Sampling of a ship’s ballast water is an essential part of checking compliance with the International Convention for the Control and Management of Ships’ Ballast Water and Sediment (BWM Convention). The BWM Convention entered in to force on the 8th September 2017 and requires ships to meet the Ballast Water Performance standards (D-2 standards).
Sampling and analysis of ballast water is a complex issue as it requires specialist equipment and a trained workforce. Sampling for compliance with the D-1 ballast water exchange standard is a forensic process, requiring a knowledge of source water parameters and comparison with measured parameters. It is comparably less complex and more of a straightforward process than sampling for D-2 performance standards. Please note, D-1 ballast water exchange sampling is not discussed in this article.
Regulation D-2 performance standards
Ballast water management (BWM) performance standards stipulate the maximum number of organism presence in the respective volumes of samples according to the organism category.
Ballast water treatment
The BWM Convention specifies the biological performance standard for ballast water discharge but does not prescribe the method(s) a ship should use to meet the requirements. The majority of ships are expected to install a Ballast Water Management System (BWMS) to treat the ballast water to be compliant with D-2 Performance standards.
Different technologies have been used in various combinations for the treatment of ballast water. BWMSs mostly consist of two phases of treatments, including physical separation followed by disinfection.
Type approval testing of BWMSs
The IMO and USCG have published their type approval requirements for BWMSs. Although there are differences, the two regulations followed similar testing regimes which includes land based and shipboard testing. Testing requires a minimum challenge condition for the test to be considered successful. Specifically, during the land-based testing, a minimum number of organism densities for organism > 10μm class categories are to be present at the uptake. The taxonomy of an organism should be consisted with at least five species across three phyla divisions. In addition, sampling should be conducted for three indicator microbes and heterotrophic bacteria plate count. The regulation also requires five test cycles (a set) to be conducted across each of the three salinities. Uptake sampling also includes analysis for physical water conditions such as temperature, particulate organic carbon (POC), dissolved organic carbon (DOC), total suspended solids (TSS), mineral matter and system relevant paraments such as UV transmittance, biochemical oxygen demand (BOD), total residual oxidants (TRO), and so on.
Type approval testing requirements consist of specific guidelines for type and method of sampling, sampling duration, sampling volume, sample handling and enumeration. These requirements can be found on Resolution MEPC. 300(72) Code for approval of BWMS and EPA/600/R-10/146 Generic protocol for the verification of ballast water treatment technology (ETV protocol).
For those BWMSs that make use of active substances are to be operated with Maximum Allowable Discharge Concentration (MADC) limit of 0.1 ppm as TRO. Depending on the type of active substances (Chemical) in use, the treated ballast water may contain Disinfected by- Products (DBP). The concentration of the DBP’s vary with the organic and inorganic constituents of the ballast water. During type approval of the BWMS and assessment of environmental acceptability is conducted for the active substances. The intention is to determine if the active substances, preparation or relevant chemicals are not persistent bio accumulative and toxic (PBT). Whole Effluent Toxicity (WET) and DBP’s are tested and evaluated during the type approval process of the BWMS in accordance with Resolution MEPC.169(57) procedure for approval of ballast water management system that make use of active substances (G9).
The collection of ballast water sampling is a skilled task; ballast water may be many thousands of cubic metres in volume held in several different and often complex shaped tanks. In addition, water and the organisms in it may not be homogeneously distributed. The collection of a representative sample with minimum mortality to the organism is the key to achieve an accurate reflection of the organism count in the sample volume. All samples and analysis are carried out to determine whether a ship complies with the BWM Convention and should be performed under reliable and verified QA/QC procedures. Any method, approach or sampling procedure should be rigorously validated, and the practicability should be assessed.
Sampling directly from ballast tanks is not recommended because of the difficulties of accurate sampling from organism population known to be heterogeneous (not mixed), as is the case with ballast water tanks. In addition to allowing for the uneven distribution in ballast water tanks, obtaining representative samples directly from tanks is more challenging because of their access, shape, size and complexity. Therefore, the samples are drawn from the discharge line.
Sampling for D-2 performance standard conducted as an inline sampling at the ballast water discharge pipe. According to Resolution MEPC. 173(58) Guideline for Ballast water sampling (G2) on the sampling of ballast water sampling points should be an isokinetic pipe and valve system that allows the taking of a representative sample of ballast water from within the discharge pipe. Installation of isokinetic sampling points is of a straight section of the discharge pipe at a point where the flow in the main pipe is fully mixed and fully developed, close to the ship overboard as practicable.
Isokinetic sampling ports installed in discharge pipes are equipped with a valve(s). Physical barriers such as valves may add undue stress to sensitive aquatic organisms. Only diaphragm valves and ball valves (fully opened during sampling) are appropriate for ballast water sampling.
It is important to ensure that samples are representative of the entire volume of interest and, if not collected throughout the entire discharge, they should be collected in a time-averaged manner in the case of multiple sample collection (for example: evenly distributed throughout a discharge).
Detailed versus indicative analysis
A detailed analysis provides direct measurement of a representative sample used to determine the viable organism concentration of a ballast water volume of interest, and should provide measurement of viable organism concentration that can be directly comparable to the D-2 standards.
