Analytical improvements and assessment of long-term performance of the oxidation-denitrifier method.

The analysis of the nitrogen (N) isotopic composition of organic matter bound to fossil biomineral structures (BB-δ15 N) using the oxidation-denitrifier (O-D) method provides a novel tool to study past changes in N cycling processes. METHODS: We report a set of methodological improvements to the O-D method, including (a) a method for sealing the reaction vials in which the oxidation of organic N to NO3 - takes place, (b) a recipe for bypassing the pH adjustment step before the bacterial conversion of NO3 - to N2 O, and (c) a method for storing recrystallized dipotassium peroxodisulfate (K2 S2 O8 ) under Ar atmosphere. RESULTS: The new sealing method eliminates the occasional contamination and vial breakage that occurred previously while increasing sample throughput. The protocol for bypassing pH adjustment does not affect BB-δ15 N, and it significantly reduces the processing time. Storage of K2 S2 O8 reagent under Ar atmosphere produces stable oxidation blanks over more than 3.5 years. We report analytical blanks, accuracy, and precision for this methodology from eight users over the course of ~3.5 years of analyses at the Max Planck Institute for Chemistry. Our method produces analytical blanks characterized by low N content (0.30 ± 0.13 nmol N, 1σ, n = 195) and stable δ15 N (-2.20 ± 3.13‰, n = 195). The analysis of reference amino acid standards USGS 40 and USGS 65 indicates an overall accuracy of -0.23 ± 0.35‰ (1σ, n = 891). The analysis of in-house fossil standards gives similar analytical precision (1σ) across a range of BB-δ15 N values and biominerals: zooxanthellate coral standard PO-1 (6.08 ± 0.21‰, n = 267), azooxanthellate coral standard LO-1 (10.20 ± 0.28‰, n = 258), foraminifera standard MF-1 (5.92 ± 0.28‰, n = 243), and tooth enamel AG-Lox (4.06 ± 0.49‰, n = 78). CONCLUSIONS: The methodological improvements significantly increase sample throughput without compromising analytical precision or accuracy down to 1 nmol of N.

Megacity development and the demise of coastal coral communities: Evidence from coral skeleton δ15 N records in the Pearl River estuary.

Historical coral skeleton (CS) δ18 O and δ15 N records were produced from samples recovered from sedimentary deposits, held in natural history museum collections, and cored into modern coral heads. These records were used to assess the influence of global warming and regional eutrophication, respectively, on the decline of coastal coral communities following the development of the Pearl River Delta (PRD) megacity, China. We find that, until 2007, ocean warming was not a major threat to coral communities in the Pearl River estuary; instead, nitrogen (N) inputs dominated impacts. The high but stable CS-δ15 N values (9‰-12‰ vs. air) observed from the mid-Holocene until 1980 indicate that soil and stream denitrification reduced and modulated the hydrologic inputs of N, blunting the rise in coastal N sources during the early phase of the Pearl River estuary urbanization. However, an unprecedented CS-δ15 N peak was observed from 1987 to 1993 (>13‰ vs. air), concomitant to an increase of NH4+ concentration, consistent with the rapid Pearl River estuary urbanization as the main cause for this eutrophication event. We suggest that widespread discharge of domestic sewage entered directly into the estuary, preventing removal by natural denitrification hotspots. We argue that this event caused the dramatic decline of the Pearl River estuary coral communities reported from 1980 to 2000. Subsequently, the coral record shows that the implementation of improved wastewater management policies succeeded in bringing down both CS-δ15 N and NH4+ concentrations in the early 2000s. This study points to the potential importance of eutrophication over ocean warming in coral decline along urbanized coastlines and in particular in the vicinity of megacities.

New Insights on the Nitrogen Footprint of a Coastal Megalopolis from Coral-Hosted Symbiodinium δ15N.

The development of megalopolises in coastal areas is often linked with severe eutrophication, requiring mitigation of anthropogenic dissolved inorganic nitrogen (DIN) pollution. Yet, identifying the DIN-sources responsible for eutrophication is challenging, hampering mitigation efforts. Here, we utilize the stable nitrogen isotope ratio of endosymbiotic dinoflagellate Symbiodinium spp. (δ15Nsym) associated with the hard coral Porites to trace DIN sources in one of the most urbanized areas of the planet: the Pearl River Delta (PRD). The mean δ15Nsym value found in the coastal waters of Hong Kong (HK), located on the eastern edge of the PRD, (7.4‰ ± 1.2‰) was +2.7‰ higher than at Dongsha Atoll, a reference site unaffected by anthropogenic-DIN (4.7‰ ± 0.4‰). The isotopic enrichment suggested a consistent dominance of DIN deriving from local and regional sewage discharges on the eastern edge of HK. Furthermore, the strong depletion of the summer δ15Nsym value (-1.6‰) observed in southern HK revealed that the Pearl River plume strongly modulates the coastal DIN pool. Our results revealed the value of benthic marine organisms' δ15N for deciphering the complex dynamics of coastal eutrophication and highlighted the pivotal role of transboundary coordination in DIN-pollution mitigation.

