A Test of Nerves
For several years now there has been discussion around the possibility that US regulators would eventually disallow the use UV ballast treatment systems. The reason for this position is not necessarily because UV systems do not work but because the methodology behind UV is different from electrochemical or mechanical systems. Mechanical systems either prevent organisms from entering the ballast tank by way of filtration or else use physical methods of killing them such as cavitation, ultrasound, deoxygenation and separation by flocculation or sedimentation. Electrochemical and chemical systems use toxins to kill organisms but UV systems work by transmitting UV light with a wavelength of 254nm which destroys nucleic acids and disrupts DNA. Some UV treatment systems for land use also produce UV light with other germicidal wavelengths but most marine systems stay with the most effective 254nm. When organism’s cells are struck by UV light, the DNA is disrupted and abnormalities known as thymine dimers, form along the DNA strand and eventually prevent DNA replication so eliminating the reproductive capability of the organism. In some cases the UV irradiation is strong enough to kill organisms outright but if this does not happen, the organism will in theory be rendered unviable since it cannot reproduce and will eventually die without procreating. This difference is put into stark contrast because of the views that exist in the use of words in IMO and US regulations and guidelines. No system has yet achieved type approval under the US system but around 60 have under IMO guidelines. Classification societies and government run or approved certifying organisations have in the main interpreted the IMO wording as allowing organisms that are unable to reproduce to be considered unviable and therefore the presence of such organisms would not cause a system to have approval withheld. By contrast the US has tended to prefer the obvious difference between living and dead as being the only choice on offer. The argument over words and their meaning is one that will probably end up being determined by lawyers and courts and a small number of UV ballast system makers have already begun a legal challenge to the USCG’s recent actions. There is however another more practical aspect of the USCG decision that will be argued over by scientists before the lawyers get involved. Now the issue has shifted to the type of testing for the condition of organisms after treatment that system makers have selected when requesting type approval. The IMO is clear that a number of options are available saying, “Viability of an organism can be determined through live/dead judgement by appropriate methods including, but not limited to: morphological change, mobility, staining using vital dyes or molecular techniques”. Under US rules the requirement for analysis of organisms in the 10 – 50μm size class requires categorisation of organisms as living or dead and demands the use of the Environmental Technology Verification (ETV) Staining Method (vital stain method). Manufacturers applying for US type approval are entitled to use test data generated during foreign type-approval testing in accordance with IMO G8 guidelines if that prior data and testing meet the USCG requirements in 46 CFR 162.060. Sometimes additional testing will be required because the IMO protocols differ from the US protocols. The attraction of using prior test data is great because it can significantly reduce the time and cost of US type-approval. In a situation where time is of the essence, early achievers in gaining US type approval clearly have a big advantage in selling into the market. Most, but not all, UV system makers have chosen to use a test method known as Most Probable Number (MPN) to gain IMO type approval and in order to keep ahead in the race of US type approval have sought to rely on those test results as detailed above. But their plans have been upset by the USCG insisting on the preferred vital stain method. Scientists are divided over the reliability of the testing methods and their relevance in testing ballast water. The campaign set in motion by four ballast water system makers to overturn the USCG decision has attracted academic support which it is using to prove its case. For its part the USCG has not yet responded to the claims and it is of course in a position of power and has not had to defend its position. The two test methods are very different. The vital stain method favoured by the US involves using a combination of two fluorescein-based stains (FDA and CMFDA) to evaluate the status of organisms in the 10 – 50μm size class in ballast water samples. The stains will penetrate into organisms, where functional esterases (a specific type of enzyme) in living organisms will convert them into fluorescent products that are retained by cellular membranes. Using epifluorescent microscopy, fluorescing organisms are enumerated as ‘living’ individuals. Any motile organisms observed are also counted as ‘living’. Those that do not fluoresce are considered as being dead. Criticism of the vital stain method have been publicised by the informal organisation that has been established to argue against the USCG decision. On its website it argues that there is a common misconception that the vital stain method gives a definitive “live” or “dead” status to an organism. In actuality, the method evaluates the presence of esterases within organisms and the integrity of the membrane. These properties do not define a cell as being “living” or “dead.” The statement says there is a common misconception that the vital stain method is highly reliable, based on the results of one study (Steinberg et al., 2011). The study reported that false negative rates were very low for the four sites investigated. Motility was used to judge whether the staining was accurate, however, there are many species and stages of phytoplankton that are non-motile. The true false negative rates could have been significantly higher, but cannot be recalculated because the species were not disclosed. Recent studies in ballast water applications have shown that for many species of phytoplankton, the fluorescence of stained living cells is not measurably higher than stained killed cells, so live and dead cells cannot be distinguished. For some species, the fluorescence is lower in stained living cells than in stained killed cells – this opposes the expectations of the assay. In addition, staining intensities can be very different between species, making evaluations of mixed assemblages prone to error; dead cells of a bright species can have a higher fluorescence than live cells of a dim species. The method used in the Most Probable Number Dilution-Culture Method (MPN method) is very different. It measures the number of viable phytoplankton cells in a sample, via their ability to reproduce. It is a formal mathematical calculation based on binary scoring data from a set of dilutions, and replicates from a sample. In a ballast water management application, the binary scoring is of reproduction or no reproduction of phytoplankton, in dilutions and replicates of a ballast water sample. In an MPN test, a ballast water sample is serially diluted and the dilutions incubated at favourable light and temperature levels together with nutrients and purified water constituents from ambient water. Over time, dilutions are monitored for chlorophyll fluorescence with a standard laboratory fluorometer. If one or more viable cells are present in any dilution, they will reproduce and increase the chlorophyll fluorescence. Dilutions are simply scored for growth or no growth, based on changes in chlorophyll fluorescence over the incubation period. Scores at each dilution are used in the MPN calculation to determine how many viable cells were present in the original sample. A criticism of the MPN method is that ballast water samples can contain a mixture of phytoplankton species that may grow at different rates, and a fast-growing species could outcompete a slow-growing species. Proponents of MPN say there is a misconception that this makes the assay blind to slow-growing species and say that viable cells of all species are distributed through the matrix of dilutions and replicates. The accuracy of the assay relies on detection of growth. In a competitive situation between two species in the same replicate, as long as one of them grows to detection, the correct scoring result will occur. At the extreme dilution where replicates contain either one or zero viable cells, there is no competition. Thus the assay is not blind to certain species due to competition. One academic, who has submitted a letter in support of the MPN method, said the MPN assay has been used in microbiology for well over a century. It can be reliably carried out by technicians with only a small degree of training, and is able to detect low concentrations of viable microorganisms. In contract, microscopic techniques require substantial training of laboratorians, and without concentration have a high detection limit; the need for concentration, which is often not required for the MPN assays, adds an additional source of variability to the assay — and may produce losses. For those outside of the scientific community it is difficult to decide on the validity of claims on the pros and cons of the two testing methods, arguably it is also difficult for those inside to do so as well otherwise there would be no dissent. It is possible that because the US Environmental Protection Agency has now been ordered by the Supreme Court to revise the US ballast requirements, some fresh thinking on a wide variety of issues will be needed and that may include the testing protocols. For UV system makers not yet embarked on US type approval, the choice is to follow the few of their peers who have decided to do things the US way or else to wait for the outcome of the appeals to the USCG and if that is successful to request their current results be accepted. Either route is likely to mean delays as the USCG has intimated that all of the authorised independent test facilities have been in high demand and new work may be out of the question.