dynamics in response to anthropogenic impacts have been previously reported in the literature but with poor repre sentation with respect to tropical estuaries (di Lascio et al., 2013; Deu dero et al., 2014). Brazilian estuaries shelter a diversity of benthic organisms and likely hold unique ecosystem services (Lana and Ber nardino, 2018; Bernardino et al., 2018b), which draws attention to the functional impacts of pollution observed in our study. Niche shifts to wards lower trophic diversity and basal resources reported here may impact a number of estuarine services with potential feedbacks on the support of coastal fisheries that are critical to the local economy and to cultural values associated with estuaries (Barbier et al., 2011). As a
Table 3 Results of the two discriminant function analysis (LDA) of i) macrofaunal trophic groups δ15N and δ13C isotopic signatures and coprostanol concentrations and ii) All macrofaunal taxa δ15N and δ13C isotopic signatures and contamination levels. LDI - coefficients of linear discriminant function between variables. δ15 N δ13 C [coprostanol] δ15 N δ13 C
LDI
Groups
Probability
Accuracy
- 0.552 - 0.056 0.0075
Omnivores Carnivores Deposit-feeders Suspension-feeders No contamination Contamination
0.19 0.09 0.53 0.19 0.37 0.25 to 0.38
25% 20% 72% 8% 61% 75%
0.499 0.093
addition, our study also supported a pronounced contribution of raw sewage nitrogen to benthic deposit-feeders and omnivores in contami nated sites of Vitoria Bay. These benthic consumers likely preferentially ingest fresh organic matter rich in ammonium nitrogen deposited in sediments near tributaries that transport effluents into the bay. In sup port of other studies that have observed multiple taxa incorporating heavy nitrogen near effluent outfalls (Connolly et al., 2013), we also detected a trend of lower δ15N signal in suspension feeders and carni vores at contaminated sites in Vitoria Bay, although these were not significant if compared to non-contaminated sites. However, the lower δ15N isotopic signatures of a broad range of benthic organisms and feeding modes strongly suggests incorporation of ammonium nitrogen from the widespread raw sewage input in Vitoria Bay. These observa tions suggest the pervasive input of nitrogen from untreated effluents in Vitoria Bay that, although are concentrated in areas with highest pollution, may reach benthic organisms and higher trophic levels even in areas that are not classified as contaminated (Hadlich et al., 2018). Our models support those observations and indicate that although coprostanol concentrations are strong predictors of benthic isotopic signatures, all organisms have a tendency of distinct isotopic signatures in contaminated sites which are clearly predominant in Vitoria Bay. This study revealed the impacts of pollution over a model estuarine 6
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result, the untreated effluent discharges in Vitoria bay are not only of ecological concern, but also the long term incorporation of ammonium nitrogen and other trace contaminants in food webs raise important health concerns for its population.
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Contribution CV, HLH and AFB participated in sampling and analyzed data. All authors have analyzed data, contributed to writting and reviewed the manuscript. All authors have approved the final article. Declaration of competing interest The authors declare no actual or potential conflict of interest. Acknowledgements Thanks to R. Servino, L. Gomes and many students that participated in field sampling and C.C. Martins for lipid analysis. HLD was supported by a FAPES graduate scholarship. RA was supported by scholarship from CAPES- DS and CAPES- PDSE (grant 19/2016, process 88881.132520/ 2016–01). AFB was supported by CNPq (PELD grant 441243/2016-9; PQ grant 301161/2017-8) and FAPES (grant 79054684/17). This is a PELD-HCES contribution #05. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ecss.2019.106410. References Anderson, M.J., Willis, T.J., 2003. Canonical analysis of principal coordinates: a useful method of constrained ordination for ecology. Ecology 84, 511–525. https://doi. org/10.1890/0012-9658(2003)084[0511:CAOPCA]2.0.CO;2. Andrades, R., Jackson, A.L., Macieira, R.M., Reis-Filho, J.A., Bernardino, A.F., Joyeux, J.C., Giarrizzo, T., 2019. Niche-related processes in island intertidal communities inferred from stable isotopes data. Ecol. Indicat. 104, 648–658. https://doi.org/ 10.1016/j.ecolind.2019.05.039. Arruda, E.P., Domaneschi, O., Amaral, A.C.Z., 2003. Mollusc feeding guilds on sandy beaches in S~ ao Paulo State, Brazil. Mar. Biol. 143, 691–701. https://doi.org/ 10.1007/s00227-003-1103-y. Barbier, E.B., Hacker, S.D., Kennedy, C., Koch, E.W., Stier, A.C., Silliman, B.R., 2011. The value of estuarine and coastal ecosystem services. Ecol. Monogr. 81, 169–193. https://doi.org/10.1890/10-1510.1. Barletta, M., Lima, A.R.A., Costa, M.F., 2019. Distribution, sources and consequences of nutrients, persistent organic pollutants, metals and microplastics in South American estuaries. Sci. Total Environ. 651, 1199–1218. https://doi.org/10.1016/j. scitotenv.2018.09.276. Bernardino, A.F., Reis, A., Pereira Filho, A.C.D., Gomes, L.E.O., Bissoli, L.B., Barros, F., 2015. Benthic estuarine assemblages of the eastern marine Brazilian Ecoregion (EME). In: Lana, P.C., Bernardino, A.F. (Eds.), Brazilian Estuaries, Brazilian Marine Biodiversity, pp. 95–116. Bernardino, A.F., Gomes, L.E.O., Hadlich, H.L., Andrades, R., Correa, L.B., 2018. Mangrove clearing impacts on macrofaunal assemblages and benthic food webs in a tropical estuary. Mar. Pollut. Bull. 126, 228–235. https://doi.org/10.1016/j. marpolbul.2017.11.008. Bernardino, A.F., Azevedo, A.R.B., Pereira Filho, A.C.D., Gomes, L.E.O., Bissoli, L.B., Barros, F.C.R., 2018. Benthic estuarine assemblages of the eastern marine Brazilian Ecoregion. In: Lana, P.C., Bernardino, A.F. (Eds.), Brazilian Estuaries: a Benthic Perspective. Springer International Publishing. Pgs 95-116. 