This research aims to link land-based sources of nitrogen pollution to changes in limu (macroalgae) in Kaua‘i’s nearshore environments. Macroalgal species readily uptake nitrogen in the marine environment, and with lack of pressure from herbivore grazing can be strong competitors with corals. On the island of Kaua‘i, there are concerns about outdated waste management systems (cesspools and septic), as well as fertilizer from farms, golf courses, and hotels entering the nearshore environment. Methods for monitoring nutrient inputs can be costly and technical, making it difficult for communities to monitor nutrient inputs and their effects on nearshore ecosystems. I focused three sites on the north eastern shorelines of Kaua‘i: Anini, Kalihiwai, and Moloa‘a. These sites once supported thriving fishing communities and were known for the diversity and health of their limu. I investigated potential sources of nitrogen pollution by collecting samples of ocean water, stream water, and macroalgae, then analyzing them for stable nitrogen isotopes. These results will be compared with land use maps of identified on-site waste disposal systems, farms, golf courses, and rubbish dumps. I will take percent cover estimates of macroalgae species at each sample location to investigate the current algal community. These will be compared to lists of historically known species for each of the sites based on macroalgal herbarium specimens from Bishop Museum as well as historical newspaper articles and interviews. This project aims to pilot new and less expensive approaches for monitoring land-based sources of pollution which may aid in community-based management.
1.Identify locations along the coastlines of specific study sites which have stable nitrogen isotope ratios (δ15N) indicating sewage
2. Identify differences in δ15N ratios of water samples and macroalgae samples
3. Identify current and historical macroalgae community diversity
4.Create a website and educational workshop to share this method with community, and changes limu in these study sites
Field Sampling
Water sampling
Water samples were collected during morning low tide around the new and full moon. Collecting during low tide reduces the amount of fresh and marine water mixing, giving a more accurate measure of individual site water quality and chemistry. Sampling will be done around the new and full moon to ensure that the low tide will be in the morning, before sunlight and wind has increased microbial activity and mixing, to give more accurate nutrient measures.
Sterile samples bottles/tubes at each site will be rinsed three times as per EPA (based on USGS) water quality sample standard procedure and filled (USGS 2006). Water samples will be filtered through 0.2 μm Nitrocellulose Mixed Ester (MCE) Membrane Filters (Sterlitech) using hand-pumped reusable filtration holder and receiver (Thermo Scientific Nalgene). Water quality characteristics (temperature, salinity, pH, and DO%) will be collected at the same time water samples are collected using a YSI-Pro. Samples will be analyzed for inorganic nutrients (in the S-LAB) and for stable nitrogen isotope ratios (at the Popp Lab for Biogeochemicial Stable Isotope Analysis). Collection of two aliquots (separate bottles) of at least 50 mL of water were recommended by the laboratories for their analysis. Sample will need to be put on ice and frozen until the analysis can take place. Denitrification and off-gassing of ammonia can occur if samples are not frozen and will skew nutrient values reported. Unfortunately, original samples were taken in March and May of 2017 were kept in a fridge instead of a freezer and are no longer viable for stable nitrogen isotope analysis. At the end of June 2017, more samples were recollected for the sites, and these samples have remained frozen. More samples were taken in April 2018, and July 2018. These are still in the process of being analyzed.
Macroalgae collection and macroalgal community identification
Macroalgae will be collected for both stable isotope analysis and identification purposes at all three sites once during the summer of 2017, spring 2018, summer of 2018, during new moon, in the morning during low tide. Macroalgae will be collected via reef-walking and snorkeling in areas too deep to walk. Each macroalgae will be rinsed to remove excess sediment and epiphytes (organisms which grows on the surface of a plant). Macroalgae will be pinched to avoid removing holdfast or other attachments of species. The following types of species, calcified algae (genus Jania, Halimeda, Neomeris, and Padina) do not report δ15N accurately and cyanophytes (genus Lyngbya and Symploca) hyper accumulate nitrogen as they are nitrogen fixers and would also not report δ15N properly. As such these species will only be collected for herbarium species identification. Species of green algae are typically used for this type of analysis and were selected from herbarium pressings collected in June 2017, and April 2018 for analysis.
To assess the community of macroalgae species, point intercept method will be used. A standard transect and quadrat will be used at each sample site at all three locations. As some areas of the reef flat are small, the transect size will be reduced to 5m. A standard 1 m2 quadrat made from PVC pipe will be used. Three quadrats will be taken per transect at 1, 3, and 5 m. The majority of macroalgal species are best identified in the field or via collection and identification via microscope in the lab for red algal or those species which overgrow each other creating canopy layers on the reef flat. Species will be identified using on the following references Huisman, Abbbot, and Smith 2007, Abbott 2004, and Abbott 1999.