An indicative analysis means a compliance test that is a relatively quick, an indirect or direct measurement of a representative sample of the ballast water volume of interest. A direct measurement that can be compared to D-2 standard may also be indicative if it has large confidence interval or high detection limits.
The qualitative difference between detailed analysis and indicative analysis often relies on the level of statistical confidence.
Detailed analysis of ballast water sample
The sample volume and number of samples will depend upon the objective of sampling, such as determining the number of organisms in different size classes; to assess the viability of organisms in different size classes; the specific analytical method to be used; and the statistical significance and certainty required. Sample handling and storage will also vary depending on the objectives and specific analytical methods. The manner sample is taken (e.g., net or pump) and the conditions in which it is stored (e.g., light, temperature, storage container) should be appropriate for the analytical method used.
Organism ≥ 50 μm size category
Inverted or simple upright microscopes are used for the enumeration of morphologically healthy organisms and motile organisms, as well as for measuring the size of organisms. This technology needs some skill and experience to evaluate the health of the individual organisms in the sample. Planktonic organisms may be fragile, and samples may need to be concentrated further to aid the accurate quantification of organisms. There are many methods to achieve this, however, care has to be taken to reduce physical stress as this may result in reduced viability levels. A simple, rapid, flexible and cautious method for concentrating zooplankton cells is the use of transparent membrane filters. If the sample analysis is performed on board the sample can be filtered directly on to this membrane, which can subsequently be placed directly under a microscope for examination. The plankton net cod-end containing the concentrated sample should be emptied into a 20-micrometre filter. Furthermore, sample concentrate has to be transferred into a 100ml container and mixed well before sub-sampling it for analysis. Sub-samples of 1ml or 2ml volume have to be transferred into counting chambers for counting the organisms.
Testing the sample for movement and response to different stimuli are simple techniques for the examination of viable/dead zooplankton under a stereomicroscope. The observation for organ activity, such as heartbeats, may also contribute to the viability assessment. Size measurement may be achieved by placing a piece of the filtering mesh (50 micrometres in diagonal dimension) underneath the stereomicroscope dish as a scale for counting all living organisms greater than or equal to 50 micrometres in minimum dimension, or the addition of 50-micron micro beads to the sample, may help with size calculations. Vital staining and electronic means of counting may also add value to these methodologies. The storage condition and time before analysis is likely to be critical to reduce mortality in the sample.
Organism ≥ 10 μm and ≤ 50 μm size category
Fluorescence generated from photosynthetic pigments can be used for more detailed analysis of the morphological health of organisms. A microscope with fluorescence capabilities is needed for the evaluation of stained organisms. However, this methodology only identifies phytoplankton (both living and dead) in the sample and makes no size differentiation. Fluorescein di-acetate (FDA), chloromethyl fluorescein diacetate (CMFDA) and Calcein-AM vital stains have both been used to determine viability. When non-specific esterase (enzymes found in live cells) are present, they cleave the acetate groups from the stains, and the resultant fluorescein molecules fluoresce green when illuminated with a blue light from an epi-fluorescence microscope. Inverted or upright microscopes are used for enumeration of the organisms. Flow cytometers are advanced technologies which can be used to determine size, and viability of organisms in ballast water with vital staining. Most Probable Number (MPN) is a method to evaluate the number of organisms in a sample by regrowth experiments, in which the visual appearance of photosynthetic organisms in a sample is followed by a specific period. At the time of writing, methods such as MPN use to evaluate viability of phytoplankton is not accepted in the United States.
10 μm < size category (indicator microbes)
There are international standards in place to analyse the bacteriological indicators in ballast water samples. Following standards should be used for the respective microbes: Enterococci, ISO 7899-1 or 7899-2; or Standard Method 9230 (in the United States), Escherichia coli, ISO 9308-3, ISO 9308-1; or Standard Method 9213D (in the United States). The methods used should be quantitative and based on a 95-percentile statistical evaluation. For Vibrio cholerae: ISO/TS 21872-1/13 is appropriate. 100 ml of ballast water should be filtered and incubated according to ISO/TS 21872-1. Analysis needs to be undertaken in a specialist laboratory.
Indicative analysis of ballast water samples As mentioned above, an indicative analysis is either an indirect measurement, or a direct measurement with a large confidence interval, a statistically low sample size, or high detection limits. However, there are currently no standardised methods for verifying indicative analysis equipment or required confidence interval or detection limits. There are several types of indicative analysis kits available in the market, the majority are portable equipment with few. being an inline measurement type.
The majority of the indicative analysis equipment that can detect Organism ≥ 10 µm and ≤ 50 µm size category, have measurement principles based on Fluorescence, Active fluorescence or Cytometry. Equipment that is based on pulse counting FDA, Mortality and Fluorescence Assay (MFA) and adenosine triphosphate (ATP) claim to detect organism ≥ 50 µm size class. For the indicator microbes category, nutrient indicators and microbial enzyme activity based on Most Probable Number (MPN) method uses to detect E. coli and Enterococci. There is only one analysis equipment based on measurement principles of ATP, an energy carrier found in all living organisms, claiming to be able to detect all three size classes, but with an estimation of total bacteria concentration.