212pp. Bissoli, L.B., Bernardino, A.F., 2018. Benthic macrofaunal structure and secondary production in tropical estuaries on the Eastern Marine Ecoregion of Brazil. PeerJ 6, e4441. https://doi.org/10.7717/peerj.4441. Borja, A., Franco, J., P� erez, V., 2000. A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments. Mar. Pollut. Bull. 40, 1100–1114. Borja, A., Muxika, I., Franco, J., 2003. The application of aMarine Biotic Index to different impact sources affecting soft-bottom benthic communities along European coasts. Mar. Pollut. Bull. 46, 835–845. Borja, A., Ranasinghe, A., Weisberg, S.B., 2009. Assessing ecological integrity inmarine waters, using multiple indices and ecosystem components: challenges for the future. Mar. Pollut. Bull. 59, 1–4. Brauko, K.M., Muniz, P., Martins, C.C., Lana, P.C., 2016. Assessing the suitability of five benthic indices for environmental health assessment in a large subtropical South
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Estuarine, Coastal and Shelf Science journal homepage: http://www.elsevier.com/locate/ecss
Tracing pollution in estuarine benthic organisms and its impacts on food webs of the Vitoria Bay estuary Cíntia S. Varzim a, Heliatrice L. Hadlich a, Ryan Andrades b, Ana Carolina de A. Mazzuco a, Angelo F. Bernardino a, * a b
Grupo de Ecologia B^entica, Departamento de Oceanografia, Universidade Federal do Espírito Santo, Vit� oria, ES, 29075-910, Brazil Laborat� orio de Ictiologia, Departamento de Oceanografia, Universidade Federal do Espírito Santo, Vit� oria, ES, 29075-910, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Sewage Coprostanol Stable isotopes Isotopic niche Macrofauna
Estuaries in South America commonly receive untreated effluents from nearby metropolitan areas demanding ecosystem-based management solutions to access pollutant impacts. In this study we investigated how organic enrichment in Vit� oria Bay changes benthic macrofaunal isotopic signatures (δ13C and δ15N) and if highly contaminated areas would exhibit lower food web diversity. Geochemical markers of sewage input (coprostanol) revealed low to high levels of pollution in the bay area. The δ15N isotope signatures of benthic deposit-feeders and omnivores were lower in contaminated sites indicating preferential intake of raw sewage and that the ef fect size of sewage input changes among benthic feeding modes. Sites with high sediment coprostanol concen trations (>0.5 μg g 1) were associated with a lower abundance of basal resources and niche shrinking of the benthic food web, suggesting that eutrophication leads to a funtional loss in the sediment-water interface in this estuary. Our study revealed that pollution in the Vit� oria Bay estuary has functional impacts to the estuarine food webs and support previous ascertions that stable isotopes may be a useful and low-cost method to indicate ecosystem health monitoring in estuarine ecosystems.
1. Introduction Estuaries are transitional ecosystems with ecological and economic relevance that are continually impacted by organic pollution. Pollution typically deteriorates water and sediment quality, with consequences for estuarine biodiversity and productivity (Lotze et al., 2006). To effi ciently track those impacts, a common practice is to look for multiple ecological indices of habitat quality that can integrate ecosystem man agement strategies in estuaries (Elliot and Quintino, 2007; Borja et al., 2009). Many ecological and biogeochemical indices can be used in estuarine management programs with accurate, reliable and direct in formation of ecosystem status (Borja et al., 2003). Macrobenthic assemblages are long used as tools to determine sediment ecological status to assess anthropogenic disturbances in estuarine ecosystems (Borja et al., 2003; Muniz et al., 2005; Bernardino et al., 2018a). Benthic indices may rely on assemblage richness, diversity and functional attributes (Borja et al., 2000; Brauko et al., 2016), which offer direct assessment of ecosystem health based on predictive re sponses of populations and assemblages to eutrophication (Pearson and
Rosenberg, 1978). Chemical markers such as sterols are used in parallel to biological indices in order to determine correspondence between sewage contamination and ecological impacts. Sterols including copro stanol (5β-cholestan-3β-ol) and epicoprostanol (5β-cholestan-3α-ol) are not natural in marine sediments, but are present in human fecal material and thus can indicate anthropogenic pollution (Martins et al., 2010). The ratio between sediment epicoprostanol and coprostanol concen trations can additionally provide information on sewage treatment before it is released into estuarine and marine ecosystems (Mudge and Duce, 2005). Multiple organic biomarkers of sediment quality have suggested that �ria Bay, a tropical estuary located in the Eastern Brazil Marine Vito Ecoregion, is predominantly eutrophic or hypertrophic given a high input of untreated sewage in the estuary (Hadlich et al., 2018). The estuary receives nearly 16,000 m 3day 1 of untreated sewage from nearby metropolitan areas through several small river tributaries, and organic and trace metal pollution have been impacting the bay for several decades (Grilo et al., 2013; Costa et al., 2015; Hadlich et al., 2018). The coprostanol concentrations in sediments of Vitoria Bay are
* Corresponding author. E-mail address: [email protected] (A.F. Bernardino). https://doi.org/10.1016/j.ecss.2019.106410 Received 27 March 2019; Received in revised form 13 September 2019; Accepted 8 October 2019 Available online 10 October 2019 0272-7714/© 2019 Elsevier Ltd. All rights reserved.
