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Test protocol land-based tests including QA/QC

1. IMO 2005 Guidelines for approval of ballast water management systems G8 Annex3 Resolution MEPC 125 53 Annex Parts 1 2 3 and 4 IMO 2008 Guidelines for approval of ballast water management systems G8 Annex 4 Resolution MEPC 174 58 Kraay G Zapata M Veldhuis MJW 1992 Separation of chlorophylls c1 c2 and c3 of marine phytoplankton by reversed phase C18 high perfomance liquid chromatography J Phycol 28 708 712 Mackey MD Mackey DJ Higgins HW Wright SW 1996 CHEMTAX a program for estimating class abundances form chemical markers application to HPLC measurements of phytoplankton Mar Ecol Progr Ser 144 265 283 Peperzak L amp CPD Brussaard 2011 Flow cytometric applicability of fluorescent vitality probes on phytoplankton J Phycol 47 692 702 Reinthaler T Herndl GJ 2005 Seasonal dynamics of bacterial growth efficiencies in relation to phytoplankton in the southern North Sea Aquat Microb Ecol 39 7 16 Schreiber U Neubauer C Schliwa U 1993 PAM fluorimeter based on medium frequency pulsed Xe flash measuring light A highly sensitive new tool in basic and applied photosynthesis Photosynth Res 36 65 72 Shapiro HM 2003 Practical flow cytometry John Wiley amp Sons Inc New Jersey Veldhuis MJW Cucci TL Sieracki ME 1997 Cellular DNA content of marine phytoplankton using two new fluorochromes taxonomic and ecological implications J Phycol 33 527 541 Veldhuis MJW Kraay GW 20
2. Settings Voltage Threshold FSC 25 SSC 198 FBG 534 FBO 394 FBR1 390 200 FBR2 547 FGO1 400 FGR2 453 FVB 341 FVG 482 e Click on syringe new specimen e Rename right click on specimen e Experiment Experiment layout Tab Acquisition Events to record fill in 10 000 000 Stopping time 300 e Carousel Carousel setup Fill in carousel number 1 2 3 or 4 under Carousel ID Recording Delay time 3 sec Mix Settings v start of carousel mix v interim mix after every 1 v tube mix duration 3 sec v Tube Pressurization Error Handling Current Run v Show error and wait OK e Acquisition dashboard Flow rate medium e Run Carousel e Every time you have to measure the flowrate with trucount beads Put 1 ml milliQ in a tube with n counts in it see label on package Use protocol Trucountbeads and measure at medium flowrate for 60 sec Fill in on the Excell file FACS Canto TruCount log xlsx with values of flowrates e Measure flowrate 59 NIOZ Land based Project Plan e Rinse the FCM according to the protocol SOP FACSCanto CALCULATIONS e Put data on your computer by using an USB stick Data processing should be done with the program FCS express e _Flowrate Total count measured count gate pi 2 x count gate p2 n count per tube see label on package Flowrate ul min Total count n x 1000 60 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Instit
3. Total 24 42 31 a The taxonomic system is as follows Kingdom Archea Bacteria Animalia Chromista Algae Plantae Subkingdom Infrakingdom Phylum Subphylum Division Class Subclass Superorder Order Family Genus Species b Division no phylum for this group The phylum unknown contains several species of unidentified phytoplankton flagellates General sampling strategy Samples are generally taken 1 In the harbour to assess test water quality before the pump Harbour water samples are analysed regularly from February onwards in order to monitor the spring plankton bloom 2 Immediately before the treatment equipment from the main pipeline but after the ballast pump that is used to pump up the test water from the harbour control TO 3 Immediately after treatment from the main pipeline treated TO and 4 During discharge from the main pipeline after the pump after 5 days control and treated T5 holding time 2 3 2 and 2 3 26 G8 guidelines and after completing a second passage through the BWTS when this step forms part of the treatment prescribed by the vendor of the BWTS i e in the case of the Cathelco BWTS treated only During ballast water tests samples will be taken sequentially covering the entire intake or discharge periods During the tests the following sample sizes will be used NIOZ Ballast Water Report 4 4 26 1 2 3 Untreated water cont
4. 2 PSU and 8 m brine 100 kg m industrial quality salt is added to increase salinity ca 2 PSU at the second set of test series At present only brackish and high salinity seawater conditions can be tested at NIOZ Figure 10 Extra natural sediment from the Mokbaai will be added when required to meet the minimum TSS concentration for the given salinity test cycle In addition per ballast tank 20 litre of mud 15 6 kg dry weight from the nearby Mokbaai Figure 1 will be added to the low salinity tests in order to reach the required TSS value of gt 50 mg L Figure 10 The organic carbon concentration is important in testing systems that use oxidizing agents as active substances DOC concentrations are usually below 5 mg L in low salinity test water but no DOC additions are made because the high POC values gt 10 mg L in the NIOZ test water are considered to compensate for that In other words the total organic load in low salinity test water is sufficiently high An overview of the required water quality with respect to the composition of total suspended solids particulate organic carbon and dissolved organic carbon is given in Table 1 NIOZ Ballast Water Report 4 2 24 Table 1 Requirements of salinity TSS POC and DOC in the test water for the high salinity and intermediate salinity and freshwater test regimes according to the G8 guidelines High Intermediate Freshwater salinity salinity units Salinit
5. The sample codes assigned a priori to the harbour tests Table 4 are also used in data handling i e the transfer of data from laboratory instruments to Excel files dedicated to specific analyses All data files are collected on a NIOZ network disk that is backed up at least once a day The separate data files are combined in one Excel file in which all appropriate calculations for D 2 and G8 will be conducted The data on the NIOZ network disk are accessible to authorised NIOZ test facility personnel only Statistical analyses will be performed in Excel version 14 Additional analyses will be performed in either SYSTAT version 13 or Primer version 6 SYSTAT and Primer allow for more sophisticated statistical analyses of the BWTS performance than the t tests that are recommended in 2 3 37 of the G8 guidelines The scientific hypothesis that will be tested the so called null hypothesis is that there are no differences between treated and control water samples Quality data are compiled in tables and when possible visualised in diagrams such as for instance Shewhart control charts NIOZ will report the total number of tests that were needed to meet the D2 standard five times for each salinity range NIOZ Ballast Water Report Table 5 Example of the assignment of coloured codes prior to tests Sample flasks contain the appropriate code on a coloured label to prevent misidentification TO is the day of treatment and Tx is the x
6. 7 Em je Treatment TE To of the ype Geopro En ander PE va as Vi RA ti EN 5 NIOZ Ballast Water Report 2012 4 PAAR A 2 MIA NIOZ Royal Netherlands Institute for Sea Research Ra D EN PROJECT PLAN FOR THE BIOLOGICAL EFFICACY TESTING OF THE CATHELCO BALLAST WATER TREATMENT SYSTEM FROM CATHELCO GmbH AS PART OF THE TYPE APPROVAL PROCESS UNDER RESOLUTION MEPC 174 58 CONFIDENTIAL Until further notice Submitted to the BSH September 2012 version 2 Signed on Texel The Netherlands on 10 September 2012 Lan L Peperzak Ph D NIOZ Royal Netherlands Institute for Sea Research P O Box 59 NL 1790 AB Den Burg The Netherlands Abstract This project plan for the testing of the Cathelco Ballast Water Treatment System consists of the following sections 1 Quality Management Plan QMP 2 Quality Assurance Project Plan QAPP 3 Standard Operating Procedures SOPs The Quality Management Program QMP addresses the quality control management structure and policies of the test facility The Quality Assurance Project Plan QAPP in section 2 is the project specific technical document reflecting the specifics of the test facility the BWTS tested and other conditions affecting the actual design and implementation of the required experiments The QAPP consists of general information information on the NIOZ test facility and the Cathelco BWTS test water quality sampling and sample storing the measurement of variables
7. The sample bottles are transferred to the laboratory and left undisturbed in the dark for all organisms and material to settle After at least 24 hours the full bottle weight is measured Using a water jet pump and specialized tubing the supernatant is aspirated The bottle is weighed once more The concentrate is transferred to small storage bottles that can be stored refrigerated for one year The bottle is weighed once more to determine the sample and concentrate volumes The samples are analyzed with an inverted microscope at 200x magnification method by Uterm hl The fixed samples or sub samples thereof are transferred into settling chambers and NIOZ Ballast Water Report neutralized using sodium thiosulfate After this the sample is stained using Bengal rose stain This stain specifically stains organic material and helps to identify organisms between sediment particles After staining the samples are left undisturbed in the dark to settle The time of settling depends on the settling chamber used but should be at least 1 hour per cm height of the settling chamber The iodide component of the Lugol s solution increases the weight of particles Therefore they settle on the bottom glass and can be counted Live dead separation in these samples is mainly based on the structural integrity of organisms This method can be applied for both zoo and phytoplankton Nevertheless certain groups are known to be affected by this standard method of
8. This Quality Management Plan QMP describes the organisation of the NIOZ quality system This consists of the quality system management and organisation the quality system components personnel qualifications and training procurement of items and activities documents and records computer hardware and software planning implementation of work processes assessment and response and finally quality improvement 2 Quality system management and organisation The quality system of the NIOZ is devoted to the reliable testing of BWT systems This means that accurate and precise measurements of the test ballast water and the treated water including the experimental control water need to be assured Accessibility of 7 NIOZ Land based Project Plan experimental and measurement protocols quality control data and data analysis should lead to a high fidelity in the conclusions reached during G8 9 tests of BWTS performance and other relevant research activities NIOZ is an IMO recognised test institute and is certified by Lloyd s Register The project leader of ballast water research is Dr Louis Peperzak He is responsible for the proper functioning of the test facility and for carrying out BWTS tests laboratory analyses production of Standard Operating Procedures SOPs new methods in ballast water research treatment enumeration vitality viability measurements compliance monitoring and the support of all staff and students that are invo
9. day of sampling Usually x 5 at discharge Test numbers are Roman numerals I II III etc using a new number for every test e g I to X Replicates are denoted with normal numbers 1 2 3 etc The example codes I T5 M 1 4 1 is for each manufacturer the first replicate sample of Test I on day 5 T5 Sample Quay Code Label colour Wadden Sea harbour inlet Navicula WSn Red Wadden Sea harbour inlet Pelagia WSp Red Orange Control tank TO Navicula Cn Green Control tank TO Pelagia Cp Green Red Treatment tanks for TO to Tx e g Manufacturer 1 Navicula I T5 M1 1 Orange Manufacturer 2 Navicula I T5 M2 1 Yellow Manufacturer 3 Pelagia I T5 M3 1 Purple Manufacturer 4 Pelagia I T5 M4 1 Blue 40 NIOZ Ballast Water Report 41 REFERENCES Cassoti R S Mazza C Brunet V Vantrepotte A Ianora amp A Miralto 2005 Growth inhibition and toxicity of the diatom aldehyde 2 trans 4 trans decadiental on Thalassirosira weissflogii baciillariophycea J Phycol 41 7 20 Falcioni T S Papa and J A Gasol 2008 Evaluating the flow cytometric nucleic acid double staining protocol in realistic situations of planktonic bacterial death Applied and Environmental Microbiology 74 1767 1779 Gasol JM del Giorgio PAD 2000 Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities Sci Mar 6
10. of BW test water together with pH measurement e Stir the salinity temperature probe slowly through the sample record when the reading is stable e Record the values in the harbour journal LITERATURE Operational Manual Conductivity Measuring Instrument version 2 0 Greisinger V O F In 2004 at the time of the Ballast Water Management Convention salinity had no unit Therefore PSU as used in G8 is not a scientifically acceptable unit http www oceanographers net forums showthread php 902 Salinity Does Not Have Physical Units Since 2010 salinity is in SI units g kg http www teos 10 org 46 l NIOZ Land based Project Plan ip Ri 10Z Royal Netherlands Institute for Sea Research gt STANDARD OPERATING PROCEDURE DATE 2012 09 04 AUTHOR Josje Snoek pH 2012 1 Goal Determination of pH during harbour testing of ballast water treatment systems in fresh brackish and seawater MATERIAL amp EQUIPMENT e Metrohm 826 pH mobile and Metrohm combined pH glass electrode Unitrode 6 0258 010 with Pt1000 for temperature e Metrohm buffer solutions pH 7 and 9 30 x 30 ml e Bucket 12 litre with Ballast Water test samples Calibration The glass electrode is calibrated at intake and discharge with 2 Metrohm buffer solutions e Turn on the pH meter by pressing the red button e Press OK to enter lt param gt go with cursor to cal settings no off buffer press 2 for a 2 point calibration Leave lt param gt by press
11. Quality system Testing RVA L154 for further analysis All analyses are carried out according to NEN ISO standards Escherichia coli Analysis for Escherichia coli is carried out according to NEN EN ISO 9308 1 for the analysis of surface waters For this the samples are filtered through membrane filters pore size 0 45 um and these filters are incubated on a selective agar plate Incubation is 4 5 0 5 hours at 37 1 C and then another 19 5 0 5 hours at 44 0 5 C After that the incubated filters are transferred on sterile filters soaked with Indol reagent For colonies of E coli this yields a red colour These red colonies are counted and set into relation to the sample volume Results are confirmed via a positive and a negative control For the latter sterilized water is incubated like a regular sample and to confirm the results it may only yield less than 1 colony forming unit cfu per mL The positive control uses a special strain of E coli also incubated like a normal sample to confirm that this species can grow and form colonies on the used media Enterococci group Analysis for this group is carried out according NEN EN ISO 7899 2 For this the samples are filtered through membrane filters pore size 0 45 um and these filters are incubated on a selective agar plate Incubation is 44 4 hours at 36 2 C on Slanetz amp Bartley medium After that red and pink colonies are counted If the presence of enterococcus bacteria can b
12. Pelagia WSp Red Orange Control tank TO Navicula Cn Green Control tank TO Pelagia Cp Green Red Treatment tanks for TO to Tx e g Manufacturer 1 Navicula I T5 M1 1 Orange Manufacturer 2 Navicula I T5 M2 1 Yellow Manufacturer 3 Pelagia I T5 M3 1 Purple Manufacturer 4 Pelagia I T5 M4 1 Blue 43 NIOZ Land based Project Plan 7 References Cassoti R S Mazza C Brunet V Vantrepotte A Ianora amp A Miralto 2005 Growth inhibition and toxicity of the diatom aldehyde 2 trans 4 trans decadiental on Thalassirosira weissflogii baciillariophycea J Phycol 41 7 20 Falcioni T S Papa and J A Gasol 2008 Evaluating the flow cytometric nucleic acid double staining protocol in realistic situations of planktonic bacterial death Applied and Environmental Microbiology 74 1767 1779 Gasol JM del Giorgio PAD 2000 Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities Sci Mar 64 197 224 Gregori G and others 2001 Resolution of Viable and Membrane Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining Appl Environ Microbiol 67 4662 4670 Jeffrey SW Mantoura RFC Wright SW 1997 Phytoplankton pigments in oceanography In S W Jeffrey R F C Mantroura and S W Wright eds Phytoplankton pigments in oceanography guidelines to modern methods SCOR UNESCO Paris pp 661