Reefs of tomorrow: eutrophication reduces coral biodiversity in an urbanized seascape.

Although the impacts of nutrient pollution on coral reefs are well known, surprisingly, no statistical relationships have ever been established between water quality parameters, coral biodiversity and coral cover. Hong Kong provides a unique opportunity to assess this relationship. Here, coastal waters have been monitored monthly since 1986, at 76 stations, providing a highly spatially resolved water quality dataset including 68 903 data points. Moreover, a robust coral species richness (S) dataset is available from more than 100 surveyed locations, composed of 3453 individual colonies' observations, as well as a coral cover (CC) dataset including 85 sites. This wealth of data provides a unique opportunity to test the hypothesis that water quality, and in particular nutrients, drives coral biodiversity. The influence of water quality on S and CC was analyzed using GIS and multiple regression modeling. Eutrophication (as chlorophyll-a concentration; CHLA) was negatively correlated with S and CC, whereas physicochemical parameters (DO and salinity) had no significant effect. The modeling further illustrated that particulate suspended matter, dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorus (DIP) had a negative effect on S and on CC; however, the effect of nutrients was 1.5-fold to twofold greater. The highest S and CC occurred where CHLA <2 µg L-1 , DIN < 2 µm and DIP < 0.1 µm. Where these values were exceeded, S and CC were significantly lower and no live corals were observed where CHLA > 15 µg L-1 , DIN > 9 µm and DIP > 0.33 µm. This study demonstrates the importance of nutrients over other water quality parameters in coral biodiversity loss and highlights the key role of eutrophication in shaping coastal coral reef ecosystems. This work also provides ecological thresholds that may be useful for water quality guidelines and nutrient mitigation policies.

Coral carbonate-bound isotopes reveal monsoonal influence on nitrogen sources in Southeastern China's Greater Bay Area from the mid-Holocene until the Anthropocene.

Most anthropogenic nitrogen (N) reaches coastal waters via rivers carrying increasing loads of sewage, fertilizer, and sediments. To understand anthropogenic N impacts, we need to understand historical N-dynamics before human influence. Stable isotope ratios of N preserved in carbonates are one way to create temporal N records. However, records that span periods of human occupation are scarce, limiting our ability to contextualize modern N dynamics. Here, we produce a fossil-bound N-record using coral subfossils, spanning 6700 years in China's Greater Bay Area (GBA). We found that during the mid-to-late Holocene, the GBA's coastal N was dominated by fluvial sources. The weakening of the Asia monsoon throughout the late-Holocene decreased river outflow, leading to a relative increase of marine nitrate. This source shift from riverine-to-ocean dominance was overprinted by anthropogenic N. During the late 1980s to early 1990s, human development and associated effluent inundated the coastal system, contributing to the decline of coral communities.

Tooth enamel nitrogen isotope composition records trophic position: a tool for reconstructing food webs.

Nitrogen isotopes are widely used to study the trophic position of animals in modern food webs; however, their application in the fossil record is severely limited by degradation of organic material during fossilization. In this study, we show that the nitrogen isotope composition of organic matter preserved in mammalian tooth enamel (δ15Nenamel) records diet and trophic position. The δ15Nenamel of modern African mammals shows a 3.7‰ increase between herbivores and carnivores as expected from trophic enrichment, and there is a strong positive correlation between δ15Nenamel and δ15Nbone-collagen values from the same individuals. Additionally, δ15Nenamel values of Late Pleistocene fossil teeth preserve diet and trophic level information, despite complete diagenetic loss of collagen in the same specimens. We demonstrate that δ15Nenamel represents a powerful geochemical proxy for diet that is applicable to fossils and can help delineate major dietary transitions in ancient vertebrate lineages.

Modification of fatty acid profile and biosynthetic pathway in symbiotic corals under eutrophication.

Symbiotic corals receive energy not only by ingesting food (e.g. plankton, inorganic/organic matter, i.e. heterotrophy), but also by endosymbiosis, which supplies photosynthates (dissolved inorganic carbon, i.e. autotrophy). These two sources of energy have distinct fatty acid (FA) profiles, which can be used to differentiate corals by their primary feeding mode. FA profiles have been applied as biomarkers to evaluate the quality of nutrition in the midst of environmental change. However, species-specific responses of coral FA profiles and biosynthetic pathway under cultural eutrophication are still unknown. We collected two coral species (Acropora samoensis, Platygyra carnosa) from sites with different levels of eutrophication to test for variations in FA profiles. Gas Chromatography-Mass Spectrometry (GC-MS) was performed to identify FA profiles and quantify their concentration. Our main findings are threefold: 1) chronic eutrophication inhibits corals' ability to synthesize essential FA; 2) PUFA:SFA ratio and certain FA biomarkers or their pathway can be successfully utilized to determine the relative degree of autotrophy and heterotrophy in corals; 3) under eutrophication, different FA profiles of coral host tissue are attributed to different feeding strategies. Thus, our research provides significant new insights into the roles of FA as a risk assessment tool in coral reef ecosystems under the pressure of eutrophication.