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within the range of other coastal metropolitan bays under heavy to moderate organic input such as Guanabara Bay (Rio de Janeiro), Cap ibaribe estuary (Pernambuco), and Barbitonga Bay (Santa Catarina) �ria (Hadlich et al., 2018). The metropolitan occupation along the Vito Bay is likely the main cause of the low environmental health of its estuarine ecosystems, which is a common reality of coastal bays and estuaries of South America (Lana and Bernardino, 2018; Barletta et al., 2019). Organic and metal pollutants are of concern as these typically accumulate in estuarine sediments and affect benthic organisms with observed changes in assemblage structure (Venturini et al., 2004; Souza et al., 2013). Although there is strong evidence for geochemical and ecological impacts of pollution on estuaries, the effects of anthropogenic pollution on benthic food webs have been rarely assessed. Stable isotopes have also been used as indicators of pollution impacts for decades (Sweeney et al., 1980). Changes in nitrogen stable isotopic signatures can determine anthropogenic organic inputs as nitrogen isotopes have end-members that vary by 9‰ between natural or sewage inputs; and yet the δ15N of raw effluents are similar to its refractory deposited fraction (Sweeney et al., 1980). Therefore, stable isotope signatures may indicate sewage input over spatial and temporal scales and additionally trace these inputs into coastal food webs (Connolly et al., 2013; Souza et al., 2018). The pollution impacts on benthic food webs are less clear and usually limited to identifying changes in organism’s isotopic signatures. Previ ous work has focused on detecting spatial-temporal changes in stable isotope signatures of selected taxa across pollution gradients (Sampaio et al., 2010; Connolly et al., 2013; Gorman et al., 2017). However, changes in faunal isotope signatures do not provide an integrated overview of food web diversity in coastal ecosystems, which is a func tional indice of interest. Otherwise, Bayesian stable isotope estimates from the SIBER model (Stable Isotope Bayesian Ellipses in R; Jackson et al., 2011), provide robust metrics allowing to test niche shrinking and trophic diversity loss in aquatic food webs under pollution and other
anthropogenic impacts (Bernardino et al., 2018a; Carvalho et al., 2019; Price et al., 2019). In this study, stable isotope signatures of benthic macrofaunal as semblages were assessed and applied in food web models to understand the long-term (scales of weeks to months) pollution impacts on benthic food webs in a tropical estuary. Benthic isotopic signatures spanning a range of feeding modes including suspensivores, deposit-feeders, car nivores and omnivores were used to estimate benthic food web trophic width and amplitude along sewage contamination gradients. The large�ria Bay were studied in detail scale gradients of organic pollution in Vito by Hadlich et al. (2018) and indicated spatially distinct contamination levels, which were used as a contemporary baseline for the food web assessment in this study. We asked if the δ13C and δ15N isotope signa tures of benthic organisms and the benthic food web diversity would be �ria spatially distinct across levels of sedimentary pollution in the Vito Bay estuary. So here we tested if the isotopic signatures of benthic or ganisms would vary spatially with respect to i) sediment coprostanol concentrations and ii) benthic feeding modes. 2. Materials and methods 2.1. Study area and sampling �ria Bay (20� 180 S, 40� 200 W; Fig. 1) is an urbanized estuary on the Vito eastern coast of Brazil with historical high levels of sewage contami nation (Bernardino et al., 2015; Grilo et al., 2013; Bissoli and Bernar �ria Bay has an area of 18.2 km2 and a catchment area of dino, 2018). Vito 1728 km2 with an average freshwater discharge of 18.7 m3 s 1 (Lessa et al., 2019). The bay is connected to the coastal ocean through two channels; the southern port channel is wider (~160 m wide) and deeper (5–24 m) than the northern passagen channel that is on average 35 m wide and 1–8 m deep. The channel bed morphology and an abundant mangrove vegetation in Vitoria Bay results in a strong tidal
Fig. 1. Sampling sites along the Vit� oria bay metropolitan estuary. River tributaries (names in yellow) and small channels with input of raw sewage to the bay are indicated by arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2
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potential preservation artifacts by adding 1‰ to δ13C macrofaunal sig natures (Sarakinos et al., 2002; Demopoulos et al., 2007).
