WEA technique saves time, money

The OSPAR Commission, the mechanism by which 15 governments of the western coasts and catchments of Europe cooperate to protect the marine environment of the northeast Atlantic, has been considering a risk-based approach to the monitoring of produced water discharges. It seems likely an approach using whole effluent assessment (WEA) alongside a single substance approach could be introduced in the near future. The essential element of WEA is the application of testing to a sample of the whole effluent rather than individual substances known to be within it.

According to OSPAR's 2007 "Practical Guidance Document on Whole Effluent Assessment," the added value of WEA is greatest in complex effluents, which contain mixtures of chemicals or require very detailed process-specific knowledge to allow characterization. Produced water would seem to fall into that category, as it consists of both naturally occurring and added chemicals, which can include biocides, defoamers, or scale and corrosion inhibitors, and its composition varies depending on location, formation type, and stage of production. Therefore, a WEA approach to produced water testing can have the potential benefit of providing a measurement of not only the effects of substances known to be present but also unknown substances as well as the combined effects of all components.

A new approach

The WEA approach involves using biological tests to determine persistence, bioaccumulation, and toxicity (PBT-criteria), and tests commonly applied to marine systems are based on the use of single species from different trophic levels. For example, in the UK the Offshore Chemical Notification Scheme requests ecotoxicity data for offshore chemical products from an algae (Skeletonema costatum), a crustacean (Acartia tonsa) and a fish species (e.g. Scophthalmus maximus). The bacteria Vibro fisheri also is commonly used as a single species test.

This LumiMARA plate image was captured after 30 minutes of exposure to a two-fold dilution series of sample. The decrease in the percentage of inhibition of bioluminescence is compared against the control row containing no sample. (Images courtesy of NCIMB Ltd.)

Researchers at the UK-based company NCIMB Ltd. have taken a new approach in developing what they believe to be the only multispecies WEA tests currently available. The Microbial Array for Toxic Risk Assessment tests, MARA and LumiMARA, are based on the use of 11 microbial species to assess toxicity, and work to date indicates that results obtained from these tests encompass the range of responses obtained from more costly and time-consuming higher order tests.

The company was formed 30 years ago to manage the UK's National Collection of Industrial Food and Marine Bacteria. It has since grown to offering a range of chemical analysis and microbiological services to industry sectors including oil and gas and houses the biggest reference collection of industrially and environmentally valuable microorganisms in the UK. There are approximately 8,000 strains in the collection, which have been isolated from a wide variety of environmental locations and sources such as the gills of marine fish and hydrothermal vents. Some of these bacteria are regularly supplied to industry for use in antibiotic production, while others could have unique properties and commercial potential that has not yet been realized.

NCIMB used strains from the collection to develop its MARA test. The microorganisms used were carefully selected to cover a wide genetic diversity and provide a spectrum of sensitivities to different components of produced water.

The MARA test comprises 10 bacterial species and one yeast. A serial dilution of the effluent is prepared, added to microbial growth media in a microtiter plate, and inoculated with the array of microorganisms. The plate is incubated for 18 hours at 30°C (86°F), with the addition of a redox dye that allows assessment of the amount of growth, and scanned. The response of the bacteria can range from no inhibition of growth to no growth, with varying degrees of growth inhibition in between. The data are imported to image analysis software and plotted; different strains will respond differently to different dilutions of the effluent, so a toxicity rating for each strain can be calculated and used to create a toxicity profile for the effluent.

LumiMARA employs 11 naturally bioluminescent bacteria from a variety of habitats, including nine marine and two freshwater species. Bioluminescent bacteria emit light as a result of a biochemical reaction. When these bacteria are exposed to toxins, their luminescence is inhibited, and reduction of light output can be used as a rapid and direct measurement of toxicity. Different dilutions of the effluent sample are added to a plate containing the 11 bioluminescent strains. The plate is incubated for 15 minutes and read using a luminometer, and results are used to calculate a toxicity rating (for both tests an EC50 value is calculated for each of the species, i.e. the concentration of toxicant which induces a response halfway between the baseline and maximum). Combined integrated evaluation of all 11 species provides a unique toxicity profile for the effluent being tested.

North Sea case study

Terry Dando, MARA R&D, and Emma MacGill, Oilfield Services, examine the analysis of a MARA plate using the MARA software.

Both MARA and LumiMARA have been tested on pro- duced water samples from 15 North Sea oil and gas installations, alongside established algae (Skeletonema costatum) and crustacean (Acartia tonsa) single species tests, as part of a wider coordinated study to assess the role of WEA in a risk-based approach to produced water monitoring. The offshore installations sampled included a mixture of oil and gas producers as well as early and late stages of production and high and low water cut.

The microbes, the algae, and the crustacean used in the test all showed different degrees of sensitivity to the different produced water samples, but for all of the produced water samples tested, the sensitivity of the algae and crustacean fell within the range of responses shown by the different microbial species. In other words, it appears that tests using a genetically diverse group of microorganisms can give results that are indicative of the response of the wider ecosystem. As higher order testing can take up to three days, the results suggest that multispecies microbial tests like MARA and LumiMARA can offer a fast, low-cost method for the WEA toolbox that could potentially be used as an initial screening test before moving on to testing that uses higher order organisms such as crustaceans and fish.

Additional benefits

The multispecies microbial approach to WEA also offers a number of additional benefits and uses. The growth of each of the different microbial species in the presence of different dilutions can be used to produce a dendogram comparing unknown chemicals to standards and can provide insight into the mode of toxic action. The fingerprints produced from the multispecies tests are ideal tools for allowing comparisons to be made between samples, locations, and time points as well as in response to chemical treatments. The tests also can be used to detect subtle changes within the toxicity profile of samples, making them a valuable tool in terms of identifying any unexpected changes in composition of the produced water before they become a bigger problem and for monitoring the effectiveness or otherwise of any remedial steps taken.