Most of this equipment uses either consumables such as regents and/ or ancillary supplies such as disposable filters to perform analysis. The equipment manufacturers may have their own guidelines for sampling, preparation and disposal. Sampling volume may also range from a couple of drops to a few litres, and the time to obtain indicative analysis test results ranges from a few seconds to one day.
Sampling for compliance
Sampling the entire discharge may have some challenges because large numbers of samples and large volumes are required over a long period of time. Sampling personnel would be required on the vessel over a significant period of time. In the case of a sequence of samples, the sampling can be developed to fit the situation on board the vessel. Different types, sizes and cargo profiles of vessels trigger very different ballast water discharge profiles and times.
Considering the practicability of SB samples, EMSA recommended the following to obtain representative sample from ballast water:
- Minimum 300 to 450 litres should be filtered and concentrated for the bigger organism standard (Organism ≥ 50 µm). For the smaller organism standard (Organism ≥ 10 µm and ≤ 50 µm size category, a “continuous drip” sample totalling approximately 5 litres (i.e., collecting approximately 0.5 litres of sample water every minute during the entire sampling. The resulting 5 litres of sample water should be mixed and sub-sampled to create two sets of samples, one alive and another preserved. It is recommended sub-sample volumes of 60 to 100 ml.
- A sample of approximately 1 litre should be taken as a sub-sample from the 5 litre “continuous drip” sample after it has been mixed for the bacterial standard.
- Longer sampling times may result in an underestimation of the viable organism concentration in the discharge, especially for bigger organisms. Bigger organisms are negatively affected by longer sampling times due to mortality caused by the concentration of the sample through a net.
- It is also assumed that the sampling flow rates may influence the results. Lower flow rates obtained by partially closed valves of the sampling line may damage organisms in the discharge, and a similar negative effect may be caused by strong flow rates affecting mainly the filtering process of the bigger organisms. Hence, the flow rate, or “valve” effect, may cause an underestimation of the organism concentration as organisms may die during the sampling process.
- Analytical measurement (biological, chemical or physical) will have a defined protocol for type, size and treatment of sample containers together with sample treatment and storage. They are essential for obtaining accurate and precise measurement from the sample.
IMO port state control guidelines require indicative analysis to be carried out to identify potential non-compliance before proceeding with detailed analysis. Recently developed BWM.2/circ.(70) Guidance for the commissioning testing of ballast water management systems, recommend indicative analysis of ballast water samples to check performance of BWMS after commissioning.
The difficulty of onboard sampling using the counting chambers is that organism counting for Organism < 50 µm size category may not be accurate as any movement, such as ship movement or movement on the microscope, induces water movements, which may lead to organisms being counted twice and some may be excluded. There are several counting chambers in the market which aim to provide greater accuracy under ship movement.
Sample processing for Organism ≥ 10 µm and ≤ 50 µm size category in minimum dimension may require concentration of samples. However, the concentration process may also damage the organisms.
The samples may be kept unpreserved for up to 24 hrs in polypropylene or polyethylene bottles and samples must be preserved directly after the collection in case of later analysis to prevent the adverse effects of light, temperature and microorganisms which might cause rapid decay of organisms. Samples can be kept alive by being stored in a fridge or preserved into some solution. Processing samples for bacteria could be prepared on board for transportation. Analysis of bacteria should be done under controlled laboratory conditions as the samples need an incubation time. V. Cholera will be analysed in a specialist laboratory. In most countries the labs will require permit/ clearance to analysis V. Cholera toxigenic types O1, O139.
Ballast water sampling often encounters logistical challenges in sampling, preservation, transportation and enumeration. As the global fleet continues to drive towards implementation of the BWM convention and an ever-increasing number of vessels will have to install a BWMS. To mitigate the risk of potential non-compliance, it is essential that shipowners and PSC rely on accurate ballast water sampling and testing, conducted using a proven methodology measured to a consistent standard.
 EMSA Study: Gollasch S., David M. Testing Sample Representativeness of a Ballast Water Discharge and Developing Methods for Indicative Analysis, Final Report, 2010
 IMO Resolution MEPC.173(58) Guidelines for Ballast Water Sampling (G2)
 IMO Resolution MEPC.252(67), Guidelines for Port State Control under the BWM Convention
 IMO Circular, BWM.2/Circ.42/Rev.1, Guidance on ballast water sampling and analysis for trial use in accordance with the BWM Convention and Guidelines (G2)
 IMO Circular, BWM.2/Circ.61, Guidance on methodologies that may be used for enumerating viable organism for type approval of ballast water
 MEPC 74-INF.18 Summary of currently available ballast water indicative analysis instruments.
 IMO Circular, BWM.2/Circ.62, Guidelines on Contingence measures under the BWM Convention
 IMO Circular, BWM.2/circ.70, Guidance for the commissioning testing of ballast water management systems
 www.biomarineservices.co.uk : Loaiza, J
 LR publication, Understanding ballast water management, Guidance for shipowner and operators