13. QUALITY SYSTEM COMPONENTS 9 PERSONNEL QUALIFICATIONS AND TRAINING 10 PROCUREMENT OF ITEMS AND ACTIVITIES 10 DOCUMENTS AND RECORDS 10 COMPUTER HARDWARE AND SOFTWARE 11 PLANNING 11 IMPLEMENTATION OF WORK PROCESSES 11 ASSESSMENT AND RESPONSE 12 QUALITY IMPROVEMENT 12 O OON OO WN o Aerial photo on cover by Pieter de Vries Photography All other photos by NIOZ INTRODUCTION The NIOZ Ballast Water Treatment System BWTS test facility is part of the department of Biological Oceanography of the Royal Netherlands Institute for Sea Research NIOZ NIOZ is an institute liaised to the Netherlands Organization for Scientific Research NWO The mission of NIOZ is to gain and communicate scientific knowledge on coastal seas and oceans for a better understanding of the system and sustainability of our planet to manage the national facilities for sea research and to support research and education in the Netherlands and in Europe NIOZ is an independent academic research institute participating in numerous international research projects in coastal seas and the oceans In addition time series of physical chemical and biological data from the Wadden Sea area are maintained in order to study long term changes in the ecology of this UNESCO World heritage site In this context the research on the efficacy of Ballast Water Treatment Systems BWTSs and related environmental questions fits within the NIOZ work field NIOZ continues to build on
14. Start stop and duration of the cleaning process are controlled by Cathelco s BW system No manual interference is needed Seawater Backflush line Cleaning line Overboard CIP unit Figure 3 Process plan of Cathelco s BWTS Water flows black lines at intake yellow lines at discharge and red lines during cleaning of the UV unit Calculation of the UV dose The UV dose is calculated directly from the water quality UV transmittance of the water and from the flow rate The use of single electronic ballast units for each individual UV lamp allows for a maximum of flexibility in the power consumption The UV lamps are operated at the most suitable dose rate at any time allowing for significant savings in overall power consumption of the system The UV dose at T10 70 will be about 135 J m2 at 200m3 h flow rate This dose is above the requirements for a 4 log reduction of many microorganisms if no photo repair mechanism occurs As there is no light inside the ballast water tanks our system accepts this dose during ballasting only If the UV T decreases further the flow will be reduced 18 NIOZ Land based Project Plan automatically to ensure that the minimum dose 133 J m2 is maintained During de ballasting the water is much clearer i e UV T is higher and the Cathelco system will dose between 400 J m2 and 700 J m which is well above the dose requirements for a 4 log reduction for the majority of organi
15. Water Report yyyy 1 2 The generic BWTS specific project plan including QAPP QMP SOPs as well as the G8 and G9 reports will also be numbered sequentially as NIOZ Ballast Water Reports These documents will be made publically available on the NIOZ web site 7 Computer hardware and software The computer hardware that is used in the NIOZ test facility consists of lap tops and personal computers that use Windows based software Both hard and software are maintained by the NIOZ Information and Presentation Centre IPC IPC is also responsible for the security of data and other information for instance by using up to date virus protection and making daily back ups The NIOZ network can only be accessed by authorised personnel and students In addition all BWTS test data are stored on a dedicated network share that is accessible only to authorised ballast water team members This authorisation needs to be granted by both IPC and one of the project leaders of the ballast water project All software used by the NIOZ test facility including Microsoft Office and dedicated software for data analysis is legally obtained 8 Planning Manufacturers of BWTSs that approach the NIOZ facility for testing of their equipment should be aware of several planning criteria 10 NIOZ Land based Project Plan First of all because NIOZ uses natural test water spring and summer are the only periods of the year in which proper testing acc
16. are usually below 5 mg L in low salinity test water but no DOC additions are made because the high POC values gt 10 mg L in the NIOZ test water are considered to compensate for that In other words the total organic load in low salinity test water is sufficiently high An overview of the required water quality with respect to the composition of total suspended solids particulate organic carbon and dissolved organic carbon is given in Table 1 26 l NIOZ Land based Project Plan Table 1 Requirements of salinity TSS POC and DOC in the test water for the high salinity and intermediate salinity and freshwater test regimes according to the G8 guidelines High Intermediate Freshwater salinity Total Suspended Solids TSS gt 50 Particulate Organic Carbon POC ee Dissolved Organic Carbon DOC to obtain a 10 unit salinity difference either brine or fresh water may be added natural mud is added to increase TSS as needed this also increases POC 4 2 Biological quality In order to establish the biological efficacy of the BWTS it should be tested with water containing a high concentration of organisms as well as a sufficient biodiversity 2 3 20 of G8 This is required by G8 to guarantee the effectiveness of the BWTS in different ecosystems across the globe The variety of organisms in the influent test water should be documented according to the size classes mentioned in Table 2 Natural water originating from the
17. be able to steadily position the pump because the freshwater surface level will change according to deballasting and tide This pump will be connected to the NIOZ installation Figure 6 by a flexible hose Figure 9 The Amarex pump to be used in pumping fresh water from a boat into the NIOZ installation BWTS test set up treatment and control tanks A typical test of a treatment system is performed with two treatment tanks and one control tank that are filled in rapid succession i e on the same day at approximately the same time in the tidal cycle Figure 6 The control tank with untreated water serves as reference to examine the effect of the treatment including holding for at least 5 days 2 3 35 G8 guidelines The control tank can also indicate an unexpected source of mortality due to the testing arrangement 2 3 37 G8 guidelines Therefore the average discharge results in the control water should not be less than or equal to 10 times the values in regulation D 2 1 2 3 36 G8 guidelines Toolbox meetings The general test set up is described in Figure 6 and in 3 2 Directly prior to each test a tool box meeting is held on the quay site with all team members During this meeting all actions such as order of tanks to be filled and flow rates are briefly discussed NIOZ Ballast Water Report 3 5 3 6 21 Ballast water book All manufacturers should log their activities in a ballast water book issued by the NIOZ Se
18. coastal zone of the North Sea high tide and the inner Western Wadden Sea low tide will be used The test period will cover the whole spring and early summer of the plankton growth season and therefore includes the natural occurring biodiversity and species succession The ambient plankton content in terms of species diversity in the relevant size classes is very high For instance in 2011 16 phyla and more than 70 species were detected during the test season Table 3 Only 5 species and 3 phyla are required 2 3 20 of G8 Table 2 Minimal numbers and species diversity required at intake for different size classes and groups of organisms 1 um 1 micron 0 001 mm Intake test water Organism unit Variet at least 5 species from at least 3 gt 10 m different phyla divisions at least 5 species from at least 3 gt 10 and lt 50 um gt 103 mL different phyla divisions heterotrophic bacteria gt 10 mL not further defined The natural waters of the test area include a large range of organisms varying in sensitivity to mechanical stress UV radiation or various active substances Besides fragile organisms also plankton that is highly adapted to harsh environmental conditions mostly hard shell organisms are present in the test water For completeness the plankton fraction lt 10 um is also included in the NIOZ analyses although this is not required by the G8 guideline 27 NIOZ Land based Project Plan Table 3
19. facilities including the two quays Navicula and Pelagia that are used in G8 tests NIOZ Ballast Water Report 3 2 18 Ballast water test site Pelagia Quay d NIOZ Ta Yg OF Q Os Om Figure 6 Piping and Instrumentation diagram of the Pelagia quay test site at the NIOZ harbour The installation to be tested is a UV treatment system The installation consists of three ballast water tanks one for control untreated water and two for treated water Brine or fresh water can be added to adjust the salinity of the test water Mud can be added to increase the concentration of Total Suspended Solids TSS Flow rates system pressures salinity and turbidity are monitored during intake and discharge P amp I diagrams are available for both Navicula and Pelagia quays and for different intake and discharge scenarios Freshwater intake in Den Helder and transport to NIOZ Fresh water in Den Helder will be taken in at location B Figure 7 by a ship that normally transports gravel and sand with a capacity of 650 m The ship will be cleaned of any remaining sand before taking in water The intake water will be pumped in the hold with tubes that are suspended at a height of maximally 1 meter below the water surface Intake will take place on the afternoon prior to the tests The Den Helder harbour is monitored by NIOZ partner IMARES According to IMARES Den Helder harbour is eutrophic and very productive In May July 2012 the concentr
20. human pathogens Live samples for microbiological analysis are taken in sterile bottles of 300 or 600 mL and sent to a special laboratory Eurofins C mark for further analysis The samples are transported immediately after sampling using a cooled transport container 4 C NIOZ Ballast Water Report 4 5 5 30 Sampling for additional incubation viability experiments Additional incubation experiments which are not required by G8 serve to better assess the effect of the different ballast water treatment systems and the five day tank storage on cell viability and concentration In case the treatment is insufficient and residual viable organisms remain present or resting stages or cysts germinate growth of the plankton will be stimulated under favourable conditions This incubation method also allows studies on the effect of the treatment over a period longer than 5 days up to 20 days The experiments usually involve control and treated water that were sampled at T5 the day of discharge They are performed in 10 L flasks in a climate room under optimal growth conditions for the plankton community including irradiance temperature and turbulence A sufficiently high level of nutrients is ensured through the addition of nitrate phosphate and silicate favouring phytoplankton growth and stimulating cyst germination throughout these experiments The standard variables measured include phytoplankton and microzooplankton abundance lt 10um
21. individual counts are recorded the difference between analysts should become lt 10 REMARKS From 2012 as well the Bogorov Gollasch dish is used chamber ends closed LITERATURE Otto Larink amp Wilfried Westerheide 2006 Costal Plankton Photo Guide for European Seas Muenchen Roger Harris 2000 CES zooplankton methodology manual San Diego Zetsche E M Meysman FJR 2012 Dead or alive Viability assessment of micro and mesoplankton J Plankton Res 34 493 509 58 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 02 23 AUTHOR Eveline Garritsen PHYTOPLANKTON Canto FCM 2012 1 Goal Determine the abundance of phytoplankton cells in Ballast Water samples MATERIAL amp EQUIPMENT e Nalgene bottles 1 litre e Flowcytometer BD Canto II e FCM tubes SAMPLING e Three 1 litre bottles are filled during de ballasting at the begin middle and end of filling deballasting the ballastwater tank e Store the bottles is the fridge till analyzing time e Take a representative sample of approximately 3 ml of every bottle in a FCM tube e Put the tubes in the carousel begin at position 1 and there should be no open places between tubes of different treatments ANALYSIS e Start up protocol Canto see protocol SOP Canto e User Ballastwater gt password ballast e Open protocol Phytoplankton dub click gt boekje open
22. marine research equipment is being designed and built according to the wishes of our individual scientists Facilities NIOZ invites marine scientists from Dutch and foreign institutes and universities to write scientific proposals involving the institute s research vessels laboratories and large research equipment Our ocean going research vessel Pelagia is shared on a European level in the Ocean Facilities Exchange Group www ofeg org The basic scientific disciplines at NIOZ are physics chemistry biology and geology Multidisciplinary sea research is regarded as one of the main strengths of the institute Therefore the research is organised in 5 multi disciplinary themes Open ocean processes Sea floor dynamics Wadden and shelf sea systems Climate variability and the sea and Biodiversity and ecosystem functioning Together with a number of oceanographic partners NIOZ also maintains the popular marine website www seaonscreen org For more information please contact our Communication amp PR department at cpr nioz nl or visit our website at www nioz nl NIOZ has extensive experiences in the field of ballast water and ballast water treatment technologies at its harbour on the island of Texel During the past seven years several pilot test for ballast water treatment were conducted in the NIOZ harbour and so far between 2007 and 2010 seven full scale land based tests were carried out for Final and Type Approval 2 2 Prof
23. maximum 6 hours in the dark and at ambient temperature Salinity is measured with a digital conductivity meter Temperature is measured with a calibrated digital thermometer pH is measured with a calibrated digital pH meter TSS POC Total Suspended Solids Particulate Organic Carbon For TSS analysis filters are dried at 60 C for at least 8 hours and weighed again The concentration of TSS per litre can be calculated from the sample volume and the weight difference of the filter before and after sampling TSS is expressed as mg L Next to determine the POC concentration the same filter is combusted overnight at 500 C and allowed to cool in a dessicator and weighed again The POC is calculated from the weight difference between this measurement and the dry TSS weight POC is expressed as mg C L Dissolved Oxygen DO Fixed samples in Winkler bottles are acidified with H2SO prior to measuring the optical density OD at 456 nm with a spectrophotometer The oxygen concentration is calculated using standards and expressed as uM O2 L or mg O2 L uM O 0 032 Since both salinity and temperature change over the season the oxygen concentrations is expressed as percentage relative to the natural saturation value for the given temperature and salinity Dissolved Organic Carbon DOC The DOC concentration is determined in the laboratory by a high temperature combustion method using a Shimadzu TOC Vcpn analyser according to Reinthaler amp Her
24. measured i e not antennae tails etc Examples are presented in Figure 15 Figure 15 Minimum dimension measurements red line in selected organism types A bivalve larvae B gastropod Potucharte Arona larvae C worm D echinodermata larvae E and F crustacean larvae and G copepod The viability of the organisms is assessed with Neutral Red which stains living organisms only and does not affect their survival rate This viability assessment remains unaffected by the possible NIOZ Ballast Water Report 5 2 2 5 2 2 1 33 death of organisms during staining or during sample analysis due to for instance warming of the sample This is because organisms that die after addition of the Neutral Red will still be clearly stained while those already dead prior to the addition will not be stained Neutral Red is pipetted in a ratio that yields an end concentration of approx 1 50 000 The Neutral Red stock solution is 1 2 000 i e approximately 4 mL of stock solution is needed to stain a sample of 100 mL The staining time is 2 hours Stained samples are filtered over a 30 um sieve and flushed into a Bogorov dish with filtered seawater Samples are analysed using a binocular with a 20x magnification for counting and up to 80x for species identification and measurements when necessary Neutral Red stains all major plankton groups including phytoplankton but it seems to have some limitations for bivalve larva
