This article examines the response of planktonic microbial communities to changes in water body nutrient inputs.
Coastal environments are subject to inputs of dissolved and particulate material from both natural and man made sources, including rivers, sewage outfalls and fish farms. These dissolved and particulate materials are often rich in carbon, nitrogen and phosphorus, providing a rich source of nutrients for phytoplankton and bacteria. They may also contain pollutants such as heavy metals or pesticides.
Marine ecosystems may have the capacity to absorb these nutrients without harm when inputs are low or are diluted by hydrographic influences. By contrast, high concentrations of nutrients may lead to detrimental effects should they be released into semi-enclosed water bodies, where water exchange with surrounding waters is weak. These detrimental effects, which often come as a result of changes in microbial growth and community composition, may include the release of toxins from harmful algae, de-oxygenation of water and sediments from excessive microbial growth, and the transfer or concentration of toxic compounds through the marine food web.
Research within the Department of Microbial and Molecular Biology at the Scottish Association for Marine Science (SAMS), examines the response of planktonic microbial communities to these changes in nutrient inputs.
Professor Keith Davison is a leading marine microbial expert and Head of the Microbial and Molecular Biology Department at SAMS. Research projects in this department investigate how nitrogenous organic nutrients affect the phytoplankton community, with a focus on species associated with Harmful Algal Blooms (HABs). This involves sampling and testing a suite of environmental parameters: temperature, salinity, light, chlorophyll a, inorganic and organic nutrients, and the phytoplankton community. The characterisation of associations between the species composition of phytoplankton blooms and the physical or chemical regime of the studied coastal systems could be used to inform future policy making and aid in the prediction of HAB events.
Temperature, salinity, light, chlorophyll a, inorganic and organic nutrients are sampled using a Conductivity, Temperature, Depth (CTD) sampler. This apparatus consists of a large metal frame holding 24 airtight bottles each with a 10 litre capacity, which can collect water from any depth in the water column. Attached to the frame are sensors that record the conductivity, temperature and depth of the water. A typical CTD instrument package comprises a stainless steel frame system with an underwater unit, which houses temperature sensors, conductivity sensors and a temperature compensated pressure sensor.
The apparatus also has a dissolved oxygen sensor, altimeter, fluorometer, transmissometer and high frequency deep-marker beacon. Samples are measured in triplicate to identify instrument precision, but the salinity and the dissolved oxygen sensors are first calibrated using independent techniques involving samples from water bottles. This CTD setup is often used to understand physical properties of the oceans such as circulation, currents and different water masses.
Basic water column dissolved nutrients, nitrate, nitrite, ammonium, phosphate, and silicate (reactive silica) are analysed from water collected from CTD casts. Samples collected directly from the CTD spigots are analysed within 24 hours of collection and stored in a fridge prior to analysis. Measurement of nutrients in the seawater is conducted using a Flow Injection Auto-analyser. This operates by utilising classical wet chemical reactions to produce colours whose intensity is proportional to the concentration of the nutrient. Detection is then performed by simple flow cell spectrophotometry.
A wide range of other analytical apparatus is also used to test seawater samples across a spectrum of environmental parameters, including:
• Induction Coupled Plasma Mass Spectroscopy (ICPMS) • ICPMS using laser ablation • Induction Coupled Plasma Optical Emission Spectroscopy (ICPOES) • Carbon and nitrogen elemental analyse • Inorganic nutrients autoanalyser – including 13c/a5n mass spectrometry and doc/n analyser • High Performance Liquid Chromatography (HPLC) • Gas chromatography • Total carbon analyser • Ion chromatograph
Analysing seawater samples
The most common types of environmental tests requested by industry are for nutrient and chlorophyll a analysis of seawater samples. These parameters are often taken as an indicator of nutrient eutrophication impacts caused by industry. With respect to the nutrients, organisations commonly measure the inorganic and organic versions of both dissolved and particulate carbon, nitrogen and phosphorous.
In order to supply environmental impact analyses for marine industries, laboratories should be accredited to ISO/IEC 17025:2005(UKAS).