amplification inside the estuary, with stronger ebb flows and higher sediment bed load transport out of the bay (Rigo, 2004; Lessa et al., 2019). On areas near the coast both channels are highy impacted by urban development that resulted in loss of intertial mangrove forests for �ria Bay also the construction of an industrial port and local marinas. Vito receives a significant amount of treated and untreated sewage from nearby cities through its river tributaries, resulting in a historical accumulation of organic and trace metal pollutants in estuarine sedi ments (Grilo et al., 2013; Costa et al., 2015; Hadlich et al., 2018). �ria Bay was sampled at 11 sites in November 2014 along a The Vito salinity gradient from the inner estuary towards the coastal ocean which are all under influence of multiple pollution discharges into the bay (Fig. 1). Based on vertical CTD casts during sampling, average salinity within the bay ranged between 23.1 and 36.2 PSU and the average temperature ranged between 22.1 and 27 � C. The sampling sites 02, 06, 07, 09 and 15 exhibited polyhaline conditions and the other sites 17, 19, 21, 24, 30 and 34 were euhaline at the time of sampling. In each site, two sediment samples (i.e., replicates) were collected with a Day Grab (0.1 m2), and the top 3 cm of undisturbed sediment from each independent replicates were mixed into one composite sam ple for sterol biomarker analyses. Samples for biomarkers were imme diately stored in pre-cleaned aluminum containers and remained frozen ( 20 � C) until laboratory analysis. Benthic macrofaunal organisms (>0.5 mm) were sampled from the remaining sediments from each in dependent replicate along the 11 stations. All faunal samples were sieved live through a 0.5 mm mesh and fixed in buffered 10% formal dehyde and transferred to 70% Ethanol until laboratory analysis.
2.3. Statistical analysis Spatial differences of macrofaunal isotopic signatures and food web niche width were compared between contaminated and noncontaminated sites using all macrofaunal taxa and by grouping taxa into feeding modes. Analysis of Variance (ANOVA) tests were used to test for spatial variation of isotopic signatures across contamination levels and benthic trophic modes. Pairwise multiple comparisons were tested by Tukey’s HSD and adjusted Bonferroni-Dunn tests with applied corrections (Quintana et al., 2015). The coprostanol concentration in each site were used to determine sewage contamination levels adapted from Hadlich et al. (2018). Sites were classified as “non contaminated” if average coprostanol concentrations were below <0.5 μg g 1; whereas “contaminated” sites had sediment coprostanol concentrations above 0.5 μg g 1. Differences in isotopic signatures among sites and the correspon dence between benthic isotopic signatures and coprostanol concentra tions were analyzed using a canonical analysis of principal coordinates (CAP) complemented by multidimensional scaling based on the Euclidean distance between sites (Anderson and Willis, 2003). Addi tionally, canonical discriminant function analyses DFA were performed in order to test the classification of samples in trophic groups and levels of contamination (Venables and Ripley, 2002; Mazzuco et al., 2019). The results from the DFAs were interpreted based on the linear discriminant coefficients, which described the relationship of the iso topic signatures and coprostanol concentrations. Jackknife re-samplings were included in the analyses to test the accuracy of the classifications by DFAs (Tukey, 1958; Ripley, 1996). All analyses were performed in R (R Core Team, 2016). Isotopic niche metrics were evaluated through the method of Stable Isotope Bayesian Ellipses in R (SIBER package; Jackson et al., 2011) based on the Layman’s niche metrics δ15N range, δ13C range, distance to centroid (CD), mean nearest neighbour distance (MNND) and standard deviation of the nearest neighbour distance (SDNND) (Layman et al., 2007). Total Area (TA) is a metric that indicates the trophic niche width or space and is highly sensitive to variations in δ13C and δ15N signatures (Brind’Amour and Dubois, 2013; Andrades et al., 2019). Niche metrics were calculated considering all macrofaunal δ13C and δ15N signatures in the studied area (i.e., all feeding modes) in order to have enough sample size and to avoid underrepresentation of feeding groups along contam ination gradients.