25. met slang TC dragergas O c glazen kolom met injectiepoort van bovenaf uit het apparaat halen en daarna de glazen buis ook van onderaf uit injectieblok trekken Pas op Indien apparaat aan staat is de kolom zo n 680 C deze buis apart neerleggen voor recycling op een schone en lege glasbuis twee streepjes zetten met zwarte viltstift voor markering van hoeveelheden a h v maatstreepjes op een kast tot aan eerste streepje vullen met Silica quartz chips daarna vullen tot volgend streepje met Platinised Alumina vervolgens injectiepoort schoonspoelen boven wasbak met milliQ denk erom dat de O ringetjes er niet uitvallen steek een pincet in de ringetjes en de opening om ze op z n plaats te houden daarna injectiepoort droog blazen met perslucht schuif glasbuis van onderaf weer in injectieblok tot hij stevig vast zit doe de glasbuis met inj blok terug op z n plaats in het apparaat en zorg dat het slangetje d in het andere slangetje past schroef a weer vast en zorg dat de dop de glasbuis ondersteund de glasbuis komt onder druk te staan 53 NIOZ Land based Project Plan daarna het perspex plaatje van de inj poort schoonspuiten met milliQ op apparaat en leg er een tissue onder en vervolgens onder de wieltjes op het inj blok e schuiven daarna schroeven b vastdraaien let erop dat de bovenkant van het plaatje in lijn staat met het achterste plaatje Als de kolom nog niet vervangen hoef
26. of each year NIOZ test water is analysed by all three staff until the difference in individual plankton concentrations is lt 10 Furthermore in the case of gt 50 um organisms the correct execution of sample and analysis procedures and identification and enumeration of the organisms is audited annually by an external independent consultant In 2012 this was performed by Mr F Fuhr of KiTe Aquatic Resources Consulting The Netherlands Additional training of staff members takes place on the job during inter comparison workshops of the ballast water test team in cooperation with the planktonlab of the Department of Biological Oceanography of NIOZ One such workshop was the comparison 9 NIOZ Land based Project Plan of flow cytometers held in January 2012 Furthermore a statistics course is planned for autumn winter 2012 A QA QC course is planned for the winter of 2012 2013 5 Procurement of items and activities The BWTS test facility is part of the NIOZ research institute and is required to use the NIOZ facilities for procuring items and services that include all sampling and laboratory equipment and chemicals The financial management assistant to the test facility ensures that all procedures are as required by internal NIOZ rules and by Dutch law Material and equipment for the test installations is procured by Mr A Smit of Smittech Den Hoorn The Netherlands In all cases of procurement the quality costs and supplier reputatio
27. organisms 250 um 2 Concentration and diversity of organisms 10 lt um lt 50 3 Concentration of heterotrophic bacteria 4 Concentration of coliform bacteria 5 Concentration of Enterococcus bacteria and 6 Concentration of Vibrio cholera The groups of organisms 250 um and 10 lt um lt 50 consist of both algae phytoplankton mainly lt 50 um and animals zooplankton mainly gt 50 um According to the Ballast Water Performance Standard Regulation D 2 and 4 7 G8 guidelines only viable organisms must be counted at the end of the test in both the treatment and the control tanks For both of the IMO relevant size classes organisms 250 um and 10 lt ym lt 50 multiple methods of enumeration and of assessing the viability are applied at NIOZ to verify the results at a high level of confidence In principle all methods should give a conclusive answer with respect to numbers and or viability of the remaining organisms At NIOZ the viability of all organisms is not only measured at discharge but during intake as well Furthermore the concentration of phytoplankton lt 10 um is measured All biological variables are measured in triplicate samples 2 3 29 G8 guidelines Sampling organisms 250 um The samples of the untreated water 20 L are taken in triplicate collected in clean buckets that are filled directly for volumetric measurements and poured through a 50 um sieve made from the same gauze as the sampling nets used for th
28. passed and the ballast water is pumped from the tanks through the UV disinfection chamber only Thus the ballast water is subjected to UV disinfection treatment prior to discharge overboard 17 NIOZ Land based Project Plan Specific features advantages of Cathelco s BWTS The filtration step Cathelco s BWTS can be operated with two different types of filters giving it the maximum possible flexibility in addressing different installation requirements The UV lamp system Cathelco s BWTS uses special medium pressure UV lamps with a reduced Mercury content These lamps are mounted to a solid flange and they are surrounded protected by a sealed robust quartz sleeve This complete UV lamp system containing two lamps is fitted to the UV reactor by a few screws Access to the UV reactor for maintenance is from a single side only The lamp recycling scheme On an annual basis the UV lamp system should be send off to Cathelco for refurbishment The renovated lamp system will be returned to the ship with a new 1 year operating guaranty if the lamp system has not been installed for a total time of more than 2 years The automated cleaning system of the UV This is the first NON chemical in place cleaning system for UV reactors CIP system It uses rubber cleaning elements that are supplied on demand to the UV reactor after the BW operation is finished The cleaning process is triggered by the intensity measurement of the individual UV lamps
29. pathogens PAM measurement for total phytoplankton viability Counting phytoplankton lt 10 um AwWwNe U U U A on A A A A A a U A UW UUNWNNNEHE WN Quality assurance Quality control QA QC 6 1 Ballast water tests 6 2 Laboratory analyses 6 3 Data analysis REFERENCES 13 NIOZ Land based Project Plan 14 15 16 16 42 42 44 1 INTRODUCTION The QAPP detailed in this document forms the basis for the biological efficacy testing of the CATHELCO Ballast Water Treatment System BWTS manufactured by Cathelco GmbH Kiel Germany in 2012 at the ballast water treatment facility of the Royal Netherlands Institute for Sea Research in The Netherlands The first version of this protocol was submitted to the BSH Bundesamt fuer Seeschifffahrt und Hydrographie or Federal Maritime and Hydrographical Agency Hamburg Germany in March 2012 in advance of the land based tests that started on April 12 of that year This Cathelco test protocol is a combination of a Quality Assurance Project Plan QAPP and a Quality Management Plan QMP In previous versions of this protocol these two were merged The QAPP is a project specific technical document reflecting the specifics of the BWTS tested the test facility and other conditions affecting the actual design and implementation of the required experiments The QMP should address the quality control management structure and policies of the test facility In 2012 the combined QAPP and the QM
30. proper procedures are followed during intake and discharge During the ballast water project all samples are taken stored and analysed according to the dedicated SOPs Prior to all tests sample codes are assigned following the rules explained in Table 4 Because the test site is within very short distance of the main NIOZ building all samples containing fresh and live material are immediately transported to the laboratory for direct analysis The sample storage flasks as well as cryovial boxes are labelled with the same coloured labels and codes Samples that are fixed for long term storage are stored in specifically designated refrigerators 4 C and freezers 20 C 80 C The samples for microbiological analysis of the presence and number of human pathogens will be taken in special bottles of 300 and 600 mL and sent to Eurofins C mark in Heerenveen accreditation certificate RvA lab no L043 The samples will be transported immediately after sampling using a cooled transport container 4 C Laboratory analyses The analyses of abiotic and biological variables are described in general in other parts of this project plan Detailed descriptions of each analysis are available These Standard Operating Procedures SOPs of sampling sample storage sample analyses data analyses and data management are part of the NIOZ Ballast Water QMP Specific quality assurance and quality control measures are contained in each SOP Data analysis
31. sample by filling a volume calibrated bottle with sea water Close the bottle and mix Open the bottle and add the reagents in reversed order C B A one by 51 NIOZ Land based Project Plan one close the bottle and mix vigorously after each addition be careful not to spill it on your clothes because of the strong acid e Open the bottle and add 100 700 ul Potassium Iodate Standard Stock solution to the blank sample and determine the Extinction at 456 nm and also measure a blank without addition of KIO3 Calculate the equivalent O2 concentration per volume KIO3 stock added 02 umol l 1000 ul stock added O2 umol ul stock bottlevolume ul stock added e Make a calibration curve E456 vs 02 umol l and calculate the slope and the intercept e Report the calibration data with those of the test samples 52 l NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 09 06 AUTHOR Eveline Garritsen DOC 2012 1 Goal This procedure describes how to analyse Dissolved Organic Carbon samples MATERIAL amp EQUIPMENT e TOC V Total Organic Carbon Analyzer Shimadzu ASI V autosampler Shimadzu 25 ml glass vials with screwcap without septum Pipets 5000 1000 200 ul tips for pipets 5000 1000 200 ul Platinised Alumina 1 Pt Granular 0 85 to 1 2 mm Elemental Microanalysis 50 gm BN 177644 e Silica Quarz Chips Granulaar 0 85 to 1 7 mm Elemental Microanal
32. the class societies that were designated by the national authorities These are the BSH Bundesamt fuer Seeschifffahrt und Hydrographie or Federal Maritime and Hydrographical Agency Hamburg for Germany and Lloyds Register London Rotterdam for The Netherlands Inspectie Leefomgeving en Transport ILT United Kingdom Maritime and Coastguard Agency MCA and Greece The BSH and LR are notified in advance of the BWTS test dates including the days of intake and discharge and are invited to witness all test related activities at the NIOZ test facility In 2012 additional oral presentations on BWTS testing at NIOZ were given to the Dutch IMO representative and ILT the BSH and to Lloyds Register NIOZ Ballast Water Report 2012 9 10 PERSONNEL QUALI FI CATIONS AND TRAINING All research personnel of the NIOZ test facility are actively involved in the development of SOPs and the QA QC measures within these SOPs The quality manager is responsible for the QA QC in each SOP and he checks the proper execution of QA QC during BWTS testing and the analysis of samples and data The quality manager will report the acquired QA QC information to the project leader of ballast water research who will if necessary instruct the personnel to adjust QA QC measures The quality manager will also use this information in his evaluation of the BWTS test results All research personnel of the NIOZ test facility is trained in such a way that at least two staff member
33. the water is still running Add 1 ml reagent A to the sample bottle with a dispenser place the dispenser tube in the sample Add 2 ml reagent B to the sample bottle with a dispenser place the dispenser tube in the sample Close the bottle with its own stopper same number Shake the bottle vigorously to ensure adequate reaction contact for the formation of the precipitate Store the closed bottle under water in a transport container filled with cold tap water Spectrophotometrical determination Preparation Turn on the Hitachi U 1100 Spectrophotometer 30 min before measurement Place the left side of the tubing attached to the flow through cuvette of the spectrophotometer in a beaker with MQ Attach a syringe to the other end of the tubing and suck the MQ through until it fills the syringe Place the tubing with syringe in a bucket on the floor and remove the syringe Fluid will keep running through the cuvette into the bucket Check regularly if it is still running Reset the blank value of the transmission at 456 nm when the reading is stable Take the bottles you want to measure out of the storage container without stirring the precipitate Dry the bottles on the outside and under the rim of the stopper with a paper towel Measurement Open the bottle by turning the stopper until it moves and slowly lift it from the bottle If the bottle will not open use an attachment which fits the hexagonal top of the stopper to increa
34. worden gedurende ca twee uur injecties met milliQ Zorg altijd voor vers milliQ slangetje van de ASI V door dop van milliQ vaatje Gebruik file aclimatization t32 Programma TOC Control opstarten Sample Table Editor aanklikken File aclimatization selecteren Edit Delete All data connect use settings Background Monitor Temp Fumace 562 C Dehumidifier 0 6 C start keep running standby vial 0 uit Erlenmeyer Bij alle regels steeds vial 0 invullen 21 regels OK External Accid Addion gt niet Start 100 ul per injectie View Sample Window rechtermuisclick Limits min max aan Y as aanpassen b v 1 min en 5 max bij milliQ View Methods Instellingen Methode bekijken NPOC Non Percal No of inj is aantal injecties per vial 5 6 No of wash 3 SDMax 0 1000 Max integration 02000 Sparge Time tijd van vooraf doorborrelen zuur toege voegd anorg C gaat eruit Datum is naam van bestand Save as file name 6 oktober 08 ballastwater t32 Edit Delete data All Bij invullen scheme Insert Auto Generate methode sample met No of samples nog 26 40 totaal OK Standby 54 NIOZ Land based Project Plan nr van vial invullen schema Sparging Acid Addition Let op geen Edit Acid Addition OK Na uur wassen met milliQ kun je de monsters draaien Sample Analysis Vul een formulier in voor het monsterschema Hierop
35. 00 Application of flow cytometry in marine phytoplankton research current applications and future perspectives Sci Mar 64 121 134 Veldhuis MJW Kraay GW Timmermans KR 2001 Cell death in phytoplankton correlation between changes in membrane permeability photosynthetic activity pigmentation and growth Eur J Phycol 36 167 177 Veldhuis MJW Fuhr F Boon JP Hallers Tjabbes C C 2006 Treatment of ballast water how to test a system with a modular concept Environ Technol 27 909 921 44 NIOZ Land based Project Plan Section 3 Standard Operating Procedures SOPs List of variables and related SOPs Supporting SOPs that are referred to in the listed SOPs are available on request Variable unit Reference SOP Page Salinity and PSU g kg Salinity and Temperature 46 Temperature C 2012 2 pH pH 2012 1 47 TSS and Particulate mg L mg L TSS POC 2012 2 48 Organic Carbon Dissolved Oxygen saturation Dissolved Oxygen 2012 1 50 Dissolved Organic mg L DOC 2012 1 53 Carbon Viable organisms 250 number per Mesozooplankton 2012 1 57 um including diversity m3 number of phyla and species Phytoplankton number per Phytoplankton Canto FCM 59 61 64 organisms 10 50 um mL 2012 1 FCM Canto operation 2012 1 FCM Canto data processing 2012 3 Phytoplankton diversity number of Koeman amp Bijkerk b v Koeman amp Bijkerk phyla and quality assessments
36. 0602 3 60 1482 162 1806 0 107 high 50602 3 60 792 84 960 0 057 med 0 057 0 001 50602 3 60 814 81 976 0 058 med Total single 2 double Flowrate total 60 counting time TCB batch beads volume sw Cathelco Phyto Cp T5 FCS FCS flowrate counted OUTPUT OUTPUT count ml sample sample date info flowrate ml min ml Large Large 17 04 12 I Cp T5 1 high 0 108 0 54 I Cp T5 1 102 189 17 04 12 I Cp T5 2 high 0 108 0 54 I Cp T5 2 140 260 17 04 12 I Cp T5 3 high 0 108 0 54 I Cp T5 3 167 310 Counted ml flowrate min counted Phyto 300sec 5 min count ml FCS OUTPUT counted volume 65 l NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 04 11 AUTHOR Eveline Garritsen Josje Snoek Phytoplankton vitality PAM 2012 1 Goal Measuring photosynthetic activity of phytoplankton MATERIAL amp EQUIPMENT e PAM Control Walz e WATER ED Emitter Detector Unit Walz e Water K Quartz glass cuvette e stopwatch CHEMICALS e 0 2 um filtered seawater SAMPLING e Place the sample in the dark for at least half a hour ANALYSIS e Switch on the computer e Start WinControl shortcut on the desktop in the centre e Select PAM settings File Load o Select folder double click on C double click on BW folder o Select file BW 2 press OK The following PAM settings will be loaded Meas