Response to Comment on Trophic strategy and bleaching resistance in reef-building corals.

Recently, we published a novel method used to assess the trophic niches of different coral species and demonstrated that their nutrition varied considerably, with some species highly dependent on their photosynthetic algal symbionts and others able to feed on plankton to meet energetic requirements. Adjustments to the use of this tool are necessary when it is applied to other scientific questions and symbiotic organisms. We respond to a comment highlighting a risk of bias in the methods, discuss suggested adjustments, and propose further refinements to improve method robustness.

Coral reef diversity losses in China's Greater Bay Area were driven by regional stressors.

Observations of coral reef losses to climate change far exceed our understanding of historical degradation before anthropogenic warming. This is a critical gap to fill as conservation efforts simultaneously work to reverse climate change while restoring coral reef diversity and function. Here, we focused on southern China's Greater Bay Area, where coral communities persist despite centuries of coral mining, fishing, dredging, development, and pollution. We compared subfossil assemblages with modern-day communities and revealed a 40% decrease in generic diversity, concomitant to a shift from competitive to stress-tolerant species dominance since the mid-Holocene. Regions with characteristically poor water quality-high chl-a, dissolved inorganic nitrogen, and turbidity-had lower contemporary diversity and the greatest community composition shift observed in the past, driven by the near extirpation of Acropora These observations highlight the urgent need to mitigate local stressors from development in concert with curbing greenhouse gas emissions.

Trophic strategy and bleaching resistance in reef-building corals.

Ocean warming increases the incidence of coral bleaching, which reduces or eliminates the nutrition corals receive from their algal symbionts, often resulting in widespread mortality. In contrast to extensive knowledge on the thermal tolerance of coral-associated symbionts, the role of the coral host in bleaching patterns across species is poorly understood. Here, we applied a Bayesian analysis of carbon and nitrogen stable isotope data to determine the trophic niche overlap between corals and their symbionts and propose benchmark values that define autotrophy, heterotrophy, and mixotrophy. The amount of overlap between coral and symbiont niche was negatively correlated with polyp size and bleaching resistance. Our results indicated that as oceans warm, autotrophic corals lose their competitive advantage and thus are the first to disappear from coral reefs.

Effects of tourism-derived sewage on coral reefs: Isotopic assessments identify effective bioindicators.

Pulau Redang and Pulau Tioman have experienced huge tourism growth over the last two decades, but minimal sewage treatment may threaten the resilience of their coral reefs. This study uses stable isotope techniques to identify suitable bioindicators of sewage nutrients (δ15N) at these islands by measuring macroalgae (Lobophora spp.), gastropods (Drupella spp.), scleractinian coral (Acropora spp.), and leather coral (Sinularia spp.). At tourist hubs using seepage septic tank systems, enrichment of Acropora δ15N (Redang, +0.7‰) and Sinularia δ15N (Tioman, +0.4‰) compared to pristine background levels indicate enhanced sewage nutrient discharge. Carbon isotopes and survey data suggest that sedimentation did not confound these δ15N trends. Potential damaging effects of sewage discharge on the coral reef communities at both islands are highlighted by strong correlations between Acropora δ15N and regional variation in coral reef community structure, and exclusive occurrence of degraded reefs at regions of high sewage influence.

Life and death of a sewage treatment plant recorded in a coral skeleton δ15N record.

We investigated the potential of coral skeleton δ15N (CS-δ15N) records for tracking anthropogenic-N sources in coral reef ecosystems. We produced a 56yr-long CS-δ15N record (1958-2014) from a reef flat in Guam that has been exposed to varying 1) levels of sewage treatment 2) population density, and 3) land use. Increasing population density (from <30 to 300ind·km-2) and land use changes in the watershed resulted in a ~1‰ enrichment of the CS-δ15N record until a sewage treatment plant (STP) started operation in 1975. Then, CS-δ15N stabilized, despite continued population density and land use changes. Based on population and other considerations, a continued increase in the sewage footprint might have been expected over this time. The stability of CS-δ15N, either contradicts this expectation, or indicates that the impacts on the outer reef at the coring site were buffered by the mixing of reef water with the open ocean.