2.2. Laboratory analysis All organic contaminant analysis (sterols) were performed on the top 3 cm of undisturbed sediment samples. Sterols were extracted from sediments with a Soxhlet apparatus for 8 h with 80 mL of n-hexane: dichloromethane (DCM) (1:1) after being spiked with surrogate stan dard (5α-androstanol, Wisnieski et al., 2016). The extracts were concentrated to 1 mL, purified and fractionated by silica and alumina liquid chromatographic column with an elution of 5 mL of ethanol/DCM (1:9, v/v), followed by 15 mL of ethanol. The purified extract fractions were dried, derivatized (BSTFA/TMCS (99:1) for 90 min at 70 � C), spiked with internal standard (5α -cholestane) before instrumental an alyses. Sterols were analyzed using an Agilent 7890A gas chromato graph equipped with a flame ionization detector (GC/FID) and an Agilent 19091J-015 capillary fused silica column coated with 5% phe nylmethylsiloxane (50 m, 0.32 mm ID and 0.17 μm film thickness). The oven temperature was programmed from 40 to 240 � C at 5 � C min 1, then to 250 � C at 0.25 � C min 1 (holding for 5 min), then to 280 � C at 5 � C min 1 and to 300 � C at 20 � C min 1 (holding for 10 min). Calibra tion was based on external standard mixtures of coprostanol at nine different concentrations between 0.25 and 15.0 ng μL 1; R2 > 0.995). Benthic organisms were counted and identified and the 12 dominant families, which contributed to over 90% of the total abundance present in the samples were classified by their feeding mode and selected for stable isotopic analysis (Arruda et al., 2003; Jumars et al., 2015). Stable isotopic signatures from sediment organic matter and macrofaunal samples were obtained after samples were dried at 60 � C for 24 h and reduced until fine powder using a mortar and pestle. Carbonate contents of samples were removed by adding 1M HCl to avoid isotopic signals from inorganic carbon. Macrofaunal individuals at each site were pooled according to taxonomic classification to ensure enough sample mass for isotopic analysis. All samples were analyzed using an isotope ratio with a mass spec trometer. Values are expressed in parts per thousand differences from the standard: δX ¼ [(Rsample/Rstandard)-1] x 103, where X is 13C, 15N. R is the corresponding ratio 13C/I2C, 15N/14N. The standard reference ma terials were Vienna Pee Dee Belemnite for carbon and air for nitrogen. Samples previously fixed with formaldehyde 4% were corrected for
3. Results Pollution along Vitoria Bay was evidenced by sediment coprostanol concentrations that ranged from 0.08 to 13.8 μg g 1 (Supplementary Table 1). The contaminated sites with coprostanol values higher than 0.5 μg g 1 included stations 2, 6, 15, 17, 19, 21 and 24 (Fig. 2). The stations with highest coprostanol concentrations (1.4–13.8 μg g 1) were located along the two oultlet channels of Vitoria Bay to the coastal ocean. The stations with lowest coprostanol concentrations and classi fied as non-contaminated were either located on the coastal areas (sta tions 30 and 34) or in the inner estuary (stations 07 and 09). The sediment isotopic signatures had limited variability along the bay indicating a mixture of organic matter sources at all sites. Sediment δ15N values ranged from 5.1‰ to 6.8‰ and were on average similar between contaminated (5.91‰ � 0.09) and non-contaminated sites (5.6‰ � 0.07; ANOVA F ¼ 0.718, p ¼ 0.577). Sediment δ13C signatures were also not directly related to contamination levels. Sediments along the contaminated sites had similar δ13C signatures ( 25.7‰ � 0.08) �ria Bay when compared to other non-contaminated sites in Vito ( 25.6‰ � 0.16; Supplementary Fig. S1). Benthic assemblages were dominated by annelid polychaetes 3
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Fig. 2. Sediment coprostanol concentrations (μg.g
1
) and contamination levels at the study sites. Values > 0.5 μg g
1
suggest sewage contamination.
(58.3%) including Capitellidae, Spionidae, Cirratulidae, Orbiniidae, Goniadidae, Onuphidae and Nereididae, with a predominance of deposit-feeding groups (Supplementary Table 2). Molluscs were also abundant (41.7% of total abundance) and included mostly suspension feeding (Mytilidae, Solecurtidae and Veneridae) and deposit feeding taxa (Hydrobiidae, Tellinidae). Carnivores were only represented by annelid Goniadidae polychaetes and omnivores were represented by annelid Onuphidae and Nereididae polychaetes. Average δ13C signa tures of benthic macrofauna were similar in non-contaminated �ria bay ( 19.7 � 0.09) and contaminated ( 20.1 � 0.05) sites in Vito (ANOVA F ¼ 0.44, p ¼ 0.516). In general, there was no distinction in δ13C signatures between feeding modes across pollution gradients in Vitoria Bay (Table 1). Average δ15N signatures of benthic macrofauna were significantly lower in contaminated sites (6.4 � 0.05; ANOVA F ¼ 17.26, p ¼ 0.0002; Table 1). Deposit feeders and omnivores had lower δ15N signatures in contaminated sites if compared to noncontaminated sites (Fig. 3; ANOVA F ¼ 18.43, p < 0.01 and ANOVA F ¼ 9.02, p ¼ 0.01, for deposit feeders and omnivores, respectively). The food web δ13C range at non-contaminated sites (2.9062) sug gests that more types of basal resources were available, compared to contaminated sites (0.9207). In a similar pattern, the δ15N range, CD, MNND and SDNND indicated a higher trophic length and diversity at Table 1 Average δ13C and δ15N signatures of deposit-feeders (DF), omnivores (Omni), carnivores (Ca) and suspension feeders (SF) in contaminated and noncontaminated sites of Vitoria Bay. Values averaged within feeding groups. Standard errors in brackets. Non-contaminated δ DF SF Omni Ca All
13
C
19.0 (0.24) 21.6 (0.1) 18.9 (0.48) 20.4 (0.27) 19.7 (0.09)
Fig. 3. Box plots (mean, 5%, 95% CI and outliers as circles) of δ15N signatures of benthic macrofaunal organisms grouped by trophic groups in noncontaminated (Non-C) and contaminated (Cont) sites in Vit� oria Bay.