37. 1000 L each Furthermore in line sampling is conducted three times with a sample volume of 10 L and 2x 1 L The 10 L samples are used to subsample for abiotic variables The 1 L samples are used 1 to subsample for phytoplankton bacteria as well as for phytoplankton lt 10 um and 2 microzooplankton 10 lt um lt 50 Additional samples of 10 L are taken for incubation experiments 3 Treated discharge T Sampling is conducted in line three times with sample volumes of 1 m gt 50 Hm using 3 IBCs of 1000 L each Furthermore in line sampling is conducted three times with a sample volume of 10 L and 2x 1 L The 10 L samples are used to subsample for abiotic variables The 1 L samples are used 1 to subsample for phytoplankton bacteria as well as for phytoplankton lt 10 um and 2 microzooplankton 10 lt um lt 50 Additional samples of 10 L each are taken for incubation experiments The sub sample volumes taken from the in line samples deviate from 2 3 32 33 G8 guideline 10 lt um lt 50 1 L for untreated water and 10 L for treated water bacteria 0 5 L Note that G8 only specifies sample sizes but not which volume of these samples should actually be analysed The reason for this deviation is that sample volumes of 10 L for organisms 10 lt um lt 50 and 0 5 L for heterotrophic bacteria are impractical much smaller volumes will eventually be analysed without compromising the analytical accuracy The sample volumes for pathogenic
38. 2012 Hamworthy Wartsila Aquarius UV NIOZ works constantly on improving its methods and technologies for the accurate and precise enumeration of aquatic organisms and the measurement of their vitality and viability These methods are compared with other organisations and test facilities in workshops and within the global network of BWTS test sites GloBal TestNet of which NIOZ is a member On a European level NIOZ is the lead beneficiary of the North Sea Ballast Water Opportunity project It is policy of the institute to communicate scientific results as much as possible through international peer reviewed publications The NIOZ BWTS test facility not only performs land based G8 or G9 tests It is also equipped for laboratory tests to investigate the principles and efficiency of new technologies as well as for bench scale tests to examine prototypes and components of BWTSs In addition the test facility is able to analyse and evaluate samples from other test facilities from ship board tests and other ballast water related activities This evaluation may range from compliance checking to IMO standards till advanced statistical analyses In co operation with IMARES Den Helder The Netherlands and Go Consult NIOZ also offers a one stop shop for complete G8 or G9 type approval including toxicity testing ship board tests and dossier formation In all activities the NIOZ test facility strives to achieve highly accurate and precise results
39. 4 197 224 Gregori G and others 2001 Resolution of Viable and Membrane Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining Appl Environ Microbiol 67 4662 4670 Jeffrey SW Mantoura RFC Wright SW 1997 Phytoplankton pigments in oceanography In S W Jeffrey R F C Mantroura and S W Wright eds Phytoplankton pigments in oceanography guidelines to modern methods SCOR UNESCO Paris pp 661 IMO 2005 Guidelines for approval of ballast water management systems G8 Annex3 Resolution MEPC 125 53 Annex Parts 1 2 3 and 4 IMO 2008 Guidelines for approval of ballast water management systems G8 Annex 4 Resolution MEPC 174 58 Kraay G Zapata M Veldhuis MJW 1992 Separation of chlorophylls c1 c2 and c3 of marine phytoplankton by reversed phase C18 high perfomance liquid chromatography J Phycol 28 708 712 Mackey MD Mackey DJ Higgins HW Wright SW 1996 CHEMTAX a program for estimating class abundances form chemical markers application to HPLC measurements of phytoplankton Mar Ecol Progr Ser 144 265 283 Peperzak L amp CPD Brussaard 2011 Flow cytometric applicability of fluorescent vitality probes on phytoplankton J Phycol 47 692 702 Reinthaler T Herndl GJ 2005 Seasonal dynamics of bacterial growth efficiencies in relation to phytoplankton in the southern North Sea Aquat Microb Ecol 39 7 16 Schreiber U Neubauer C Sch
40. Biodiversity as number of species in NIOZ test water according to phylum and to size class based on data from the 2011 spring and early summer test season The test water contains at least 18 phyla with a total of 16 phyla in the 10 50 and gt 50 um size classes The total number of species in each G8 size class is 42 8 phyla for the 10 lt um lt 50 and 31 10 phyla for the 250um size classes Organisms lt 10um that are not bacteria are not part of the D 2 regulation The high test water biodiversity largely exceeds the G8 quideline 82 3 20 Phylum Number lt 10 um Number 10 50 um Number gt 50 um Amoebozoa 1 Annelida 5 Arthropoda 13 Cercozoa 1 Chlorophyta 1 1 Choanozoa 1 Ciliophora p85 Cnidaria El 2 Cryptophyta ee Ctenophora Echinodermata Euglenozoa RE 7 Haptophyta En Mollusca Myzozoa 2 6 Ochrophyta 12 28 Rotifera We A Unknown AN Total 4 1 Nematoda e RI 1 2 1 HH 24 42 31 The taxonomic system is as follows Kingdom Archea Bacteria Animalia Chromista Algae Plantae Subkingdom Infrakingdom Phylum Subphylum Division Class Subclass Superorder Order Family Genus Species P Division no phylum for this group The phylum unknown contains several species of unidentified phytoplankton flagellates 4 3 General sampling strategy Samples are generally taken 1 In the harbour to assess test water quality before the pum
41. OZ Ballast Water Project The QMP forms the basis of the project s quality assurance and quality control QA QC The Quality Assurance Project Plan QAPP is the project specific technical document reflecting the specifics of the NIOZ test facility the BWTS tested and other conditions affecting the actual design and implementation of the required experiments The QAPP consists of general information specific information of the test facility and the BWTS tested test water quality sampling and sample storing the measurement of IMO required variables and a general outline of the QA QC Variable specific QA QC measures are contained in each individual SOP that together form section three of the QAPP Notebooks are used to record data observations and deviations from SOPs that are made during BWTS tests as well as during laboratory analyses All notebooks are stored in the NIOZ Ballast Water archive In a number of cases such as in monitoring NIOZ harbour test water temperature and salinity and in the in line measurement of turbidity and salinity of test water at intake data loggers are used to store information electronically All digital information is stored on a dedicated server that is accessible to Ballast Water project personnel only Specific forms are used to record data during specific routine monitoring activities These activities include monitoring of meteorological and environmental data and equipment functioning during BWTS test
42. P collectively known as the quality control quality assurance QA QC program was extensively extended when compared to previous years Nevertheless the BSH invited dr A Cangelosi Northeast Midwest Institute USA to review the QA QC section of the protocol In addition NIOZ commissioned a review of the entire protocol to dr S Kools Grontmij The Netherlands The present version of the protocol is revised in reaction to these two reviews In the summer of 2012 the BSH decided that a sufficient number of marine saline tests had been performed earlier that year and that five successful freshwater tests were needed for type approval This meant that additional information had to be added on freshwater sampling and testing in the relevant chapters of this protocol The tests outlined in this protocol will evaluate the biological efficacy as outlined in the Guidelines for Approval of Ballast Water Management Systems G8 adopted on 22 July 2005 as Resolution MEPC 125 53 IMO 2005 and as adopted in revised form on 10 October 2008 as Resolution MEPC 174 58 23 Annex 4 of the Ballast Water Convention of the IMO IMO 2008 The test protocol will be submitted for Type Approval by the applicant Cathelco GmbH to the German National Administration BSH The Cathelco BWTS is composed of two main modules to disinfect ballast water of ships One module is a filtration system the second a UV reactor Both are operated during ballast water int
43. S i ferry Mokbaai Figure 1 Aerial view of the NIOZ harbour lower right NIOZ and the TESO ferry connecting the island of Texel with the main land top The Mokbaai is the source for additional suspended solids Photo Simon Smit Photography Den Burg Texel NI OZ profile All tests of the Cathelco BWTS will be carried out under supervision of the Royal Netherlands Institute for Sea Research Landsdiep 4 1797 SZ t Horntje Texel The Netherlands from here on NIOZ for details see www nioz nl NI OZ Royal Netherlands Institute for Sea Research is the National Oceanographic Institute of the Netherlands NIOZ is an institute of the Netherlands Organization for Scientific Research NWO The institute employs about 340 people at locations on the island of Texel on the border of the North Sea and the Wadden Sea main location and in Yerseke in the southwest of the country The annual budget is approximately 30 million The mission of NIOZ is to gain and communicate scientific knowledge on coastal seas and oceans for a better understanding of the system and sustainability of our planet to manage the national facilities for sea research and to support research and education in the Netherlands and in Europe In order to fulfil its mission the institute performs tasks in four specific fields Research The emphasis is on innovative and independent fundamental research in continental seas and open oceans Increasingly the ins
44. S test dates including the days of intake and discharge and are invited to witness all test related activities at the NIOZ test facility In 2012 additional oral presentations on BWTS testing at NIOZ were given to the Dutch IMO representative and ILT the BSH and to Lloyd s Register 4 Personnel qualifications and training All research personnel of the NIOZ test facility are actively involved in the development of SOPs and the QA QC measures as part of these SOPs The quality manager is responsible for the QA QC in each SOP and he checks the proper execution of QA QC during BWTS testing and the analysis of samples and data The quality manager will report the acquired QA QC information to the project leader of ballast water research who will if necessary instruct the personnel to adjust QA QC measures The quality manager will also use this information in his evaluation of the BWTS test results All research personnel of the NIOZ test facility is trained in such a way that at least two staff members are able to perform a specific sampling or analysis This should prevent personal bias in sample and data analysis In addition this procedure ensures that in case of unexpected staff absence sampling or sample analysis can still be carried out by trained personnel In case of non automated sample analysis the microscopic identification and enumeration of planktonic organisms the research personnel is trained in groups of three At the beginning
45. System BWTS test facility is part of the Department of Biological Oceanography of the Royal Netherlands Institute for Sea Research NIOZ NIOZ is an institute liaised to the Netherlands Organization for Scientific Research NWO The mission of NIOZ is to gain and communicate scientific knowledge on coastal seas and oceans for a better understanding and sustainable use of our planet to manage the national facilities for sea research and to support research and education in the Netherlands and in Europe NIOZ is an independent academic research institute participating in numerous international research projects in coastal seas and the oceans In addition time series of physical chemical and biological data from the Wadden Sea area are maintained in order to study long term changes in the ecology of this UNESCO World heritage site In this context the research on the efficacy of Ballast Water Treatment Systems BWTSs and related environmental questions fits within the NIOZ work field NIOZ continues to build on its many years of experience since 2007 NIOZ has tested nine BWTSs Table 1 Table 1 Ballast Water Treatment Systems tested at NIOZ year Company System 2007 Hamann SEDNA 2008 EcoChlor EcoChlor 2008 Hyde Marine Hyde Guardian 2009 Mahle Ocean Protection System 2009 Severn Trent De Nora BallPure STDN 2010 Aquaworx AquaTricomb 2010 Erma First ESK SA ErmaFirst 2011 Hamworthy Wartsila Aquarius EC
46. The Netherlands The first version of this protocol was submitted to the BSH Bundesamt f r Seeschifffahrt und Hydrographie or Federal Maritime and Hydrographical Agency Hamburg Germany in March 2012 in advance of the land based tests that started on April 12 of that year The QAPP is a project specific technical document reflecting the specifics of the BWTS tested the test facility and other conditions affecting the actual design and implementation of the required experiments On the other hand the QMP should address the quality control management structure and policies of the test facility In 2012 the combined QAPP and the QMP collectively known as the quality control quality assurance QA QC program was extensively extended when compared to previous years Nevertheless the BSH invited dr A Cangelosi Northeast Midwest Institute USA to review the QA QC section of the protocol In addition NIOZ commissioned a review of the entire protocol to dr S Kools Grontmij The Netherlands The present version of the protocol is revised in reaction to these two reviews In the summer of 2012 the BSH decided that a sufficient number of marine saline tests had been performed earlier that year and that five successful freshwater tests were needed for type approval This meant that additional information had to be added on freshwater sampling and testing in the relevant chapters of this protocol The tests outlined in this protocol will evalu
47. Turbidity is a function of TSS and because turbidity is difficult to measure reliably at relatively low TSS concentrations this variable is only measured in line and not in separate samples Vibrio cholerae is not present in NIOZ test water cannot be added and hence is not sampled for An additional measurement is that of phytoplankton lt 10 um which is not an IMO requirement Table 5 1 All measurements are described in the Standard Operating Procedures SOPs that are listed in section 3 of this project plan Samples for E coli and enterococci are outsourced and analysed according to NEN ISO standards Table 5 1 A brief description of all relevant methods is given in the following paragraphs 5 1 Abiotic variables 5 1 1 Salinity Temperature and pH Water samples for salinity temperature and pH are collected in 10 L buckets Measurements are either done immediately or after storage maximum 6 hours in the dark and at ambient temperature Salinity is measured with a digital conductivity meter Temperature is measured with a calibrated digital thermometer pH is measured with a calibrated digital pH meter 5 1 2 TSS POC Total Suspended Solids Particulate Organic Carbon For TSS analysis filters are dried at 60 C for at least 8 hours and weighed again The concentration of TSS per litre can be calculated from the sample volume and the weight difference of the filter before and after sampling TSS is expressed as mg L Next to de
48. a five day holding period as a measure of viability In UV systems this means after a second UV treatment on discharge Samples for PAM and microzooplankton are taken on the first day of incubation and up to seven days on working days only On day 7 complete sampling is performed including samples for phytoplankton bacteria and microzooplankton MATERIAL amp EQUIPMENT e 10 L polycarbonate bottle Nalgene e Magnetic stirrer and stir bar rotation 130 rpm e Climate room at ambient temperature and irradiance at 100 umol photons m st L D 18 6 h h PAM fluorimeter Canto flow cytometer Inverted microscope 100 mL sample bottles Greiner tube 50 mL with black tape 2 5 mL cryovials Flow cytometer tubes 1 mL pipets CHEMICALS e Phytoplankton nutrient stocks N P Si Eveline add one bottle of each nutrient to 10L sample e Lugol s iodine solution e Formaldehyde hexamine 18 v v METHOD e Incubate 10 L sample on T5 discharge day in the climate room add nutrients and stir e Sample on each work day for 7 days T12 SAMPLING e Pour 100 mL sample into a 100 mL sample bottle with 0 4 mL Lugol and store at 4 C in a refrigerator microzooplankton e Pour 50 mL sample into a 50 mL Greiner tube e Pipette 2x 1 5 mL bacteria en 3 5ml phytoplankton sample from the Greiner tube in two cryovials with 18 formaldehyde 100 ul phytoplankton en 150 ul bacteria e snap freeze e store at 80 C bacteria phytoplank
