The effects of a PSII inhibitor on phytoplankton community structure as assessed by HPLC pigment analyses, microscopy and flow cytometry
Introduction
Irgarol 1051® is an s-triazine photosynthetic inhibitor used in antifouling paints to prevent attachment and growth of algae on the hulls of ships or boats. After painting, the chemical is continuously released to the surrounding water and remains active for approximately 1–2 years (HSE, 2002a), posing a threat to non-target organisms in the environment. Concerns over the use of antifouling ‘booster’ biocides have increased since TBT restrictions were introduced in the late 1990s (HSE, 2002b, Readman et al., 1993) and their use became more widespread. Irgarol 1051® was, until recently, one of the most commonly used booster biocides in the UK (Thomas et al., 2002), with levels up to 1421 ng l−1 being reported in UK marinas (Thomas et al., 2001). As a consequence, concerns have been raised about the environmental impact they may have on indigenous organisms (Readman et al., 2004).
Algal species comprising the phytoplankton are highly susceptible to herbicidal antifouling compounds which damage photosynthetic pathways. Due to their short generation times and differential responses and susceptibilities to the biocides, algae can, potentially, be affected over time scales from hours to days (McCormick and Cairns, 1994, Jørgensen and Christoffersen, 2000), with resultant changes in algal communities (Hamilton et al., 1988). As modifications to phytoplankton communities can alter the functional structure of the food web, the detection of such changes imposed by toxicants is crucial to determine the extent of damage to the ecosystem.
Several techniques have been employed to investigate phytoplankton community composition. Light microscopy is the classical method for identifying and enumerating phytoplankton, allowing estimations of carbon content from biovolume determinations (Strathmann, 1967, Montagnes and Franklin, 2001). While detailed information about species and size can be acquired, this method is very time consuming and requires considerable taxonomic expertise. Moreover, light microscopy examination does not easily lend itself to identification of small-sized organisms (e.g. nanoplanktonic flagellates). Furthermore, fixation can alter the structure of fragile cells of many species (Gieskes and Kraay, 1983, Simon et al., 1994). Analytical flow cytometry (AFC) provides a more rapid assessment and quantification of phytoplankton communities, but lacks detailed compositional information. Based on the optical properties of cells, AFC can only discriminate phytoplankton communities into picoplanktonic prokaryotes, picoeukaryotes, and nanoeukaryotes (Readman et al., 2004).
A chemotaxonomic approach has provided an additional tool that overcomes some of the limitations of microscopy and AFC. Based on analyses of pigments using high performance liquid chromatography (HPLC), taxon-specific markers can discriminate between the main algal classes contributing to the phytoplankton community (e.g. Barlow et al., 1993, Jeffrey et al., 1997). For example, peridinin has been used as a diagnostic pigment marker for dinoflagellates, 19′-hexanoyloxyfucoxanthin for prymnesiophytes, alloxanthin for cryptophytes, while other pigments are shared within more than one algal class, e.g. chlorophyll b which occurs in chlorophytes, prasinophytes and prochlorophytes (Andersen et al., 1996, Jeffrey et al., 1997). However, the use of specific biomarkers in coastal waters is more complicated than in open ocean, since inland inputs can change the chemical, physical and consequently the biological characteristics more intensively.
Several recent studies have compared microscopic and chemotaxonomic approaches on natural communities (Breton et al., 2000, Rodriguez et al., 2002, Carreto et al., 2003, Garibotti et al., 2003, Fietz and Nicklish, 2004), with some also including flow cytometry analyses (e.g. Gin et al., 2003). Studies have demonstrated that HPLC pigment analysis is a valuable tool for phytoplankton studies and that it can generate a good approximation of carbon biomass. However, more research is needed to assess the chemotaxonomic approach, since the use of chlorophyll a as a biomass indicator must be undertaken with caution as it is susceptible to changes in environmental conditions. To date, few investigations on community changes under anthropogenic stressors have been reported (Readman et al., 2004). Data on single species exposures and preliminary data on natural phytoplankton community changes have been reported by Devilla et al. (in press). In this study, we examine the consequences of environmental disturbances on phytoplankton composition by manipulating a natural algal community in microcosms exposed to the PSII inhibitor Irgarol 1051®. The aim is to compare and contrast changes in community structure as determined by HPLC-class specific pigment analyses, optical microscopy and AFC and use these techniques to monitor the effects of Irgarol 1051® in microcosms.
Section snippets
Sampling and experimental design
One hundred litres of coastal surface water was collected from the well-characterised L4 station (Pingree et al., 1978, Rodriguez et al., 2000) off Plymouth, UK (50°15′N, 04°13′W) on 11 August 2003. The in situ temperature was 16 °C and salinity 35 PSU. At the laboratory, the seawater was filtered through a 150 μm nylon mesh net and 4 l were transferred into each of 15 microcosms comprising 5 l borosilicate glass Erlenmeyer flasks.
A stock solution of 0.503 μg μl−1 Irgarol 1051®
Analytical flow cytometry
Growth of nanophytoplankton and picophytoplankton was monitored by AFC for 120 h. Nanoplanktonic cryptophytes were at insignificant numbers and were therefore omitted in the results. Picoeukaryotes were exponentially growing until 96 h in the controls (Fig. 2), therefore data resulting from this day were selected for comparisons. Results showed a decline in cell numbers relative to controls with increasing concentrations of Irgarol 1051® throughout the experimental period (Fig. 2). After 96 h,
Discussion
Addition of the PSII inhibitor Irgarol 1051® to the microcosms induced marked changes in the phytoplankton community as revealed by microscopy, class-specific pigments and AFC. Nanoeukaryotes appeared to be more affected by Irgarol 1051® than picoeukaryotes. This higher sensitivity could be related to the presence of species- or class-specific algae which are more vulnerable to antifoulant stress. The decline in cyanophytes with time, as measured by AFC, may have resulted from the experimental
Conclusions
The toxicity of the PSII inhibitor Irgarol 1051® towards the phytoplankton community studied induced a restructuring of the community and therefore, changes in taxonomic composition. Group-specific sensitivity was detected through microscopy and was corroborated with CHEMTAX estimations. Results revealed that chlorophytes and dinoflagellates are comparatively resistant to Irgarol 1051®. Estimates from HPLC–CHEMTAX pigment analyses were correlated with chlorophytes (R2 = 0.53), which increased in
Acknowledgements
This research was supported by a Brazilian Research Council (CNPq) studentship to R.A. Devilla, and was also supported by the European Commission under the ACE (Assessment of Antifouling Agents in Coastal Environments) contract (MAS3-CT98-0178) (a component of the EC IMPACTS cluster). The authors thank H.W. Higgins for a copy of CHEMTAX program and D. Harbour for microscopic analysis.
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