Organic versus inorganic
One nutrient parameter of particular interest is the relative proportions of inorganic nitrogen (N) and organic nitrogen (ON) in seawaters. Traditionally, the importance of N for the nutrition and growth of marine phytoplankton has been recognised, while the utilisation of ON has received less attention. ON is, however, often the largest pool of N, contributing up to 90% of the total, of which 70% may be in the form of dissolved organic nitrogen (DON).
DON is an important nitrogen source for bacteria and can be a source of N to phytoplankton. Neither the extent to which ON serves as source to plankton assemblages nor its influence on trophic dynamics is well understood. Such perturbations have the potential to modify total nutrient concentrations and inorganic: organic nutrient stoichiometry, particularly in areas of reduced exchange such as Scottish sea lochs.
Research in the Department of Microbial and Molecular Ecology seeks to better understand the role of DON (as well as dissolved organic carbon). It is thought that this balance may modify phytoplankton community dynamics and contribute to HABs.
Identifying and culturing phytoplankton
Identification and enumeration of phytoplankton species, including potentially toxic species, from within seawater samples is done using, among other techniques, the Utermöhl method. This involves the examination of water samples using light microscopy.
SRSL is accredited to ISO/IEC 17025:2005 (UKAS) for this process, so that the laboratory can work with industry to assess their eutrophication impacts on the marine environment.
Many of the phytoplankton species that are commonly encountered in commercial and research projects at SAMS are cultured and stored as part of the on-site Culture Collection of Algae and Protozoa (CCAP). This national facility houses the most diverse culture collection in the world, with more than 3,000 strains of marine and freshwater algae and protists from across the globe. Some of these cultures have been in cultivation for over a century while others have been added in the past few months.
The collection is made up of a wide range of taxa including cyanobacteria, eukaryotic phytoplankton, thalloid red algae, free-living non-pathogenic protozoa, and a small number of potentially pathogenic protozoa. Strains are primarily maintained sale or storage by serial sub-culture, although about 30% of the algal strains and 2% of the protozoan strains are cryopreserved to maximise genetic stability.
Harmful algal blooms
An HAB is a rapid increase or accumulation in the population of toxic algae in an aquatic system. Algal blooms may occur in freshwater as well as in marine environments. Typically, only one or a small number of phytoplankton species are involved and some blooms may be recognised by discolouration of the water resulting from the high density of pigmented cells. HABs in marine waters present a problem that is of increasing concern in UK waters and worldwide through their negative effect on human health, the environment and the economy, particularly in the form of shellfish poisoning events or preventative fishery closures.
The shellfish industry is especially vulnerable to HABs, as bivalve molluscs filter feed on phytoplankton and can accumulate phytoplankton derived toxins within their tissue. The poisoning that results from the ingestion of these shellfish may manifest in illnesses ranging from mild gastrointestinal problems to respiratory and neurological disorders and even death.
Phytoplankton genera Alexandrium , Pseudo-nitzschia and Dinophysis are most important in this respect, due to the production of toxins that result in paralytic shellfish poisoning (PSP), Amnesic shellfish poisoning (ASP) and Diarrhetic shellfish poisoning (DSP), respectively. Other lipophilic shellfish toxins such as yessotoxins (YTX), pectenotoxins (PTX) and azaspiracids (AZA) have also been detected in low levels in shellfish from UK waters.
Recently, blooms of Karenia mikimotoi have been evident in Scottish Waters. Although this species is not of concern in terms of shellfish toxicity, it could lead to mortalities of farmed and wild fish, as well as benthic organisms. To prevent human illness from the ingestion of contaminated shellfish an extensive phytoplankton monitoring programme is conducted in Scottish waters.
HABs are perceived to be increasing worldwide. Monitoring ensures the safety of shellfish for human consumption, with active research programmes attempting to provide better early warning of HABs to better protect human health and the sustainable development of aquaculture and other coastal activities.
There are numerous well-known cases of HABs worldwide including in the US and Canada. In the EU a number of ‘new’ HAB threats have appeared or become more prevalent in recent years, including the shellfish toxin producing genus Azadinium and the aerosol generating Ostreopsis.