49. against a lab reference with the approximate ambient salinity value For freshwater 3 g KCI or NaCl is dissolved in 997 g mQ For freshwater 22 g KCI or NaCl is dissolved in 978 g mQ For freshwater 32 g KCI or NaCl is dissolved in 968 g mQ KCI or NaCl is dried for overnight at 60 C and cooled down in an exsiccator before weighing Reference samples are made in a large volume and distributed over small 60 ml plastic bottles Each bottle is used once e Reading is correct if the difference with the reference solution is between 5 e Correct the GMH 3430 reading by changing the Cell Correction factor Press the Set menu key for 2 sec and shortly press the same key a few times until Cell and a number between 0 40 and 1 00 appears on screen Increase or decrease the correction factor value by pressing max arrow upwards or min arrow downwards key set the value by pressing the Store quit key Check if the reading is correct if not repeat the sequence Record the measured values reference value before and after the correction in the harbour journal e Temperature is calibrated with a mercury precision thermometer before and after each test cycle e Record the measured values mercury and sensor values in the harbour journal Measurement e Turn on the GMH 3430 by pressing the on off key e Press Set Menu key shortly until arrow in top of screen is at SAL e Salinity and temperature are measured directly in each sample bucket 3 per test
50. ake During de ballast operations the ballast water from the tanks passes again through the UV reactor before discharge The applicant gives a brief description of the main components of the BWT system in chapter 2 A detailed description of the test facility and the design of the test sampling sample storage and descriptions of the measurements of abiotic as well as biological variables are given in ensuing chapters Because NIOZ is an academic research institute additional methods to count organisms and to establish their viability are continuously being developed These methods may be applicable to efficacy testing according to the present as to potentially future D 2 Ballast Water Performance Standards and G8 guidelines 14 NIOZ Land based Project Plan 2 GENERAL DESCRIPTIONS Figure 1 Aerial view of the NIOZ harbour lower right NIOZ and the TESO ferry connecting the island of Texel with the main land top The Mokbaai is the source for additional suspended solids Photo Simon Smit Photography Den Burg Texel 2 1 NIOZ profile All tests of the Cathelco BWTS will be carried out under supervision of the Royal Netherlands Institute for Sea Research Landsdiep 4 1797 SZ t Horntje Texel The Netherlands from here on NIOZ for details see NIOZ Royal Netherlands Institute for Sea Research is the National Oceanographic Institute of the Netherlands NIOZ is an institute of the Netherlands Organization for Scientific Researc
51. ampling dissolved gasses such as dissolved oxygen In special cases gastight tubing which is fitted to the sampling tubing is used to avoid exchange of gasses such as CO or oxygen between water and surrounding atmosphere Figure 13 All environmental variables are measured in triplicate samples 8 2 3 29 G8 guidelines From 2012 onwards additional continuous measurements of temperature salinity flow rate system pressure and turbidity will be made at intake and discharge by sampling via a specific sample port equipped with the appropriate monitoring instrumentation e g S and T in Figure 3 The data will be logged electronically and will be used to monitor basic system variables during intake and discharge of the BWTS tests 4 5 Biological variables sampling and storing Biological variables that need to be considered Regulation D 2 and 2 3 20 21 G8 guidelines are 1 Concentration and diversity of organisms 250 um 2 Concentration and diversity of organisms 10 lt um lt 50 3 Concentration of heterotrophic bacteria 4 Concentration of coliform bacteria 5 Concentration of Enterococcus bacteria and 6 Concentration of Vibrio cholera The groups of organisms 250 um and 10 lt um lt 50 consist of both algae phytoplankton mainly lt 50 um and animals zooplankton mainly gt 50 um According to the Ballast Water Performance Standard Regulation D 2 and 4 7 G8 guidelines only viable organisms must be counte
52. and QA QC Detailed Standard Operating Procedures SOPs for each of the analyses are provided in section 3 This is the second version of the project plan The first version contained information for intermediate and high salinity range testing only This new version includes information on freshwater testing scheduled for autumn 2012 and is split in the three sections outlined above 2 NIOZ Land based Project Plan Section 1 Ouality Management Plan MP 8 9 Introduction Quality system management and organisation Quality system components Personnel qualifications and training Procurement of items and activities Documents and records Computer hardware and software Planning Implementation of work processes 10 Assessment and response 11 Quality improvement 3 NIOZ Land based Project Plan 10 10 10 10 11 11 11 Section 2 Quality Assurance Project Plan APP INTRODUCTION GENERAL DESCRIPTIONS 2 1 NIOZ profile 2 2 Profile of the CATHELCO group 2 3 Technical overview of CATHELCO s BWTS TEST FACILITY NIOZ test facility Freshwater intake at Den Helder and transport to NIOZ BWTS test set up treatment and control tanks Toolbox meetings Ballast water book Test season time planning WWW WWW AuURWNEH TEST WATER QUALITY SAMPLING STORING 4 1 Abiotic quality 4 2 Biological quality 4 3 General sampling strategy 4 4 Abiotic variables sampling and storing 4 5 Biological va
53. and 10 lt um lt 50 the viability of phytoplankton PAM fluorimetry and the abundance of bacteria Usually the incubation time is seven days In more elaborate experiments samples are taken daily and may include multiple analyses of phytoplankton and heterotrophic bacteria NIOZ Ballast Water Report 5 1 5 1 1 5 1 2 5 1 3 5 1 4 ch MEASUREMENT OF VARI ABLES A list of all abiotic and biotic variables that are required by G8 is provided in Table 5 1 All variables are measured in samples of test water taken during intake and discharge as required Turbidity is a function of TSS and because turbidity is difficult to measure reliably at relatively low TSS concentrations this variable is only measured in line and not in separate samples Vibrio cholerae is not present in NIOZ test water cannot be added and hence is not sampled for An additional measurement is that of phytoplankton lt 10 um which is not an IMO requirement Table 5 1 All measurements are described in the Standard Operating Procedures SOPs that are listed in section 3 of this project plan Samples for E coli and enterococci are outsourced and analysed according to NEN ISO standards Table 5 1 A brief description of all relevant methods is given in the following paragraphs Abiotic variables Salinity Temperature and pH Water samples for salinity temperature and pH are collected in 10 L buckets Measurements are either done immediately or after storage
54. arch gives permission to revise quality documents or to produce new documents 10 Assessment and response During BWTS tests inspections may be made by inspectors of Lloyd s Register or the BSH depending on the national authority that will apply for type approval at IMO All ballast water team members are obliged to report deviations from quality standards or procedures to the quality manager At the end of each BWTS test the quality manager will report on all deviations from the QAPP or from QA QC measures in SOPs The quality manager also checks the BWTS test data that are compiled by the project leader of ballast water research The project leader of ballast water research will report any deviations in harbour test water monitoring data and intake test water data from the QAPP to the quality manager 11 Quality improvement All team members are motivated to enhance specific QA QC measures and methods Regular feedback between team members and the quality manager will enhance self improvement In addition the NIOZ quality objectives are submitted to independent external reviewers NIOZ test facility methods are compared and discussed in NSBWO workshops with other test facilities and experts in the field of ballast water research Proficiency tests for the enumeration of aquatic organisms will improve the quality of analyses and will enhance the comparison of results between test facilities The NIOZ test facility invites comments to im
55. asurement of IMO required variables and a general outline of the QA QC Variable specific QA QC measures are contained in each individual SOP that together form section three of the QAPP Notebooks are used to record data observations and deviations from SOPs that are made during BWTS tests as well as during laboratory analyses All notebooks are stored in the NIOZ Ballast Water archive In a number of cases such as in monitoring NIOZ harbour test water temperature and salinity and in the in line measurement of turbidity and salinity of test water at intake data loggers are used to store information electronically All digital 8 NIOZ Land based Project Plan information is stored on a dedicated server that is accessible to Ballast Water Project personnel only Specific forms are used to record data during specific routine monitoring activities These activities include monitoring of meteorological and environmental data and equipment functioning during BWTS tests as well as the identification and enumeration of gt 50 um organisms All forms are stored on paper in the NIOZ Ballast Water archive or in electronic format on the Ballast Water Project server Specific forms for sample custody are also used in case of the analyses of pathogenic bacteria or specific chemical toxicological analyses which are carried out in commercial laboratories of third parties Standard Operating Procedures or SOPs are available for each IMO variable that
56. at least 5 days 82 3 35 G8 guidelines The control tank can also indicate an unexpected source of mortality due to the testing arrangement 82 3 37 G8 quidelines Therefore the average discharge results in the control water should not be less than or equal to 10 times the values in regulation D 2 1 82 3 36 G8 guidelines 3 4 Toolbox meetings The general test set up is described in Figure 6 and in 3 2 Directly prior to each test a tool box meeting is held on the quay site with all team members During this meeting all actions such as order of tanks to be filled and flow rates are briefly discussed 24 NIOZ Land based Project Plan 3 5 Ballast water book All manufacturers should log their activities in a ballast water book issued by the NIOZ Several books may be issued during the entire test period These books remain at NIOZ in the appropriate dossier 3 6 Test season time planning The intermediate and high salinity range test season at the NIOZ harbour is restricted to spring and summer In this period of the year sufficiently high numbers of organisms are naturally present in the North and Wadden Sea At NIOZ the test water is not enriched with organisms neither artificially cultured nor collected at sea In general early spring sea water has a lower salinity then in summer due a decrease in river discharge A decrease in wind speed during spring will lead to diminishing concentrations of total suspended solids TSS and t
57. ate the biological efficacy as outlined in the Guidelines for Approval of Ballast Water Management Systems G8 adopted on 22 July 2005 as Resolution MEPC 125 53 IMO 2005 and as adopted in revised form on 10 October 2008 as Resolution MEPC 174 58 23 Annex 4 of the Ballast Water Convention of the IMO IMO 2008 The test protocol will be submitted for Type Approval by the applicant Cathelco GmbH to the German National Administration BSH The Cathelco BWTS is composed of two main modules to disinfect ballast water of ships One module is a filtration system the second a UV reactor Both are operated during ballast water intake During de ballast operations the ballast water from the tanks passes again through the UV reactor before discharge The applicant gives a brief description of the main components of the BWT system in chapter 2 A detailed description of the test facility and the design of the test sampling sample storage and descriptions of the measurements of abiotic as well as biological variables are given in ensuing chapters Because NIOZ is an academic research institute additional methods to count organisms and to establish their viability are continuously being developed These methods may be applicable to efficacy testing according to the present as to potentially future D 2 Ballast Water Performance Standards and G8 guidelines NIOZ Ballast Water Report 10 NIOZ Ballast Water Report 2 1 11 GENERAL DESCRI PTI ON
58. ater related activities This evaluation may range from compliance checking with IMO standards to advanced statistical analyses In co operation with IMARES Den Helder The Netherlands and Go Consult it also offers a one stop shop for complete G8 or G9 type approval including toxicity testing ship board tests and dossier formation In all activities the NIOZ test facility strives to achieve highly accurate and precise results This Quality Management Plan QMP describes the organisation of the NIOZ quality system This consists of the quality system management and organisation the quality system components personnel qualifications and training procurement of items and activities documents and records computer hardware and software planning implementation of work processes assessment and response and finally quality improvement NIOZ Ballast Water Report 2012 9 QUALITY SYSTEM MANAGEMENT AND ORGANISATION The quality system of the NIOZ is devoted to the reliable testing of BWT systems This means that accurate and precise measurements of the test ballast water and the treated water including the experimental control water need to be assured Openness of experimental and measurement protocols quality control data and data analysis should lead to a high fidelity in the conclusions reached during G8 9 tests of BWTS performance and other relevant research activities NIOZ is an IMO recognised test institute and is certified by Lloyd s Reg
59. ation of gt 50 um organisms was between 68 000 and 22 100 000 m For the 10 50 um organisms this was 397 to 8 040 per mL IMARES experience at another location in The Netherlands learned that organism concentrations can remain high up till winter Only when water temperature drops below ca 7 C the zooplankton production will stop Such low temperatures can be expected in November December but not during the planned G8 tests in September October The freshwater test water is transported over a relatively short distance across the Marsdiep tidal inlet of the Wadden Sea within 12 hours by ship to the NIOZ harbour Figure 8 The day following intake at Den Helder i e within 24 hours the test water is available in the NIOZ harbour and ready to be pumped into the NIOZ harbour installation NIOZ Ballast Water Report 19 Figure 7 Freshwater intake at B Spoorhaven the IMARES monitoring site in Den Helder harbour The red horizontal bar is 0 5 km Site A was the regular monitoring site site B is the new IMARES monitoring site where fresh water for BWTS testing at NIOZ will be collected NIOZ harbour Freshwater Intake site Figure 8 The test water will be transported to the NIOZ harbour over a distance of 6 5 km in less than 12 hours NIOZ Ballast Water Report 3 3 3 4 20 The pump to be used is an Amarex KRTK 100 401 354WG S that will be suspended by a crane from the NIOZ Pelagia quay Figure 9 The crane will
60. bacteria are 300 and 600 mL 4 4 Abiotic variables sampling and storing Figure 11 Navicula quay laboratory for first line handling of samples The measurement of the following abiotic environmental variables is required during sampling G8 2 3 25 1 salinity 2 temperature 3 Particulate Organic Carbon POC 4 Total Suspended Solids TSS 29 NIOZ Land based Project Plan 5 pH 6 Dissolved Oxygen DO and 7 Dissolved Organic Carbon DOC Turbidity is also mentioned in 2 3 25 G8 but experience has learned that it is difficult to reliably measure this variable in water samples of limited volume taken during intake or discharge Furthermore turbidity is a function of TSS a variable that is measured accurately and with far better precision In addition NIOZ research has shown that at low TSS values a standard turbidity instrument underestimates the actual solid content of the water Rapid measurement and processing of samples for the basic variables is ensured by the NIOZ harbour laboratory on the Navicula and Pelagia quays of the test facility Figure 11 A number of measurements is performed after sampling e g temperature salinity and pH while the other samples DOC POC TSS DO is processed for later analysis DOC samples are filtered through GF C filters and sealed in pre combusted glass ampoules or special screw cap bottles after adding hydrochloric acid HCI Sealed ampoules or bottles are st
