Issue
Knowl. Manag. Aquat. Ecosyst.
Number 421, 2020
Topical Issue on Fish Ecology
Article Number 41
Number of page(s) 15
DOI https://doi.org/10.1051/kmae/2020033
Published online 30 October 2020

© J. Hong and B. Gu, Published by EDP Sciences 2020

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC-BY-ND (https://creativecommons.org/licenses/by-nd/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. If you remix, transform, or build upon the material, you may not distribute the modified material.

1 Introduction

A number of environmental indicators have been used to assess ecological processes in aquatic ecosystems. Current indicators of environmental changes for freshwater include nutrient loading such as soil and water total phosphorus (TP), total nitrogen (TN) concentrations, biodiversity and primary productivity (Jeppesen and Sammalkorpi, 2002). These indicators often reveal the changes in the impacted systems at the basal resource levels (nutrients and primary producers). Identification of signs of environmental change along higher trophic levels in aquatic food webs is critical for the restoration of disturbed systems and wildlife protection (Vander Zanden et al., 2005).

Nitrogen stable isotope compositions of organic matter may be a complementary means to detect environmental changes in aquatic ecosystems. The ratios of 15 N/14N (defined as δ15N) may provide insight into the sources, sinks and cycling of nitrogen in biota that interact with their physical and chemical environments (Peterson and Fry, 1987). The use of δ15N as an indicator of aquatic eutrophication is based on the fact that increases in ecosystem productivity controlled by nutrient enrichments will lead to decreases in isotope fractionation by primary producers and the transfers of organic matter from one trophic level to another will result in predictable isotope enrichment along food chain (Post, 2002; Vander Zanden et al., 2015). At present, the use of consumer δ15N largely focuses on the source of nitrogen contaminations (Lake et al., 2001; Vander Zanden et al., 2005; Schlacher et al., 2005; Robinson et al., 2016; Souza et al., 2018) and trophic interactions (Post, 2002; Vander Zanden et al., 2015; Wang et al., 2018). Very few studies link consumer isotope composition to primary productivity in freshwater ecosystems (Woodland et al., 2012; Hou et al., 2013).

In this study, we compared δ15N ratios in 10 species of freshwater fish collected along a nutrient gradient in the Everglades, Florida, USA. The purposes of this study were (1) to understand the responses and mechanisms controlling the isotope variations along the nutrient gradient, and (2) to evaluate if fish δ15N is a reliable and feasible candidate for human-induced eutrophication in freshwater wetlands. This study provides insight into isotopic responses to changing water quality.

2 Materials and methods

2.1 Site description

The Everglades is the largest subtropical peatland in the United States with its historic geochemistry and biological community characterizing an oligotrophic ecosystem (Wright et al., 2008; Richardson, 2010). Since human settlement, a large portion of the Everglades peatland immediately south of Lake Okeechobee was converted into farmlands, i.e., Everglades Agricultural Area (EAA). The remaining Everglades has been divided by drainage canals, levees and water control structures into three Water Conservation Areas (WCA-1, WCA-2 and WCA-3), and Everglades National Park (ENP). Discharge of EAA runoff which contains high concentrations of total phosphorus (TP) and total nitrogen (TN) has led to cattail (Typha spp.) invasion and replacement of the native macrophytes and periphyton (Sklar et al., 2005). Increased P loads in surface water runoff have shifted portions of the ecosystem from oligotrophic to eutrophic states. As a result, TP and TN concentrations in the water column and soil near the inflow regions are elevated (Wright et al., 2008).

2.2 Sources of data

Stable isotope data on fish collected from 1994 to 1999 were downloaded from the United States Geological Survey South Florida Information Access (Appendix Tab. A1). A total of 16 sites, with three sites in Stormwater Treatment Area-1 West (STA-1W) and 13 sites in the WCAs and ENP (Fig. 1) were sampled, often on multiple field trips. These sites include canals, near levee inflow and outflow structures and interior marshes. Fish were caught randomly and brought to a laboratory where muscle tissue from each fish was removed, dried and ground to fine powder for stable isotopes analysis. Additional samples of mosquitofish were collected along the nutrient gradient in WCA-2A in 2007 (Fig. 1 and Tab. 1). Muscle tissue from 3 to 5 mosquitofish was composed into a single sample per site and processed as above prior to stable isotope analysis.

Select environmental data were downloaded from DBHYDRO, a hydrometeorologic, water quality, and hydrogeologic data retrieval system managed by the South Florida Water Management District (West Palm Beach Florida, USA). Water quality data recorded one year before fish collection date were averaged to reflect environmental conditions of each habitat. When environmental data were not available from the same fish collection site, data from closely located sites were used.