Contaminated δ
15
N
8.5 (0.14) 5.3 (0.18) 10.7 (0.29) 11.2 (0.43) 8.7 (0.1)
δ
13
C
20.2 (0.09) 22.0 (0.48) 19.0 (0.11) 18.5 (0.54) 20.1 (0.05)
δ
15
non-contaminated sites, if compared to contaminated sites (Fig. 4), with more niche diversification at unpolluted sites. The macrofaunal δ15N and δ13C isotopic signatures followed sedimentary patterns with higher niche width at non-contaminated sites (TA ¼ 0.3841), compared to contaminated sites (TA ¼ 0.3033). Isotopic niche width suggest that non-contaminated sites had more resources being exploited by the
N
6.1 (0.07) 4.3 (0.17) 7.7 (0.2) 9.5 (0.26) 6.4 (0.05)
4
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probabilities of groups (pg) and levels of accuracy (ac) of the classifi cation varied between groups, and were higher for deposit-feeders (pg ¼ 0.53, ac ¼ 72%) and lower for carnivores (pg ¼ 0.09, ac ¼ 8%). Similarly, samples were separated according to the contamination levels using only the isotopic signatures with an accuracy of 51%. Lower levels of contamination had higher probability and accuracy of classification (Table 3). 4. Discussion Integrated approaches are valuable tools to assess ecosystem health and our study supported previous assertions that nitrogen stable iso topes are greatly useful to identify and track pollution in estuaries. Based on sediment coprostanol concentrations that are specific biomarkers indicative of sewage contamination, we showed that increased pollution decreased δ15N isotope signatures of benthic macrofaunal deposit feeders and omnivores in Vitoria Bay. More importantly, benthic food web niche width and mean isotopic signatures of dominant benthic trophic groups were spatially associated with geochemical markers of sediment contamination. Our results showed a strong contrast between contaminated and non-contamined sites based on these metrics, sup porting that stable isotope data can generate useful indices for moni toring estuarine ecosystems (Connolly et al., 2013; Gorman et al., 2017; Souza et al., 2018). �ria Bay receives significant inputs of sewage and other pollutants Vito from several river tributaries, local marinas and other anthropogenic sources, which lead to chronic contamination in some areas (Hadlich et al., 2018). High coprostanol sediment concentrations and other geochemical indices support a strong input from untreated sewage into Vitoria Bay, as observed in other major metropolitan areas in Brazil (Carreira et al., 2002; Hadlich et al., 2018). Untreated sewage is mostly composed of ammonium and has low δ15N signatures (<3‰; Sweeney et al., 1980), which is in contrast to the high nitrogen isotope signatures of treated effluents typically discharged into coastal regions (Gorman et al., 2017). Although receiving high volumes of untreated sewage daily, the sediments of Vitoria Bay did not exhibit strong differences in carbon or nitrogen isotope signatures between contaminated and non-contaminated sites, suggesting a strong mixing with estuarine organic matter sources and supporting the use of biomarkers to identify sewage contamination. �ria Bay indicated The sedimentary and faunal δ13C signatures in Vito strong mixing of freshwater, estuarine and marine organic matter sources. Carbon isotopes did not clearly indicate sediment contamina �ria Bay, which supports previous observations about un tion in Vito specific δ13C signatures in coastal ecosystems and partially rejects our hypothesis. Sites with marked contrast in coprostanol concentrations had similar sedimentary δ13C, suggesting a strong input of natural organic matter sources and effective mixing processes in this bay, which was previously observed in other estuaries of the region (Bernardino et al., 2018a). This complex mixing of organic sources limits the use of sediment carbon isotopic signatures to trace pollution sources into estuarine ecosystems. However, in coastal areas that are very close to point sources of effluents, carbon signatures seem to be additionally useful to detect antropogenic organic matter into food webs (Waldron et al., 2001; Sampaio et al., 2010; Gorman et al., 2017; Souza et al., 2018). Benthic organisms can ingest a range of food particles deposited in estuarine sediments and their isotopic signatures will reflect the incor poration of organic carbon and nitrogen in the area. Our results partially supported our hypothesis of lower δ15N in benthic macrofaunal organ isms within contaminated sites and suggests the incorporation of raw sewage with predominantly light nitrogen. This is in contrast to previous work indicating incorporation of heavier nitrogen from treated effluent outfalls (Connolly et al., 2013; Mazumder et al., 2015; Gorman et al., 2017), and to our knowledge this is the first evidence of incorporation of light nitrogen into benthic organisms from untreated effluents. In
Fig. 4. The six Layman metrics (NR; CR; TA; CD; MNND; SDNND) applied to all macrofaunal data from contaminated (A) and non-contaminated sites (B). Black dots represent their mean, and the red “x”, the corrected mean. Shaded boxes represent the 50, 75 and 95% confidence intervals from dark to light grey. NR ¼ δ15N range; CR ¼ δ13C range; TA ¼ total area of the convex hull; CD ¼ distance to centroid; MNND ¼ mean nearest neighbour distance; SDNND ¼ standard deviation of the nearest neighbour distance. (For interpre tation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