61. ballast water is directed into the disinfection chamber where a cross flow arrangement with two medium pressure ultraviolet lamps delivers the high intensity irradiation The UV light intensity is continuously monitored during system operation so that intensity is maintained above pre set values to ensure delivery of the required dose The ultraviolet lamps are housed within quartz sleeves and an automatic mechanical cleaning system minimizes bio fouling and controls the accumulation of deposits on the UV lamp sleeves k i LS Gai por Figure 2 The Cathelco ballast water treatment system to be tested at NIOZ in 2012 This design allows for an easy exchange of the filters NIOZ Ballast Water Report At discharge the automatic backwash filter is by passed and the ballast water is pumped from the tanks through the UV disinfection chamber only Thus the ballast water is subjected to UV disinfection treatment prior to discharge overboard Specific features advantages of Cathelco s BWTS 14 The filtration step Cathelco s BWTS can be operated with two different types of filters giving it the maximum possible flexibility in addressing different installation requirements The UV lamp system Cathelco s BWTS uses special medium pressure UV lamps with a reduced Mercury content These lamps are mounted to a solid flange and they are surrounded protected by a sealed robust quartz sleeve This complete UV lamp system contain
62. blem e Optimale temperature for the flow cytometer 16C 31C e Doesn t function well lt 5C and gt 40C when exposed for longer times e Power laser 20 mW e Pressure in system 4 5 psi in system e Speed particles trough flowcell at 6 m s e Maximal flowspeed 10 000 events sec if higher than coincidence melding abort frequentie info benelux europe bd com CST beads Ref 641319 119 euro per kit 21 03 11 Trucount tubes 50 tests Ref 340334 157 euro 21 03 11 BD FACS Shutdown Solution Ref 334224 63 l NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 09 06 AUTHOR Josje Snoek Louis Peperzak FCM Canto data processing 2012 3 Goal Calculation cells ml from CANTO data based on new FCS Express lay out MATERIAL amp EQUIPMENT e BD Canto II Flowcytometer e FCS express 4 FCM analyses program the lay out for this software has been adjusted after size and concentration measurements of beads and phytoplankton cultures Exporting data from the CANTO e Switch on the computer The window Log On to Windows appears Username Administrator The password is BDIS gt OK e Double click on the icon BD FACSDiva Software on the Desktop Username Ballastwater password ballast gt OK e Open BW 2012 test results and select Stephan Gollash folder e Go to File Export FCS files a Export FCS Files window will open press OK Put th
63. c variables sampling and storing 26 Biological variables sampling and storing 28 Sampling organisms 250 um 28 Sampling and storage of 10 lt um lt 50 organisms 29 Sampling and storage of total and viable heterotrophic bacteria 29 Sampling of human pathogens 29 Sampling for additional incubation viability experiments 30 MEASUREMENT OF VARIABLES 31 Abiotic variables 31 Salinity Temperature and pH 31 TSS POC Total Suspended Solids Particulate Organic Carbon 31 Dissolved Oxygen DO 31 Dissolved Organic Carbon DOC 31 Biological variables 32 Counting of organisms 50 um 32 Counting of organisms 10 lt um lt 50 33 5 2 2 1 Counting viable microzooplankton 33 5 2 2 2 Counting viable phytoplankton 34 5 2 3 5 2 4 Counting total heterotrophic bacteria 35 Human pathogens 36 5 2 5 2 PAM measurement for total phytoplankton viability 37 5 2 5 3 Counting phytoplankton lt 10 um 37 6 1 6 2 6 3 QUALI TY ASSURANCE QUALI TY CONTROL QA QC 39 Ballast water tests 39 Laboratory analyses 39 Data analysis 39 REFERENCES 41 Aerial photo on cover by Pieter de Vries Photography All other photos except on page 11 by NIOZ INTRODUCTION The QAPP detailed in this document forms the basis for the biological efficacy testing of the CATHELCO Ballast Water Treatment System BWTS manufactured by Cathelco GmbH Kiel Germany in 2012 at the ballast water treatment facility of the Royal Netherlands Institute for Sea Research in
64. croscope Figure 19 resulting in nearly identical results However because the flow cytometer method is much faster results are obtained within 100 seconds and over 100 samples can be analyzed per day and highly reproducible this counting method is to be preferred above the far more time consuming and labour intensive microscopic observations Human pathogens lt eurofins Cemark The samples for microbiological analysis are taken in special bottles of 300 or 600 mL and send to a special laboratory Eurofins C mark Quality system Testing RVA L154 for further analysis All analyses are carried out according to NEN ISO standards Escherichia coli Analysis for Escherichia coli is carried out according to NEN EN ISO 9308 1 for the analysis of surface waters For this the samples are filtered through membrane filters pore size 0 45 um and these filters are incubated on a selective agar plate Incubation is 4 5 0 5 hours at 37 1 C and then another 19 5 0 5 hours at 44 0 5 C After that the incubated filters are transferred on sterile filters soaked with Indol reagent For colonies of E coli this yields a red colour These red colonies are counted and set into relation to the sample volume Results are confirmed via a positive and a negative control For the latter sterilized water is incubated like a regular sample and to confirm the results it may only yield less than 1 colony forming unit cfu per mL The positive control use
65. cting the specifics of the test facility the BWTS tested and other conditions affecting the actual design and implementation of the required experiments The QAPP consists of general information information on the NIOZ test facility and the Cathelco BWTS test water quality sampling and sample storing the measurement of variables and QA QC In September 2012 this QAPP was sent to the BSH as section 2 of the Project Plan QAPP for the Biological Efficacy Testing of the Cathelco Ballast Water Treatment System from Cathelco GmbH as part of the Type Approval Process under Resolution MEPC 174 58 version 2 In August 2013 on request by the BSH the text was formatted as a stand alone document This stand alone document includes the correct process diagram of the Cathelco BWTS 2 1 2 2 2 3 3 1 3 2 3 3 3 4 3 5 3 6 4 1 4 2 4 3 4 4 4 5 4 5 1 4 5 2 4 5 3 4 5 4 4 5 5 5 5 1 5 1 1 5 1 2 5 1 3 5 1 4 5 2 5 2 1 5 2 2 TABLE OF CONTENTS INTRODUCTION 9 GENERAL DESCRIPTIONS 11 NIOZ profile 11 Profile of the CATHELCO group 12 Technical overview of CATHELCO s BWTS 13 TEST FACILITY 17 NIOZ test facility 17 Freshwater intake at Den Helder and transport to NIOZ 18 BWTS test set up treatment and control tanks 20 Toolbox meetings 20 Ballast water book 21 Test season time planning 21 TEST WATER QUALITY SAMPLING STORING 23 Abiotic quality 23 Biological quality 24 General sampling strategy 25 Abioti
66. d at the end of the test in both the treatment and the control tanks For both of the IMO relevant size classes organisms 250 um and 10 lt um lt 50 multiple methods of enumeration and of assessing the viability are applied at NIOZ to verify the results at a high level of confidence In principle all methods should give a conclusive answer with respect to numbers and or viability of the remaining organisms At NIOZ the viability of all organisms is not only measured at discharge but during intake as well Furthermore the concentration of phytoplankton lt 10 um is measured All biological variables are measured in triplicate samples 2 3 29 G8 guidelines 4 5 1 Sampling organisms 250 um The samples of the untreated water 20 L are taken in triplicate collected in clean buckets that are filled directly for volumetric measurements and poured through a 50 um sieve made from the same gauze as the sampling nets used for the 1 m IBC samples Discharge water samples are collected using Hydrobios 50 um diagonal mesh size nets as recommended in MEPC 54 Inf 3 that are fitted into 1m IBC s Figure 14 Sampling is 31 NIOZ Land based Project Plan conducted via flexible hoses which are connected to the sampling points To sample treated water the hose is put into the Hydrobios net The whole sampling procedure will be timed in a way to cover the whole period of filling the ballast water tank with ca 250 m water For practical reaso
67. d flow speed for 60s Finish with high flow for 60s Please write the number of beads you ve analyzed in the log book on the computer In Browser click on already made folder gt right mouse button gt Copy Go to Administrator gt right mouse button gt Paste Change in Inspector the name of the copied experiment Threshold FL 5 660 20 rode laser value 1200 Change to FL 1 530 30 blue laser In left kantlijn Browser you find arrows in front of each tube If you want to remove a tube you need to click on the arrow left en delete Carousel setup gt p 124 manual Carousel ID 1 If you change from global to normal worksheet you can combine several samples Select in browser all samples Draw a plot in normal worksheet Experiment layout gt Acquisition Enter Events to Record gt click 60 s measurement recording delay time 3 sec aanvinken start of carousel mix aanvinken interim mix after every 1 tube mix duration 3 sec Cytometer gt Degas flowcel and bubble filter waar zit het bubble filter Replace sheath vessel When sheet vessel is replaced Cytometer gt Cleaning modes gt Prime after Tank refill voor ontluchten gt FACSFlow gt OK Cytometer Setup and Tracking Setup Control Research Use Only Define Baseline wordt per machine 1x gedaan of na veranderen filter configuratie Load tube Manually Flowrates LOW 12 MED 60 HIGH 120 ul min sample pressure 4 7 CST Mismatch Kies altijd Us
68. e For the latter movement including that of hart and gill is used to verify viability This is dependent on the expertise of the person analysing the samples Therefore only persons with a dedicated training period will analyse samples Organisms that are able to swim are also considered alive In doubt the organism can be poked with a dissection needle The procedure is outlined in Figure 16 Organisms larger 50pm landbased tests Viability testing Filling emptying of test tanks Structural integrity undisturbed Structural integrity disturbed Sampling with 50um net Stained pg Movement Unstained or light Transfer of organisms into colour and or no filtered water amp transport to lab movement Adjustment of volume to 100 Physical ipulati 200 ml and addition of stain in ot ne a 1 50 000 ratio Staining time needle or observation 1 hrs at higher magnificaton Filter stained sample over 30 um sieve Transfer of organisms into Bogorov dish with filtered Alive Viable unstained water Count at 20x magnification Retain filtered water for further analysis of organisms lt 50um Figure 16 Sampling and viability assessment for organisms larger than 50 um during land based tests Counting of organisms 10 lt um lt 50 Counting viable microzooplankton
69. e 1 m IBC samples Discharge water samples are collected using Hydrobios 50 um diagonal mesh size nets as recommended in MEPC 54 Inf 3 that are fitted into 1m IBC s Figure 14 Sampling is conducted via flexible hoses which are connected to the sampling points To sample treated water the hose is put into the Hydrobios net The whole sampling procedure will be timed in a way to cover the whole period of filling the ballast water tank with ca 250 m water For practical reasons all organisms quantitatively retained on the 50 um net are considered as larger than 50 um in minimum dimension NIOZ Ballast Water Report 4 5 2 4 5 3 4 5 4 29 The organisms retained in the cod end of the Hydrobios net are flushed into a beaker using a squeeze bottle containing filtered seawater Organisms are kept in approximately 100 to 200 mL of filtered lt 0 2 um filter sterile water of the relevant salinity Samples are transferred to the lab directly after sampling treated for 2 hours with the viability stain Neutral Red and counted These samples are not stored Figure 14 Intermediate Bulk Containers IBCs of 1 m3 each A 50 um Hydrobios plankton net is fitted in the containers through the lid for easy sampling Sampling and storage of 10 lt ym lt 50 organisms Samples for the 10 lt um lt 50 fraction are taken as undisturbed unfiltered one litre samples This size fraction is not separated from the organisms lt 10 um o
70. e BW USB in the computer and browse to the USB stick select a folder press Choose Directory make a file name and press Save Exporting will start e When exporting is ready close the CANTO program take out the USB and turn of the computer e Copy the files to Flowcyt BW 2012 Gollash Go consult FCM Files Facs Canto Processing data with FCS express e If you don t have FCS express on your computer already copy the FCS express exe shortcut from the bio L FCS FCS Express exe Shortcut version 4 e Open FCS express version 4 with the shortcut on your computer e A empty Layout will appear e Left mouse click on the round symbol with nr 4 in it on the top left side of the FCS screen e Open Layout browse to Flowcyt BW 2012 Gollash Go consult FCM Files Facs Canto select BW 2012 CANTO Phyto SG Louis 16 8 2012 fey e Import the data into the Layout go to Batch Data List Select all the files and press on the red cross to erase all data Press on the green plus and browse to Flowcyt BW 2012 Gollasch Go consult FCM Files Facs Canto open the folder s with your data and select the fcs files press open Select the first file in the list go to the header of the Data List window and press Change File and press Change Data On All Plots Close the Data List window e Check if all data fits well into the gates go to Data and Click on Next or previous to check all selected files e If all files are OK go to Batch Batch Actions rig
71. e CST Settings Deze melding krijg je alleen na een baseline calibratie Experimenten aanmaken in Browser Inspector gt Folder Symbool boekje aanklikken Via Inspector naam geven recht muisknop Name Beads aanvinken Use global cytometer setting Click in Browser Cytometer settings Wich detectoren ga ik gebruiken 62 NIOZ Land based Project Plan e Inspector e Global Sheet e Name e Number of Pages e Hoe meten spuitje New specimen e Via Inspector naam aanpassen e Aquisition Dashboard gebruik je voor starten stoppen metingen e Acquire Data e Sample beschrijving dmv Inspector Tube manually Acquisition Dashboard e Right mouse button e Show all e Basic Control e Manual Rinse needle e Cytometer gt Cleaning modes gt SIT flush Statistics e Statistiek gt Click on plot gt Right mouse button gt Create statistic view Batch analysis e Click on booklet Experiment gt right mouse button gt batch analysis gt Manual dan kan je nog gates aanpassen e Plotjes saven als pdf Je kan ook plotjes slepen naar excel sheet Shutdown procedure e Fill four tubes with the following solutions FACSClean milliQ FACSRinse milliQ e Cytometer gt Cleaning Modes gt place tube under needle gt Clean Flowcel e Repeat this step for all the tubes e Cytometer gt Fluidics Shutdown gt File gt Quit gt Shutdown In general e BD Technische dienst 020 582 9424 e Tube empty air in system no pro
72. e of several planning criteria First of all because NIOZ uses natural test water spring and summer are the only periods of the year in which proper testing according to IMO regulations and NIOZ quality objectives can be performed Second as part of a research institute it is of interest to the facility that the BWTS to be tested is of a different design as previously tested BWTSs The two project leaders of the test facility jointly decide on the admission of a BWTS for land based testing In case of requests for combined land based and ship board tests the admission will be discussed with the NIOZ partners IMARES and GoConsult Contracts on behalf of the NIOZ will be signed by one of the NIOZ directors The provisional project plan and planning for testing the BWTS is made by the project leader of ballast water research Next the provisional project plan and planning is discussed in the ballast water team where all practical issues including those related to quality management will be dealt with IMPLEMENTATI ON OF WORK PROCESSES Quality management objectives as specified in this QMP and in the QAPP and SOPs will be implemented in the work process At the lowest level individual members of the facility s test team are responsible for carrying out quality checks as detailed in SOPs on a daily basis The quality manager is responsible for supervising QA QC activities during all test and laboratory activities The quality manager will give hi