Despite ongoing research on nutrient pollution and extensive phytoplankton monitoring efforts, there is little robust understanding of where blooms will appear, their magnitude and their toxicity in Scottish and UK waters. A number of ongoing research programmes are attempting to address this.
EU member states are required to monitor both the presence and geographic distribution of marine biotoxin-producing phytoplankton in shellfish harvesting areas. As the Competent Authority in Scotland, the Food Standards Agency Scotland (FSAS) is responsible for carrying out monitoring of classified production areas in Scotland for the presence of phytoplankton in samples of water and for marine biotoxins.
In Scotland, phytoplankton monitoring of seawater has been carried out by SRSL on behalf of the FSAS since September 2005. Similar monitoring programmes are carried out in England, Wales and Northern Ireland.
Water samples are collected from designated shellfish growing areas around the Scottish coast and analysed, by light microscopy, for seven potentially toxic genera or species of phytoplankton. Most harmful phytoplankton are dinoflagellates such as Alexandrium and Dinophysis , with only one genus of diatom, Pseudo-nitzschia , producing toxin. As the toxicity of harmful species may vary due to a number of (as yet poorly understood) environmental factors, the presence of potentially toxic phytoplankton in a water sample does not necessarily indicate that toxins will be present. Rather, the data collected from the phytoplankton monitoring will provide an early warning for the potential occurrence of shellfish toxin events and hence alert the industry and, as well as safeguard health, minimise product recalls and subsequent financial loss.
Approximately 40 sites, the majority of which are in classified shellfish growing areas, are monitored weekly in Scotland for the presence of potential toxin producing phytoplankton, as part of the statutory monitoring programme. Results are reported to FSA Scotland and other relevant parties on a daily basis and contribute to their biotoxin risk assessment strategy.
Monitoring programmes such as this are vitally important to the Scottish economy, since aquaculture is one of the Scottish Government’s priority areas, with ambitions to increase shellfish production (especially mussels) to 13,000 tonnes by 2020, from 6,757 tonnes in 2013. Currently, the Scottish shellfish farming industry is worth £8-10 million at first sale value (Scottish Government statistics) and is estimated to be worth more than £30 million at final sale value.
The FSA monitoring programme has led to further seawater quality monitoring projects. In June 2012, SRSL in collaboration with the Centre for Environment, Fisheries and Aquaculture Science (CEFAS) and Shetland Seafood Quality Control (SSQC) began providing Shellfish Sanitary Surveys for the Food Standards Agency. The project involves a complex programme of field surveys to assess sources of contamination in the harvesting areas around shellfish farms in Scotland, excluding Orkney and Shetland.
The project involves shoreline visual surveys of 29 sites around shellfish farms to assess potential sources of contamination such as sewerage outfalls and farm run-off. Water samples and other measurements are then taken around the shoreline and at the shellfish farm areas. These samples are analysed for several parameters including E.coli . As part of the surveys, the extent of the shellfish farming operations is also verified.
These sanitary surveys are an essential first step towards establishing a microbiological monitoring programme for the classification of bivalve production areas. All new bivalve production areas require a sanitary survey of this kind.
Ongoing research aims to deliver more efficient and effective monitoring of water quality in areas of aquaculture through the supply of:
• Prediction models for harmful algal events – research in this area now often focuses on satellite-derived chlorophyll images and physical oceanographic modelling techniques
• Training courses targeted to agencies and industry, which will include outcomes of HABs research, as well as relevant new technologies – such as the rapid analysis of the presence of biotoxins in shellfish and simple model-based procedures for the prediction of harmful phytoplankton events
• Establishment of new technologies for rapid analysis of the presence of biotoxins in shellfish and also sustainable procedures for local analysis of these toxins
Effective forecasting models of HABS would enable better risk analysis and in turn more successful mitigation of potential negative impacts on industry and the environment. This kind of information will be of great importance to regulators, monitoring bodies, industry and coastal zone managers throughout Europe.
Published: 17th Dec 2014 in AWE International