Fish samples collected in the 1990s were analyzed for stable isotopic composition (δ13C and δ15N) using a Micromass Optima continuous flow mass spectrometer coupled to a Carlo Erba elemental analyzer at US Geological Survey. Results are reported in the usual delta notation relative to V-PDB for 13C and air for 15N. Analytical precision (1σ) based on repeat analysis of both samples was generally in the range of 0.1‒0.2‰ for both C and N, but for some samples replication was no better than ± 0.5 ‰ due to sample heterogeneity (Kendall et al., 2005). Mosquitofish samples collected in 2007 were analyzed using a Carlo Erba Elemental Analyzer interfaced to a Finnigan MAT Delta Plus XP stable isotope ratio mass spectrometer (IRMS) at Florida State University. The precision of the C and N isotope analysis was ±0.2‰ (1σ) or better on the basis of repeated analysis of different laboratory standards.

thumbnail Fig. 1

Map showing sampling sites in Stormwater Management Area 1 West (STA-1W), Water Conservation Areas (WCAs) and Everglades National Park (ENP) of south Florida. The insert indicates location of the study site in the USA.

Table 1

Averages of environmental variables for each study site during the study period. Sites are generally listed from north to south.

2.3 Statistics

Because not all fish were found in each site, only fish found in at least 6 sites were used in this analysis. Ten species belonging to different trophic levels met this criterion. The δ15N ratios of select fish species from different years of collection were pooled from a given site (Appendix Tab. A1). Spearman Rank Correlation analysis was used to establish relationships between TP, TN and molar TN:TP ratios and δ15N of each fish species. All statistics were performed using SAS JMP (Version 7, SAS Institute). Statistics were considered significant at P < 0.05.

3 Results

3.1 Environmental conditions

The 16 study sites received water with considerably different concentrations of nutrients and other chemical compounds (Tab. 1). Study sites at Cell 3 and Cell 4 of the STA-1W, and E0 and F1 of WCA-2A, which received direct EAA discharges are highly enriched with TP (>40 μg L‒1). Study sites located immediately downstream of STA-1W, WCAs or close to interior canals are moderately enriched with TP (>10 and <30 μg L‒1). Interior marshes in WCAs and ENP typically maintain the oligotrophic state indicated by a low TP concentration (<10 μg L‒1). Total nitrogen (TN) and dissolved inorganic nitrogen (DIN) concentrations were also higher at the near inflow sites than the marsh interior sites, except for U3, the interior site of WCA-2A, which is enriched with ammonium and TN. Theses study sites are also generally characterized by above-neutral pH, high alkalinity, and low dissolved oxygen (DO) concentrations, with an exception of an interior site in WCA-1. This is a rain-driven system, where pH values are low (Tab. 1).

3.2 Fish ecology and δ15N ratios

Species described in Table 2 represent major fish assemblage in the subtropical wetlands. They are either omnivorous, feeding on both algae, aquatic macrophytes and invertebrates (killifish, golden topminnow, mosquitofish and sailfin molly), primary consumers, feeding on aquatic insects (bluegill and spotted sunfish), snails (redear sunfish) or piscivores (largemouth bass and Florida gar).

Average δ15N ratios for each species collected from multiple sites and years ranged from 8.4‰ in sailfin molly to 11.6‰ in Florida gar. In general, δ15N ratios reflect the trophic position of each species. For example, both the Florida gar and largemouth bass which are piscivores displayed higher δ15N ratios than all other species preying on lower trophic levels. Omnivorous species depending on both primary producers and invertebrates typically show low δ15N ratios. It is surprising that the least killifish, bluefin killifish and mosquitofish which are reported depending partially on primary producers had higher δ15N ratios than those reportedly true primary consumers such as the three sunfish species.

Table 2

List of fish species and mean δ15N ratios used in this analysis.

3.3 Patterns of fish δ15N along the nutrient gradient

Fish δ15N ratios selected in this analysis generally increased with the increases in TP concentrations (Figs. 2 and 3). Except for the golden topminnow, all other fish had significant correlation between water column TP concentrations and δ15N ratios (Tab. 3). Significant correlation between TN concentrations and fish δ15N ratios were found in five species (Tab. 3). Nearly all fish displayed a decline in δ15N ratios with increases in molar TN/TP ratio although only seven species displayed significant correlation (Tab. 3). Highly significant correlations (p < 0.001) between TP, TN, TN/TP ratio and δ15N ratios were found in the sailfin molly, mosquitofish, least killifish, largemouth bass and Florida gar (Tab. 3). The δ15N ratios of mosquitofish samples collected from seven study sites in 2007 were plotted against TP concentrations (Fig. 4). A highly significant relationship between nutrients and δ15N ratios (p < 0.001) was also found in these samples.