73. e suspected after the colour of the colonies the filter is transferred to a pre heated selective agar plate and incubated for another 2 hours at 44 0 5 C After that the medium is examined whether or not a brown to black colour can be found in it Results are confirmed via a positive and a negative control For the latter sterilized water is incubated like a regular sample and to confirm the results it may only yield less than 1 colony forming unit cfu per 100 mL The positive control uses a strain of Enterococcus faecium 5 2 5 2 PAM measurement for total phytoplankton viability The photochemical efficiency of photosystem II is an indicator of the physiological health condition of phytoplankton cells It is a bulk variable that is measured using a Pulse Amplitude Modulated PAM fluorimeter Schreiber et al 1993 Figure 20 The simple fluorescence ratio Fv Fm gives a qualitative indication of the photosynthetic efficiency of the phytoplankton community In addition the maximum fluorescence value Fm is an indication of phytoplankton biomass Prior to the measurement the sample is kept in the dark for at least 30 minutes 3 mL of unfiltered sample water control and treated each in triplicate are filled into a glass cuvette and analysed within 2 minutes In the case of a high photosynthetic efficiency of the bulk phytoplankton community samples can be filtered using 50 and 10 um Hydrobios gauze to determine the exact size class of
74. e to be carried out at specific water qualities as defined in the G8 guidelines The NIOZ harbour represents a brackish water environment with a varying salinity 20 35 PSU High salinity water originating from the North Sea is taken in around high tide Low salinity water from the Wadden Sea is taken in around low tide The salinity of the Wadden Sea water is dependent on the discharge of freshwater from Lake IJssel which itself depends on the amount of rainfall and on the flow rates in the rivers Rhine and IJssel In an effort to maintain a minimum 10 PSU salinity difference as requested under 2 3 17 of G8 per tank 15 m freshwater is added in the pipelines to the natural water prior to the pump to reduce the ambient salinity ca 2 PSU and 8 m brine 100 kg m industrial quality salt is added to increase salinity ca 2 PSU at the second set of test series At present only brackish and high salinity seawater conditions can be tested at NIOZ Figure 10 Extra natural sediment from the Mokbaai will be added when required to meet the minimum TSS concentration for the given salinity test cycle In addition per ballast tank 20 litre of mud 15 6 kg dry weight from the nearby Mokbaai Figure 1 will be added to the low salinity tests in order to reach the required TSS value of gt 50 mg L Figure 10 The organic carbon concentration is important in testing systems that use oxidizing agents as active substances DOC concentrations
75. elco CS Sample Point f Pre Filtration Turbidity UV Meter i Filter Drain required Overboard Discharge Figure 4 Piping and Instrumentation diagram of Cathelco s BWTS for a TRC treatment related capacity of 200 m3 h 15 NIOZ Ballast Water Report 16 NIOZ Ballast Water Report 3 1 17 TEST FACILITY NIOZ test facility The land based tests will be carried out on the island of Texel NIOZ harbour NL from March to July spring and early summer season The NIOZ test site is equipped with two times Navicula and Pelagia quay three silos or underground storage tanks of 300 m3 each to simulate ship s ballast water tanks Figure 5 The NIOZ harbour is located at the Marsdiep tidal inlet between the North Sea and the Wadden Sea By sampling in different phases of the tidal cycle waters from different seas with different abiotic and biological characteristics can be used in G8 tests The area as a whole is characterized by a rich and varying biodiversity and high number of various planktonic organisms especially in the spring and early summer period During the test cycles the numbers of organisms present in the water will continuously be monitored to assure that the validity of the test cycles is in accordance with 8 2 3 20 of the G8 guidelines RANN N ee TRS N ik A g Hoogtematen tov NAP Situatie 1 1000 Staalmaten in mm Overige maten in meters Figure 5 Schematic of the NIOZ harbour test
76. en analysed Measurement e With FCM BD CANTO see SOP FACS CANTO Operation 2012 1 and FACS CANTO data processing 2012 3 e Use the BD Canto Bact SYTOX protocol measuring time is 100 sec CALCULATIONS e See SOP FCM Data Processing with FCS Express 4 0 2012 1 e Use FCS Express Layout for Phyto SYTOX 73 NIOZ Land based Project Plan ity Assurance Proje A of the Ca n pe a Li gt 4 as thy Fy al LA UNE i a gt Cs i tro A ET A NN bi A e tra lt Ta n i pr vi J si fe dd aks Wie G i E AUD elek fp zn MST SATE gt CE r 4 ct Plan APP for thes thelco Ballast Water IP i Fi ih sr Log er z 3 iN tu cat pas a rr VA gt SN S K gt co R wf l gt ds E Pa i fo 2 fi Ox A E OS Ii Ae 4 ci FA 7 TEN iF Per ps f Li gy VAIN Say HAY SYK a a SS ee SS QUALITY ASSURANCE PROJECT PLAN QAPP FOR THE BIOLOGICAL EFFICACY TESTING OF THE CATHELCO BALLAST WATER TREATMENT SYSTEM FROM CATHELCO GMBH AS PART OF THE TYPE APPROVAL PROCESS UNDER RESOLUTION MEPC 174 58 CONFIDENTIAL UNTIL FURTHER NOTICE September 2012 version 2 Signed on Texel The Netherlands on 10 September 2015 A Va f L Pepefzak Ph D NIOZ Royal Netherlands Institute for Sea Research P O Box 59 NL 1790 AB Den Burg The Netherlands ABSTRACT This Quality Assurance Project Plan QAPP is the project specific technical document refle
77. en the North Sea and the Wadden Sea By sampling in different phases of the tidal cycle waters from different seas with different abiotic and biological characteristics can be used in G8 tests The area as a whole is characterized by a rich and varying biodiversity and high number of various planktonic organisms especially in the spring and early summer period During the test cycles the numbers of organisms present in the water will continuously be monitored to assure that the validity of the test cycles is in accordance with 8 2 3 20 of the G8 guidelines NEN I i i I I i e 1 BITENvE DJE 4 Hoogtematen tov NAP Situatie 1 1000 Staalmaten in mm Overige maten in meters Figure 5 Schematic of the NIOZ harbour test facilities including the two quays Navicula and Pelagia that are used in G8 tests 20 NIOZ Land based Project Plan Ballast water test site Pelagia Quay d NIOZ Figure 6 Piping and Instrumentation diagram of the Pelagia quay test site at the NIOZ harbour The installation to be tested is a UV treatment system The installation consists of three ballast water tanks one for control untreated water and two for treated water Brine or freshwater can be added to adjust the salinity of the test water Mud can be added to increase the concentration of Total Suspended Solids TSS Flow rates system pressures salinity and turbidity are monitored during intake and dischar
78. fixation and therefore they will be systematically underestimated if present 5 2 2 2 Counting viable phytoplankton 34 Organisms in the 10 lt um lt 50 size class will be analyzed via flow cytometry Figure 17 a semi automated method used in the NIOZ for the counting of phytoplankton bacteria and viruses In principle flow cytometry allows to assess a complete view of the effectiveness of the applied treatment technique for all organisms irrespective of their size Veldhuis amp Kraay 2000 By applying special staining techniques the numerical abundance of heterotrophic as well as autotrophic bacteria can also be estimated The vitality of the different organisms present will be addressed by using a specific fluorescent dye method Veldhuis et al 2001 Cassoti et al 2005 Veldhuis et al 2006 Peperzak amp Brussaard 2011 In addition the viability will be assessed by incubating discharge water see 4 5 5 The diversity of the phytoplankton is measured by microscopy of Lugol fixed samples of the NIOZ test water by Koeman amp Bijkerk The Netherlands This company is actively involved in international quality assessments http www planktonforum eu Three replicate samples from both control and treated water are placed in the carousel of a bench top flow cytometer Beckman Coulter XL MCL or Becton Dickson Canto II of which the Canto flow cytometer has an elaborate quality control system All procedures and handling are co
79. ganic Carbon mg L Y SOP Dissolved Oxygen saturation Y SOP Dissolved Organic Carbon mg L Y SOP Viable organisms 250 um number per m Y SOP including diversity number of phyla and species Organisms 10 50 um number per mL Y SOP phytoplankton Phytoplankton diversity number of phyla Y SOP and species Phytoplankton viability Fv Fm Y SOP PAM fluorimetry Phytoplankton viability number per mL Y SOP SYTOX Green Organisms 10 50 um number per mL Y SOP microzooplankton Microzooplankton diversity number of phyla Y SOP and species Microzooplankton viability or Y SOP Organisms lt 10 um number per mL SOP phytoplankton Heterotrophic bacteria number per mL SOP E coli cfu per 100 mL NEN EN ISO 9308 1 Enterococci cfu per 100 mL 35 NIOZ Land based Project Plan NEN EN ISO 7899 2 5 2 Biological variables 5 2 1 Counting of organisms 250 pm For minimum dimension measurements the body of the organism should be measured i e not antennae tails etc Examples are presented in Figure 15 Figure 15 Minimum dimension measurements red line in selected organism types A bivalve larvae B gastropod larvae C worm D echinodermata larvae E and F crustacean larvae and G copepod The viability of the organisms is assessed with Neutral Red which stains living organisms only and does not affect their survival rate This viability assessment remains unaffected by the possible death of orga
80. ge P amp I diagrams are available for both Navicula and Pelagia quays and for different intake and discharge scenarios 3 2 Freshwater intake in Den Helder and transport to NIOZ Freshwater in Den Helder will be taken in at location B Figure 7 by a ship that normally transports gravel and sand with a capacity of 650 m The ship will be cleaned of any remaining sand before taking in water The intake water will be pumped in the hold with tubes that are suspended at a height of maximally 1 meter below the water surface Intake will take place on the afternoon prior to the tests The Den Helder harbour is monitored by NIOZ partner IMARES According to IMARES Den Helder harbour is eutrophic and very productive In May July 2012 the concentration of gt 50 um organisms was between 68 000 and 22 100 000 m For the 10 50 um organisms this was 397 to 8 040 per mL IMARES experience at another location in The Netherlands learned that organism concentrations can remain high up till winter Only when water temperature drops below ca 7 C the zooplankton production will stop Such low temperatures can be expected in November December but not during the planned G8 tests in September October 21 NIOZ Land based Project Plan Figure 7 Freshwater intake at B Spoorhaven the IMARES monitoring site in Den Helder harbour The red horizontal bar is 0 5 km Site A was the regular monitoring site site B is the new IMARES monitoring site where fresh
81. h NWO The institute employs about 340 people at locations on the island of Texel on the border of the North Sea and the Wadden Sea main location and in Yerseke in the southwest of the country The annual budget is approximately 30 million The mission of NIOZ is to gain and communicate scientific knowledge on coastal seas and oceans for a better understanding of the system and sustainability of our planet to manage the national facilities for sea research and to support research and education in the Netherlands and in Europe In order to fulfil its mission the institute performs tasks in four specific fields Research The emphasis is on innovative and independent fundamental research in continental seas and open oceans Increasingly the institute also carries out research based on societal issues The senior scientists at NIOZ all participate in international research projects Several of them also hold a professorship at Dutch or foreign Universities Education The institute educates PhD students and master students of universities and schools for professional education Together with several universities NIOZ also organises courses for PhD students and master students in the marine sciences A number of our senior scientists is also appointed as professor at Dutch and foreign universities 15 NIOZ Land based Project Plan Marine Technology NIOZ has its own workshops for mechanical instrumental en electronical engineering Here
82. h screen The screen is arranged in cone shaped filter candles where the number of filter candles varies with the treatment related capacity TRC of the BWTS During the NIOZ G8 tests to be performed at 200 m h the number of filter candles will be nine The automatic cleaning cycle of the filter is activated by an increased pressure drop across the filter The filter candles are cleaned one after the other without interruption of the filtration process The frequency of the filter back flushes depends on the quality of the water e g the TSS content The concentrate is discharged over board This ensures that the screen is kept clean and the filtration process maintained at maximum efficiency at all times 2 UV disinfection The filtered ballast water is directed into the disinfection chamber where a cross flow arrangement with two medium pressure ultraviolet lamps delivers the high intensity irradiation The UV light intensity is continuously monitored during system operation so that intensity is maintained above pre set values to ensure delivery of the required dose The ultraviolet lamps are housed within quartz sleeves and an automatic mechanical cleaning system minimizes bio fouling and controls the accumulation of deposits on the UV lamp sleeves Figure 2 The Cathelco ballast water treatment system to be tested at NIOZ in 2012 This design allows for an easy exchange of the filters At discharge the automatic backwash filter is by
83. herefore sediment from the nearby Mokbaai Figure 1 is added to increase TSS to the required value of 50 mg l for brackish water The freshwater test season according to IMARES data begins in March and probably ends in November December In February sampling and measurements in the harbour start in order to monitor the start of the spring plankton bloom In March the first G8 tests may be performed depending on natural circumstances such as water temperature and underwater light climate that affect the plankton development The first set of tests is carried out at the intermediate salinity range of G8 82 3 17 because in early spring the freshwater content of the Wadden Sea is relatively high Test water is pumped from the harbour at low tide when low salinity Wadden Sea water flows towards the North Sea The second set of high salinity tests is performed in late spring or early summer Test water is pumped from the harbour at high tide when relatively saline North Sea water flows towards the Wadden Sea After consultation with the BSH the Cathelco BWTS tests carried out in spring and early summer of 2012 were combined to one salinity saline range This meant that an additional series of freshwater tests had to be performed The first appropriate months for these freshwater tests were September and October 25 NIOZ Land based Project Plan 4 TEST WATER QUALITY SAMPLING STORING 4 1 Abiotic quality The land based test cycles hav
84. horised NIOZ test facility personnel only Statistical analyses will be performed in Excel version 14 Additional analyses will be performed in either SYSTAT version 13 or Primer version 6 SYSTAT and Primer allow for more sophisticated statistical analyses of the BWTS performance than the t tests that are recommended in 2 3 37 of the G8 guidelines The scientific hypothesis that will be tested the so called null hypothesis is that there are no differences between treated and control water samples Quality data are compiled in tables and when possible visualised in diagrams such as for instance Shewhart control charts NIOZ will report the total number of tests that were needed to meet the D2 standard five times for each salinity range 42 NIOZ Land based Project Plan Table 5 Example of the assignment of coloured codes prior to tests Sample flasks contain the appropriate code on a coloured label to prevent misidentification TO is the day of treatment and Tx is the x day of sampling Usually x 5 at discharge Test numbers are Roman numerals I II III etc using a new number for every test e g I to X Replicates are denoted with normal numbers 1 2 3 etc The example codes I T5 M 1 4 1 is for each manufacturer the first replicate sample of Test I on day 5 T5 Sample Quay Code Label colour Wadden Sea harbour inlet Navicula WSn Red Wadden Sea harbour inlet
