Using nitrogen isotopes in macroalgae to determine and monitor sources of nitrogen pollution in estuarine and coastal areas: an in vitro study and case study in North East England, UK.

The growing human population in coastal areas, combined with the intensification of agricultural activity have increased the amount of dissolved inorganic nitrogen (DIN) delivered to estuaries and coastal waters. These high concentrations of DIN can cause eutrophication which in turn causes the growth of opportunistic macroalgae (seaweed) and anoxic conditions both in the water column and in the sediment. Stable nitrogen isotope ratios (δ15N) can be used to discern sources of excess nitrogen pollution in water. The δ15N values of nitrate in water often do not reflect the true δ15N source value owing to high temporal variation, and there are high analytical costs associated with obtaining δ15N values from water nitrate. As such, δ15N values can be measured in macroalgae samples to identify the bioavailable nitrogen inputs in an area. Macroalgae have been previously reported to be accurate biomonitors for sources of nitrogen in the environment, with fractionation assumed to be negligible; however, some workers report varying environmental and biological conditions can cause fractionation between the water column and macroalgal δ15N values. Tips of Fucus vesiculosus (F. vesiculosus) were incubated in isotopically-labelled artificial seawater solutions with varying temperatures and salinities to determine whether these factors affected equilibration of macroalgal δ15N values with δ15N-DIN values. Temperature and salinity were found to have a significant effect on the uptake of nitrogen isotopes by F. vesiculosus tips, suggesting varying these environmental conditions is likely to cause fractionation of nitrogen isotopes between the water column and macroalgal tissue. After 14 days of incubation there was a greater than 1 ‰ difference between δ15N and δ15N-DIN. Tips of F. vesiculosus were also collected from around the North East of England coastline every 2-3 months from October 2020 to July 2021. The tips were analysed for δ15N values. There was a significant difference in macroalgal δ15N values between sampling locations for each collection period in both the River Wear and Tyne and also along the North East coastline. For F. vesiculosus tips collected from the River Wear, the dominant source of nitrogen pollution was found to be treated sewage or manure with point source pollution from untreated sewage discharged from Combined Sewage Overflows (CSO’s). The River Tyne macroalgal tips appear to be impacted by untreated sewage, given the low δ15N values and the temporal variations. Tips collected from the North East coastline look to be affected by wastewater pollution, i.e., treated sewage or manure but are regulated by the unpolluted marine nitrogen. Tips from all sites record δ15N values in the unpolluted range in the winter months, potentially as a result of increased river discharges diluting any pollution δ15N values with lower terrestrial nitrogen δ15N values. There are also environmental and physiological factors that could influence the δ15N values recorded by macroalgae such as biogeochemical cycles, temperature and salinity. All sites followed the same temporal trend of δ15N values, suggesting an impact from environmental factors, rather than source changes. Therefore, δ15N values in F. vesiculosus tips can be used as an indicator of sources of nitrogen loading in the environment, including for point source pollution. However, δ15N values in F. vesiculosus tips are significantly affected by changes in temperature and salinity, and so more work is needed to quantify fractionation between water column and macroalgal δ15N caused by different factors. These data demonstrate that macroalgae can provide an efficient, low-cost alternative to current analytical methods for determining and monitoring nitrogen pollution