benthic macrofauna (Table 2; Supplemental Fig. S1). We detected a significant association between trophic group δ15N and δ13C isotopic signatures and coprostanol concentrations (CAP, F ¼ 7.26, p ¼ 0.004). The spatial ordination of the samples according to the CAP analysis suggests a separation of the stations along gradients of pollution and benthic trophic groups, explaining 20–60% of the vari ability (Fig. 5). Macrofaunal taxa sampled in contaminated areas had lower δ15N signatures in contrast to non-contaminated areas. The segregation of macrofaunal taxa was also partially driven by feeding modes, with deposit-feeders exhibiting lower δ15N isotopic signatures in sites with higher coprostanol concentrations (Fig. 5). It is possible to distinguish trophic groups by isotopic signatures and concentrations of coprostanol through linear functions, with an overall accuracy of 47%. LDs confirmed the negative relationship between the isotopic signatures and coprostanol in the samples (Table 3). The Table 2 Layman metrics results applied to δ13C and δ15N isotopic signatures of benthic macrofaunal taxa from contaminated and non-contaminated sites. TA ¼ total area of the convex hull; CD ¼ distance to centroid; MNND ¼ mean nearest neighbour distance; SDNND ¼ standard deviation of the nearest neighbour distance. δ13C range δ15N Range TA CD MNND SDNND
Contaminated
Non-contaminated
0.9207 1.4139 0.3033 0.7161 0.7755 0.6313
2.9062 2.8488 0.3841 1.4140 1.9863 0.1985
5
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Fig. 5. Canonical analyses of principal coordinates (CAP) of samples according to differences in isotopic signatures (δ15N e δ13C) and sediment coprostanol con centrations. Vector is based on Spearman correlation values higher than 0.5 (p < 0.05). Proportion of data explained by axis 1 and 2 are in parenthesis. Significance test result: F ¼ 7.26, p ¼ 0.004.
food web that has been rarely directly assessed. Isotopic niche analysis further captured functional impacts on benthic food webs from sewage input, supporting our hypothesis. Contaminated areas had a 2-times shorter food chain length (δ15N range), lower diversity of resources (based δ13C values) and higher trophic redundancy (CD and MNND values) among their benthic fauna, if compared to non-contaminated sites. These food web metrics support that pollution reduces feeding group niche diversity in estuarine sediments by excluding non-tolerant taxa (Pearson and Rosenberg, 1978). The high trophic redundance in benthic assemblages from contaminated sites revealed an impaired food web structure among benthic consumers that is in contrast to non-contaminated sites. In addition, niche analysis revealed that both δ13C and δ15N values were significantly associated (47% accuracy) with coprostanol levels, even in an ecosystem with a broad environmental variability. These results give additional support to far-reaching impacts that chronic raw sewage discharges have in the Vitoria Bay. In contaminated sites, benthic consumers also had lower δ15N signatures which could be traced to higher trophic levels (Connolly et al., 2013), and could have ecological implications in areas under raw or treated effluent discharges. Contaminated areas can therefore be traced by ni trogen stable isotopes in benthic organisms with accuracy, and more importantly, they could also trace food web integrity and represent functional changes as a result of eutrophication in estuaries and coastal bays. The food web niche dynamics in response to anthropogenic impacts have been previously reported in the literature but with poor repre sentation with respect to tropical estuaries (di Lascio et al., 2013; Deu dero et al., 2014). Brazilian estuaries shelter a diversity of benthic organisms and likely hold unique ecosystem services (Lana and Ber nardino, 2018; Bernardino et al., 2018b), which draws attention to the functional impacts of pollution observed in our study. Niche shifts to wards lower trophic diversity and basal resources reported here may impact a number of estuarine services with potential feedbacks on the support of coastal fisheries that are critical to the local economy and to cultural values associated with estuaries (Barbier et al., 2011). As a
Table 3 Results of the two discriminant function analysis (LDA) of i) macrofaunal trophic groups δ15N and δ13C isotopic signatures and coprostanol concentrations and ii) All macrofaunal taxa δ15N and δ13C isotopic signatures and contamination levels. LDI - coefficients of linear discriminant function between variables. δ15 N δ13 C [coprostanol] δ15 N δ13 C
LDI
Groups
Probability
Accuracy
- 0.552 - 0.056 0.0075
Omnivores Carnivores Deposit-feeders Suspension-feeders No contamination Contamination
0.19 0.09 0.53 0.19 0.37 0.25 to 0.38
25% 20% 72% 8% 61% 75%
0.499 0.093
addition, our study also supported a pronounced contribution of raw sewage nitrogen to benthic deposit-feeders and omnivores in contami nated sites of Vitoria Bay. These benthic consumers likely preferentially ingest fresh organic matter rich in ammonium nitrogen deposited in sediments near tributaries that transport effluents into the bay. In sup port of other studies that have observed multiple taxa incorporating heavy nitrogen near effluent outfalls (Connolly et al., 2013), we also detected a trend of lower δ15N signal in suspension feeders and carni vores at contaminated sites in Vitoria Bay, although these were not significant if compared to non-contaminated sites. However, the lower δ15N isotopic signatures of a broad range of benthic organisms and feeding modes strongly suggests incorporation of ammonium nitrogen from the widespread raw sewage input in Vitoria Bay. These observa tions suggest the pervasive input of nitrogen from untreated effluents in Vitoria Bay that, although are concentrated in areas with highest pollution, may reach benthic organisms and higher trophic levels even in areas that are not classified as contaminated (Hadlich et al., 2018). Our models support those observations and indicate that although coprostanol concentrations are strong predictors of benthic isotopic signatures, all organisms have a tendency of distinct isotopic signatures in contaminated sites which are clearly predominant in Vitoria Bay. This study revealed the impacts of pollution over a model estuarine 6
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result, the untreated effluent discharges in Vitoria bay are not only of ecological concern, but also the long term incorporation of ammonium nitrogen and other trace contaminants in food webs raise important health concerns for its population.