The δ15N ratios of mosquitofish and least killifish were available in all monitoring sites from WCA-2 (Fig. 1) and plotted along with TP concentrations collected for each site (Fig. 5). The δ15N ratios of both fish and TP concentrations were considerably higher in both inflow (E0) and near inflow (F1) sites than those in the interior site (U3) of WCA-2A and near a canal site (L35B) and interior (2BS) WCA-2B which displayed similar δ15N ratios but variously low TP concentrations.

thumbnail Fig. 2

δ15N ratios (mean ± SD) of ten fish species along the TP concentration gradient in the Everglades wetlands. Each dot represents data from a specific date of sample collection in the 1990s.

thumbnail Fig. 3

δ15N values (mean ± SD) of ten fish species along the TN concentration gradient in the Everglades wetlands. Each dot represents data from a specific date of sample collection in the 1990s.

Table 3

Result of Spearman Rank Correlation between fish δ15N ratios, water-column TP and TN concentrations and molar TN/TP ratios in this study.

thumbnail Fig. 4

Relationship between total P (TP) concentration and δ15N ratios in mosquitofish collected from the Everglades Protection Area in 2007.

thumbnail Fig. 5

Changes in the δ15N ratios of mosquitofish and least killifish and TP concentrations along the nutrient gradient in the WCA-2.

4 Discussion

Findings from this analysis are consistent with previous studies, which reveal positive relationship between nutrient concentrations and biota δ15N ratios (Cole et al., 2004; Inglett and Reddy, 2006; Gu et al., 2009). However, the results from this analysis may be complicated by several factors, including study design and variability of nutrient data selected for this analysis. For example, biological characteristics of fish including age, size, gender, growth rate and feeding habits at each site will certainly introduce additional variations. Without data for fish age and tissue turnover time, we used the average TP concentrations measured one year prior to fish collection, which may not accurately reflect the growth condition of fish.

Half of the species also had significant relationship between TN concentrations and δ15N ratios. Nitrogen from human and animal wastes is often enriched in 15N (McClelland et al., 1997). Therefore, the increasing pattern of δ15N ratios along the nutrient gradient may also be caused by increases in wastewater loading. Enriched δ15N of various flora and fauna has been used as an indicator for sewage influence in the freshwater and coastal marine environments (Cole et al., 2004; Rožič et al., 2014; Souza et al., 2018; de Carvalho et al., 2019). There have been no reports of any significant wastewater contributions from human or animal sources to the Everglades. Inglett et al. (2005) reported that the δ15N ratios of porewater NH4+ (the dominant N species in reduced soils) is similar at both the eutrophic and nonaffected WCA-2A sites. The high δ15N at the impacted sites is unlikely the result of the uptake of wastewater enriched with 15N and subsequent transfers to the consumer community (Cabana and Rasmussen, 1995).

Other nitrogen cycling processes (nitrification, denitrification and volatilization) may impact on the isotope composition of DIN in natural wetlands. However, ammonium, not nitrate, was the dominant species of DIN in this region (Tab. 1). Under low DO concentrations in this shallow wetland, nitrification is not likely the main process. Denitrification occurs under low DO concentrations and may result in significant changes in isotope composition the substate and products. However, nitrate was not the major form of DIN in the Everglades. Finally, volatilization occurs only under high pH and the nearly neutral pH found in the south Florida wetlands (Tab. 1) makes this process highly unlikely. Along with the findings from a previous study showing the similar N signatures in both affected and unaffected area of Everglades (Inglett et al., 2006), site-specific N transformation would not likely be the major process leading to the differences in fish δ15N.

Nitrogen concentration may also influence biota δ15N ratios through a substrate-mediated isotope effect (Peterson and Fry, 1987). When N is not a limiting nutrient in a system, an increase in N concentration will normally cause an increase in isotopic fractionation and therefore a decrease in δ15N ratios. This would be evidenced by a negative correlation between TN concentration and fish δ15N ratios in this study. Instead, the majority of the fish in this study displayed positive relationship between TN and δ15N ratios. This implies that N is not a limiting nutrient to plant growth in the Everglades. This is also supported by the high molar ratios of TN:TP ratios. Some negative correlation between the water column TN:TP ratio and the fish δ15N ratios also indicate that P, not N, is the limiting nutrient in the Everglades.