To prepare the limu for stable isotope analysis the samples must be dried in the field before reaching the laboratory. The drying method typically used for δ15N analysis is conventional oven at 60 – 70 °C for 24 hrs. Samples used in this research were dried using standard herbarium pressing methods. To address the difference in method, a small study looking at differences in drying methods between δ15N ratios was employed by myself to ensure that samples that were dried via herbarium were not significantly different in ratio values than those dried a conventional oven. Methods tested included conventional oven, herbarium, silica gel desiccant, and salt for relative water content and δ15N ratios. This research was conducted from October 2017 to January 2018 to assess potential variation from the standard oven method. Preliminary results from this test showed that there was no significant difference in the δ15N values of the macroalgae sampled between drying methods (oven, herbarium pressing, silica gel, salt). This indicates that although the samples for this research were not dried using the standard methods, as they were dried via herbarium, there should be no significant difference between the δ15N values received.
Laboratory Analysis
Analysis of Water Samples
Water samples will be kept frozen until time of analysis. Water will be tested for inorganic nutrient amounts and total nitrogen (NO3- + NO2-, PO43-, NH4+, H4SiO4) at the S-LAB at the University of Hawai’i at Mānoa. Inorganic nutrients and total nitrogen values are necessary to accurately reflect δ15N (Table 2).
Table 2. Inorganic nutrients measured at the S-LAB, University of Hawai‘i at Manoa.
Inorganic Nutrient |
Common Name |
Source/Importance |
NO3- |
Nitrate |
Source of N for plants |
NO2- |
Nitrite |
Intermediate between ammonium and nitrate |
PO43- |
Phosphate |
Weathering of rocks, limiting nutrient in land plants |
NH4+ |
Ammonium |
Waste product |
H4SiO4 |
Silicic Acid |
Diatoms convert to silica Groundwater |
After receiving the amounts of inorganic nutrients (specifically the nitrate+nitrite amounts), δ15N can be analyzed via Stable Nutrient Isotope analysis for water at the Biogeochemical Stable Isotope Facility run by the Popp Lab at the University of Hawai‘i at Mānoa. Bacterial cultures will be made for the fractionation of the water samples to estimate the relative δ15N ratio.
Isotopic compositions of N in samples will be normalized to reference materials NIST 3, USGS- 32, USGS-34, and GA NaNO3 relative to AIR. Ratios of15N:14N are expressed as δ15N in per mil (‰).
Analysis of Macroalgae
After drying macroalgae samples in the field, samples will be analyzed in the Biogeochemical Stable Isotope Facility run by the Popp Lab at the University of Hawai‘i at Mānoa. Samples will be ground using a mortar and pestle. The mortar and pestle will be cleaned with ethanol between samples. Glass sample vials will be used for storage at the lab in case more sample is needed for the analysis, excess macroalgae will be saved for reruns and comparisons between species. Samples will be stored in desiccant until analysis of tissue δ15N (‰), using a Costech ECS 4010 Elemental Combustion System (Costech Analytical Technologies, CA, USA) interfaced with a ThermoFinnigan DeltaXP (Thermo Fisher Scientific Inc., MA, USA).
Approximately 2 mg of sample is typically used at the Biogeochemical Stable Isotope Facility. Ground sample will need to be weighed into a tin capsule (3 x 5 mm) using a microbalance. Exact weight will be recorded for reference. Tin capsules should be folded to contain the material so no powder will exit, and be folded into a small ball to fit into the Elemental Combustion System tray.
Analysis of Macroalgae Community Structure
To estimate community structure of macroalgae, at each of the sites, a quadrat will be used to determine % benthic cover. A photo will be taken at each quadrat to calculate percent cover. Identification will be semi-automated via online program CoralNet which can be programed to identify species at random points in the photo for analysis. This automated process will assist with the image analysis, reducing the need for manual analysis, but will be calibrated by initial manual analysis to program the software.
A database of archived species from theses field sites was collected from the bishop museum's online herbarium collection: http://macroalgae.org/portal/index.php. Kalihiwai and Anini were combined in the dataset. Also species that were known historically at Moloa'a (Aparagopsis taxiformis) were not found in the herbarium dataset. In this case the herbarium data is supplemented by information from Hawaiian dictionary, and historical Kaua'i interviews and sources. It was probably not included in the original herbarium collection because of its economic value at the time.
'Anini:
http://olakawaiolakalimu.weebly.com/anini.html
Kalihiwai:
http://olakawaiolakalimu.weebly.com/kalihiwai.html
Moloa'a:
http://olakawaiolakalimu.weebly.com/moloaa.html