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Contribution CV, HLH and AFB participated in sampling and analyzed data. All authors have analyzed data, contributed to writting and reviewed the manuscript. All authors have approved the final article. Declaration of competing interest The authors declare no actual or potential conflict of interest. Acknowledgements Thanks to R. Servino, L. Gomes and many students that participated in field sampling and C.C. Martins for lipid analysis. HLD was supported by a FAPES graduate scholarship. RA was supported by scholarship from CAPES- DS and CAPES- PDSE (grant 19/2016, process 88881.132520/ 2016–01). AFB was supported by CNPq (PELD grant 441243/2016-9; PQ grant 301161/2017-8) and FAPES (grant 79054684/17). This is a PELD-HCES contribution #05. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ecss.2019.106410. References Anderson, M.J., Willis, T.J., 2003. Canonical analysis of principal coordinates: a useful method of constrained ordination for ecology. Ecology 84, 511–525. https://doi. org/10.1890/0012-9658(2003)084[0511:CAOPCA]2.0.CO;2. Andrades, R., Jackson, A.L., Macieira, R.M., Reis-Filho, J.A., Bernardino, A.F., Joyeux, J.C., Giarrizzo, T., 2019. Niche-related processes in island intertidal communities inferred from stable isotopes data. Ecol. Indicat. 104, 648–658. https://doi.org/ 10.1016/j.ecolind.2019.05.039. Arruda, E.P., Domaneschi, O., Amaral, A.C.Z., 2003. Mollusc feeding guilds on sandy beaches in S~ ao Paulo State, Brazil. Mar. Biol. 143, 691–701. https://doi.org/ 10.1007/s00227-003-1103-y. Barbier, E.B., Hacker, S.D., Kennedy, C., Koch, E.W., Stier, A.C., Silliman, B.R., 2011. The value of estuarine and coastal ecosystem services. Ecol. Monogr. 81, 169–193. https://doi.org/10.1890/10-1510.1. Barletta, M., Lima, A.R.A., Costa, M.F., 2019. Distribution, sources and consequences of nutrients, persistent organic pollutants, metals and microplastics in South American estuaries. Sci. Total Environ. 651, 1199–1218. https://doi.org/10.1016/j. scitotenv.2018.09.276. Bernardino, A.F., Reis, A., Pereira Filho, A.C.D., Gomes, L.E.O., Bissoli, L.B., Barros, F., 2015. Benthic estuarine assemblages of the eastern marine Brazilian Ecoregion (EME). In: Lana, P.C., Bernardino, A.F. (Eds.), Brazilian Estuaries, Brazilian Marine Biodiversity, pp. 95–116. Bernardino, A.F., Gomes, L.E.O., Hadlich, H.L., Andrades, R., Correa, L.B., 2018. Mangrove clearing impacts on macrofaunal assemblages and benthic food webs in a tropical estuary. Mar. Pollut. Bull. 126, 228–235. https://doi.org/10.1016/j. marpolbul.2017.11.008. Bernardino, A.F., Azevedo, A.R.B., Pereira Filho, A.C.D., Gomes, L.E.O., Bissoli, L.B., Barros, F.C.R., 2018. Benthic estuarine assemblages of the eastern marine Brazilian Ecoregion. In: Lana, P.C., Bernardino, A.F. (Eds.), Brazilian Estuaries: a Benthic Perspective. Springer International Publishing. Pgs 95-116. 212pp. Bissoli, L.B., Bernardino, A.F., 2018. Benthic macrofaunal structure and secondary production in tropical estuaries on the Eastern Marine Ecoregion of Brazil. PeerJ 6, e4441. https://doi.org/10.7717/peerj.4441. Borja, A., Franco, J., P� erez, V., 2000. A marine biotic index to establish the ecological quality of soft-bottom benthos within European estuarine and coastal environments. Mar. Pollut. Bull. 40, 1100–1114. Borja, A., Muxika, I., Franco, J., 2003. The application of aMarine Biotic Index to different impact sources affecting soft-bottom benthic communities along European coasts. Mar. Pollut. Bull. 46, 835–845. Borja, A., Ranasinghe, A., Weisberg, S.B., 2009. Assessing ecological integrity inmarine waters, using multiple indices and ecosystem components: challenges for the future. Mar. Pollut. Bull. 59, 1–4. Brauko, K.M., Muniz, P., Martins, C.C., Lana, P.C., 2016. Assessing the suitability of five benthic indices for environmental health assessment in a large subtropical South
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