The major external source of N in the Everglades is agricultural runoff (Richardson, 2010). Because manufactured fertilizers are depleted in 15N (Kohl et al., 1971), assimilation of this 15N-depleted N will not result in 15N enrichment in the impacted sites. We conclude that the 15N enrichment in the Everglades is the result of increased primary production stimulated by P availability. The average δ15N ratios for fish increase progressively along the TP gradient. The low δ15N ratios in fish at the unimpacted sites was the result of low TP concentration and large 15N fractionation by primary producers during DIN uptake under P stress. The high N availability at the unimpacted sites also allowed selective assimilation of 14N by aquatic plants. In contrast, the high δ15N ratios at the impacted sites were due to P enrichment which leads to high N demand and low 15N fractionation. Many studies have demonstrated that P is the limiting nutrient in the Everglades (e.g., Newman et al., 1996). Recent studies using stable isotopes found a positive relationship between the TP and δ15N ratios of periphyton, sawgrass and cattail in WCA-1 and WCA-2A, which was attributed to P-driven plant growth and reduced isotope fractionation (Inglett and Reddy, 2006; Chang et al., 2009; Wang et al., 2015).

The widespread significant correlation between TP concentrations and fish δ15N ratios is the consequence of the transfers of plant protein and the associated 15N/14N signal to the consumers, with 15N enrichment along the food chain. The consumer δ15N ratios increases along the TP gradient, which is consistent with the pattern of increases in the primary producers. Furthermore, the relationship between TP concentration and fish δ15N ratios seems to improve as the trophic level increases. For example, Florida gar, which is positioned at the highest trophic level in our samples, had the highest correlation coefficient (r = 0.80) with a moderate sample size (n = 21) among fish selected for this study. In general, fish δ15N is a better indicator of the eutrophication process because they integrate temporal and spatial variations in source δ15N ratios over longer time periods (Cabana and Rasmussen, 1996; Vander Zanden et al., 2005).

5 Conclusions

The eutrophication process resulting from excessive P loading from the agricultural runoff to the Everglades is demonstrated using the nitrogen stable isotopic ratios of fish. The δ15N ratios of nearly all fish species responded positively to the increases in TP concentration. This is considered to be caused by increasing the N uptake and decreasing the 15N fractionation by primary producers stimulated by P enrichment. The 15N enrichment along the nutrient gradient is evident in fish that reliably transfer the isotope signals from primary producers along the trophic level. The significant correlations between TP concentration and δ15N ratios in mosquitofish in the 1990s and 2007 suggest that the eutrophication trend along the nutrient gradient persisted after almost two decades. Results from this study indicate that aquatic consumers such as fish are the better environmental indicators because they are capable of integrating biogeochemical changes over time.

Compliance with ethical standards

Conflict of interest: On behalf of all authors, the corresponding author states that there is no conflict of interest.

Acknowledgements

We appreciate the United States Geological Survey and South Florida Water Management District for providing stable isotope and water quality data.

Appendix

Table A1

Stable isotope data used in this analysis.

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Cite this article as: Hong J, Gu B. 2020. Responses of nitrogen stable isotopes in fish to phosphorus limitation in freshwater wetlands. Knowl. Manag. Aquat. Ecosyst., 421, 41.

All Tables

Table 1

Averages of environmental variables for each study site during the study period. Sites are generally listed from north to south.

Table 2

List of fish species and mean δ15N ratios used in this analysis.

Table 3

Result of Spearman Rank Correlation between fish δ15N ratios, water-column TP and TN concentrations and molar TN/TP ratios in this study.

Table A1

Stable isotope data used in this analysis.

All Figures

thumbnail Fig. 1

Map showing sampling sites in Stormwater Management Area 1 West (STA-1W), Water Conservation Areas (WCAs) and Everglades National Park (ENP) of south Florida. The insert indicates location of the study site in the USA.

In the text
thumbnail Fig. 2

δ15N ratios (mean ± SD) of ten fish species along the TP concentration gradient in the Everglades wetlands. Each dot represents data from a specific date of sample collection in the 1990s.

In the text
thumbnail Fig. 3

δ15N values (mean ± SD) of ten fish species along the TN concentration gradient in the Everglades wetlands. Each dot represents data from a specific date of sample collection in the 1990s.

In the text
thumbnail Fig. 4

Relationship between total P (TP) concentration and δ15N ratios in mosquitofish collected from the Everglades Protection Area in 2007.

In the text
thumbnail Fig. 5

Changes in the δ15N ratios of mosquitofish and least killifish and TP concentrations along the nutrient gradient in the WCA-2.

In the text

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