Enumerating viable phytoplankton using a culture-based Most Probable Number assay following ultraviolet-C treatment

Hugh L Macintyre,John J Cullen,Trina J Whitsitt,Brian Petri

doi:10.1007/s10811-017-1254-8

Hugh L Macintyre, John J Cullen + Show 2 more

Open Access

https://doi.org/10.1007/s10811-017-1254-8

Copy DOI

Abstract

Ballast water management systems (BWMS) must be tested to assess their compliance with standards for the discharge of organisms, for example in the ≥ 10- and < 50-μm size category, which is dominated by phytoplankton. Assessment of BWMS performance with the vital stains fluorescein diacetate + 5-chlorofluorescein diacetate, required by regulations in the USA, is problematic in the case of ultraviolet-C (UVC) radiation. This is because UVC targets nucleotides—and thus reproduction, hence viability—rather than membrane integrity, which is assayed by the stains. The Serial Dilution Culture-Most Probable Number (SDC-MPN) method, long used to enumerate fragile phytoplankton from natural communities, is appropriate for counting viable phytoplankton. We developed QA/QC “best practice” criteria for its application as a robust and repeatable assay of viable cells in cultures of phytoplankton before and after experimental treatment, then constructed dose-response curves for UVC-induced loss of viable cells in 12 species of phytoplankton from seven divisions. Sensitivity to UVC, expressed as the dose required to reduce viability by 99%—the criterion for type approval of treatment systems—varied more than 10-fold and was not correlated with cell size. The form of the dose-response curves varied between taxa, with most having a threshold dose below which there was no reduction in viability. Analysis of the patterns of growth indicates that if recovery from treatment occurred, it was complete in 1 or 2 days in > 80% of cases, long before the assays were terminated. We conclude that the SDC-MPN assay as described is robust and adaptable for use on natural phytoplankton.

Highlights

Harmful algal blooms (HABs) can be costly in both economic and ecological terms
Regulatory authority resides with the United States Coast Guard (USCG), which entered into a formal Memorandum of Understanding with the US Environmental Protection Agency to develop procedures for evaluating ballast water management systems (BWMS)
Growth responses for each treatment were consistent over all temperatures and light intensities tested, i.e., controls and cultures subjected to the low dose of UVC always grew, and cultures subjected to the high dose of UVC always failed to grow and bleached

Summary

Introduction

Harmful algal blooms (HABs) can be costly in both economic and ecological terms. There is much debate over the conditions that give rise to them (e.g., Davidson et al 2012; Glibert et al 2012), but one contributory factor is fundamental: viable cells of the bloom-forming organism must be present for a bloom to develop. There is a significant potential for transport and introduction of HAB taxa through ships’ ballast water (e.g., Doblin et al 2004; Bolch and de Salas 2007; Roy et al 2012), which was one stimulus for the International Maritime Organization (IMO) adopting the International Convention for the Control and Management of Ships’ Ballast Water and Sediments at a Diplomatic Conference in London in 2004 (IMO 2004). National regulation is already in effect in the USA, governed by the Nonindigenous Aquatic Nuisance Prevention and Control Act (1990) and the National. Regulatory authority resides with the United States Coast Guard (USCG), which entered into a formal Memorandum of Understanding with the US Environmental Protection Agency to develop procedures for evaluating ballast water management systems (BWMS)

Methods

Results

Discussion

Conclusion