85. ht mouse click Excel Column Mode go to Properties go to Save to a new fi and change the name of the file or even change the directory by browsing with the yellow folder sigh right of the window with the file name Press OK e Do the same with Export To Powerpoint Choose the same name as the Excel file press OK e Go to Batch Run and all data will be calculated and stored e Repeat the same with the files of the True Count Beads TCB to calculate the flow rate 64 NIOZ Land based Project Plan Use the FCS Layout for TCB beads go to Flowcyt BW 2012 Gollasch Go consult FCM Files Facs Canto select BW 2012 CANTO TCB GS fey e Open the excel Phyto output of FCS express and copy all data into a FCM data calculation file e Open the excel TCB output of FCS express and copy counts of single and double counts into a flow rate calculation file Calculate flow rate and Phyto counts ml see below e Examples of data calculation files FCS OUTPUT TCB Layout singl Trucount I II K T5_High 1 fcs compensated e 1510 double 158 singl Trucount I II K T5_High 2 fcs compensated e 1482 double 162 singl Trucount I II K T5_Medium 1 fcs compensated e 792 double 84 singl Trucount I II K T5_Medium 2 fcs compensated e 814 double 81 EXCEL calculation sheath flowrate TCB TCB batch volume counting counts flowrate SW flow beads ml time s single dubble total ml min rate average sdtdev 50602 3 60 1510 158 1826 0 108 high 0 108 0 001 5
86. ile of the Cathelco Group Cathelco Ltd was formed in 1956 and has become a world leading supplier of cathodic protection equipment to the shipping and offshore marine markets The parent company based in Chesterfield United Kingdom manufactures marine pipe work anti fouling AF and impressed current cathodic protection ICCP systems The Cathelco Group has grown through a series of acquisitions beginning with Corrintec Ltd in 1995 Today Corrintec Marine continues to operate as a wholly owned subsidiary serving the military sector worldwide In 2005 the company established Cathelco Korea a subsidiary engaged in manufacturing and distribution serving the Korean shipbuilding market More recently in 2010 Seafresh Desalinators Ltd was acquired which specialises in reverse osmosis water makers from a manufacturing facility in Bournemouth United Kingdom To serve the shipbuilding and repair industry in South East Asia Cathelco S E A based in Singapore became a subsidiary in 2011 In addition to its marine engineering activities the Cathelco Group encompasses Casting Repairs Ltd specialising in the repair of architectural cast ironwork It also has an active property division focusing on property development and rental via the Broomco Ltd subsidiary Cathelco GmbH was established in 2010 in Kiel Germany to research and develop ballast water treatment equipment for the worldwide market The aim is to develop a chemical free two s
87. illiQ vanwege de te nauwe opening van de ampul en de afwijking in centrering van de naalden van de autosampler Gebruik b v een 5 ml pipet en gebruik ong 1 ml monster om de vial mee te spoelen Er moet nl ongeveer 18 ml in de vial zitten Spoel de buitenkant van de pipetpunt goed af met milliQ en spoel nog 2x in Erlenmeyer met milliQ alvorens het volgende monster wordt overgedaan Om twee CRM vials te vullen gebruik je 2 ampullen van 30 ml en spoel beide vials 3x met ca 0 5 ml CRM vloeistof Carrousel pas vullen met de vials indien deze allemaal gevuld zijn en er spoedig met het meten van de monsters kan worden begonnen er moet niet veel tijd zitten tussen het acclimatiseren en de eigenlijke metingen dus niet het apparaat lang stil laten staan Bereiding ijklijn uit stockoplossing Stock 1000 ppm C in koelkast 500 ml maatkolven goed gespoeld met milliQ OMC 0 ul stock 25 150 50 300 100 600 200 1200 55 NIOZ Land based Project Plan Bij gebruik van electronische pipet de pipetpunt schuin tegen de wand van de erlenmeyer houden bij het uitspuiten totdat de het 2 piepje en de trilling voorbij zijn Maatkolven met milliQ aanvullen tot 500 ml onderkant meniscus raakt de lijn En daarna 2 ml HCI toevoegen Het volume wordt dus 502 ml Het volume van je monster is ook 20 ml 4 druppels HCI Let op dit phosphorzuur is erg stroperig en moeilijk te pipetteren Sample analyses abort are you sure g
88. in outsourced species www planktonforum eu Phytoplankton vitality Fv Fm Phytoplankton vitality PAM 66 PAM fluorimetry 2012 1 Phytoplankton vitality number per Phytoplankton vitality 68 SYTOX Green mL SYTOX FCM 2012 1 Phytoplankton viability or Plankton viability T5 71 incubation Microzooplankton number per Microzooplankton 2012 2 69 organisms 10 50 um mL and including diversity number of phyla and species Microzooplankton or Plankton viability T5 71 viability incubation Phytoplankton number per Phytoplankton Canto FCM 59 61 64 organisms lt 10 um mL 2012 1 FCM Canto operation 2012 1 FCM Canto data processing 2012 3 Heterotrophic bacteria number per Bacteria count PicoGreen 73 mL 2012 1 E coli cfu per 100 NEN EN ISO 9308 1 Eurofins C mark mL outsourced Enterococci cfu per 100 NEN EN ISO 7899 2 Eurofins C mark mL outsourced 45 NIOZ Land based Project Plan ip N 10Z Royal Netherlands Institute for Sea Research wy STANDARD OPERATING PROCEDURE DATE 2012 09 05 AUTHOR Josje Snoek Salinity and temperature 2012 2 Goal Determination of salinity and temperature MATERIAL amp EQUIPMENT Greisinger Digital Conductivity Meter GMH 3430 with Pt sensor e Laboratory Salinity References 3 22 and 32 g KCI or NaCl kg e KCl or NaCl e Dry oven BINDER ED 115 e Bucket 12 litre with Ballast Water test water Calibration e At intake and at discharge the GMH 3430 is calibrated for salinity
89. ing quit button e Press the Call button e Open anew bag with buffer solution for each calibration Start with pH 7 and stir slowly until instrument decides the reading is stable and asks for the next buffer e Repeat with pH 9 e Information will be shown if the calibration in succeeded if not repeat the whole process e Record the Calibration data in the harbour journal press OK when lt param gt is shown at the bottom of the screen or use arrow left or right until lt param gt is shown go with arrow down to cal data Measurement e pH is measured in each bucket 3 per test with Ballast Water test water e Move the glass pH electrode slowly through the water until the reading is stable e Be careful not to break the glass electrode e Record the value in the harbour journal LITERATURE e See Applikon Metrohm for specifications and instructions http www metrohm nl PDFdownloads Metrohminfo M Info 1 05 E pdf 47 l NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research eee _____ o STANDARD OPERATING PROCEDURE DATE 2012 09 06 AUTHOR Eveline Garritsen Josje Snoek TSS POC 2012 2 Goal Determination of Total Suspended Solids and Particulate Organic Carbon MATERIAL amp EQUIPMENT e Balance e Dried and pre weighed GFC filters Whatman Glass microfiber filters 47 mm cat no 1822 047 in numbered 5 cm plastic petri dishes e Dry oven 60 C BINDER ED 115 e Exsicatto
90. ing two lamps is fitted to the UV reactor by a few screws Access to the UV reactor for maintenance is from a single side only The lamp recycling scheme On an annual basis the UV lamp system should be send off to Cathelco for refurbishment The renovated lamp system will be returned to the ship with a new 1 year operating guarantee if the lamp system has not been installed for a total time of more than 2 years The automated cleaning system of the UV This is the first NON chemical in place cleaning system for UV reactors CIP system It uses rubber cleaning elements that are supplied on demand to the UV reactor after the BW operation is finished The cleaning process is triggered by the intensity measurement of the individual UV lamps Start stop and duration of the cleaning process are controlled by Cathelco s BW system No manual interference is needed Ballast Water Tank Filter by pass _ CIP cleaning after BW operation CIP unit Overboard Backflush line mums discharge Woverboard Figure 3 Process diagram of Cathelco s BWTS Calculation of the UV dose The UV dose is calculated directly from the water quality UV transmittance of the water and from the flow rate The use of single electronic ballast units for each individual UV lamp allows for a maximum of flexibility in the power consumption The UV lamps are operated at the most suitable dose rate at any time allowing for significant savings in overall powe
91. ister The project leader of ballast water research is dr Louis Peperzak He is responsible for the proper functioning of the test facility and for carrying out BWTS tests laboratory analyses production of Standard Operating Procedures SOPs new methods in ballast water research treatment enumeration vitality viability measurements compliance monitoring and the support of all students that are involved in this research In cooperation with members of the ballast water team he analyses the BWTSs test results and writes the G8 G9 reports as first author Co project leader is dr Jan Boon He manages the overall Ballast Water Project at NIOZ especially in relation to the North Sea Ballast Water Opportunity NSBWO project of the European Union of which the NIOZ is the lead beneficiary He is also responsible for the external relations of the Ballast Water project Because of his experience in chemical quality assurance programs Dr Boon also acts as the quality manager He is responsible for the QMP and will evaluate all QA QC activities of BWTS tests He is co author of the G8 G9 reports Both project leaders are assisted by Mrs D rte Poszig M Sc general management Mr Marcel van der Linden financial management and Mrs Marieke Holthuizen Vloemans external communication Sampling sample analyses and first data analyses are carried out by Mrs Josje Snoek Mrs Eveline Garritsen Mrs Eva Immler Mr Dennis Mosk and Mr Alex Bli
92. its years of experience since 2007 NIOZ has tested nine BWTSs Table 1 Table 1 Ballast Water Treatment Systems tested at NIOZ Year Company System 2007 Hamann SEDNA 2008 EcoChlor EcoChlor 2008 Hyde Marine Hyde Guardian 2009 Mahle Ocean Protection System 2009 Severn Trent De Nora BalPure STDN 2010 Aquaworx AquaTricomb 2010 Erma First ESK SA ErmaFirst 2011 Hamworthy Wartsila Aquarius EC 2012 Hamworthy Wartsila Aquarius UV NIOZ works constantly on improving its methods and technologies for the accurate and precise enumeration of aquatic organisms and the measurement of their vitality and viability These methods are compared with other organisations and test facilities in workshops and within the global network of BWTS test sites GloBal TestNet of which NIOZ is a member On a European level NIOZ is the lead beneficiary of the North Sea Ballast Water Opportunity project It is policy of the institute to communicate scientific results as much as possible through international peer reviewed publications The NIOZ BWTS test facility not only performs land based G8 or G9 tests It is also equipped for laboratory tests to investigate the principles and efficiency of new technologies as well as for bench scale tests to examine prototypes and components of BWTSs In addition the test facility is able to analyse and evaluate samples from other test facilities from ship board tests and other ballast w
93. liwa U 1993 PAM fluorimeter based on medium frequency pulsed Xe flash measuring light A highly sensitive new tool in basic and applied photosynthesis Photosynth Res 36 65 72 Shapiro HM 2003 Practical flow cytometry John Wiley amp Sons Inc New Jersey Veldhuis MJW Cucci TL Sieracki ME 1997 Cellular DNA content of marine phytoplankton using two new fluorochromes taxonomic and ecological implications J Phycol 33 527 541 Veldhuis MJW Kraay GW 2000 Application of flow cytometry in marine phytoplankton research current applications and future perspectives Sci Mar 64 121 134 Veldhuis MJW Kraay GW Timmermans KR 2001 Cell death in phytoplankton correlation between changes in membrane permeability photosynthetic activity pigmentation and growth Eur J Phycol 36 167 177 Veldhuis MJW Fuhr F Boon JP Hallers Tjabbes C C 2006 Treatment of ballast water how to test a system with a modular concept Environ Technol 27 909 921 NIOZ Ballast Water Report TD SAY y if AKG NL L Peperzak EN NIOZ Ballast Water Report 2012 9 NIOZ Royal Netherlands Institute for Sea Research Quality Management Plan QMP Pd Signed on Texel The Netherlands on 29 August 2013 L Peperzak Ph D NIOZ Royal Netherlands Institute for Sea Research P O Box 59 NL 1790 AB Den Burg The Netherlands TABLE OF CONTENTS INTRODUCTION 7 QUALITY SYSTEM MANAGEMENT AND ORGANISATION 8
94. low the surface by using dispenser units to fix the oxygen concentration A stopper secured with a rubber band is put on the bottle and the bottle is gently mixed Bottles are stored in a dark container filled with water of the same temperature as the samples until further analysis in the laboratory Figure 12 Figure 12 Glass bottles for Dissolved Oxygen DO measurements are stored submerged prior to analysis In special cases gastight tubing which is fitted to the sampling tubing is used to avoid exchange of gasses such as CO or oxygen between water and surrounding atmosphere Figure 13 All environmental variables are measured in triplicate samples 2 3 29 G8 guidelines From 2012 onwards additional continuous measurements of temperature salinity flow rate system pressure and turbidity will be made at intake and discharge by sampling via a specific sample port equipped with the appropriate monitoring instrumentation e g S and T in Figure 3 NIOZ Ballast Water Report 4 5 4 5 1 28 The data will be logged electronically and will be used to monitor basic system variables during intake and discharge of the BWTS tests Figure 13 Sampling point on a tank at the Navicula quay for sampling dissolved gasses such as dissolved oxygen Biological variables sampling and storing Biological variables that need to be considered Regulation D 2 and 8 2 3 20 21 G8 guidelines are 1 Concentration and diversity of
95. lved in this research In cooperation with members of the ballast water team he analyses the BWTSs test results and writes the G8 G9 reports as first author Co project leader is Dr Jan Boon He manages the overall Ballast Water Project at NIOZ especially in relation to the North Sea Ballast Water Opportunity NSBWO project of the European Union of which the NIOZ is the lead beneficiary He is also responsible for the external relations of the Ballast Water Project Because of his experience in chemical quality assurance programs Dr Boon also acts as the quality manager He is responsible for the QMP and will evaluate all QA QC activities of BWTS tests He is co author of the G8 G9 reports Both project leaders are assisted by Mrs D rte Poszig M Sc M A general management Mr Marcel van der Linden financial management and Mrs Marieke Holthuijsen Vloemans external communication Sampling sample analyses and first data analyses are carried out by Mrs Josje Snoek Mrs Eveline Garritsen Mrs Eva Immler Mr Dennis Mosk and Mr Alex Blin Mrs Eva Immler is responsible for the correct operation of the first NIOZ test installation on the Pelagia quay and for correct sampling sample handling and storage there Mr Dennis Mosk is responsible for the correct operation of the NIOZ test installation on the Navicula quay and for correct sampling sample handling and storage there The operation and maintenance of both NIOZ test installation
96. m the in line samples deviate from 2 3 32 33 G8 guideline 10 lt um lt 50 1 L for untreated water and 10 L for treated water bacteria 0 5 L Note that G8 only specifies sample sizes but not which volume of these samples should actually be analysed The reason for this deviation is that sample volumes of 10 L for organisms 10 lt um lt 50 and 0 5 L for heterotrophic bacteria are impractical much smaller volumes will eventually be analysed without compromising the analytical accuracy The sample volumes for pathogenic bacteria are 300 and 600 mL Abiotic variables sampling and storing Figure 11 Navicula quay laboratory for first line handling of samples The measurement of the following abiotic environmental variables is required during sampling G8 2 3 25 1 salinity 2 temperature 3 Particulate Organic Carbon POC 4 Total Suspended Solids TSS 5 pH 6 Dissolved Oxygen DO and 7 Dissolved Organic Carbon DOC NIOZ Ballast Water Report 27 Turbidity is also mentioned in 8 2 3 25 G8 but experience has learned that it is difficult to reliably measure this variable in water samples of limited volume taken during intake or discharge Furthermore turbidity is a function of TSS a variable that is measured accurately and with far better precision In addition NIOZ research has shown that at low TSS values a standard turbidity instrument underestimates the actual solid content of the water Rapid meas
97. man amp Bijkerk CALCULATIONS Sample weight full sample weight empty bottle weight Concentrated sample weight concentrated sample weight empty bottle weight Ciliate count sample weight concentrated sample weight ml analyzed REMARKS Prior to 2012 4 full series of the certification tests done for a company in one year were analyzed From 2012 onwards for the intake samples a mixture subsample of triplicates at every sampling point are analyzed for all certification tests Same applies for the control samples of the discharge day Treated discharge samples are all analyzed Before 2012 the micro zooplankton data was entered and processed in the counting program Koeman en Bijkerk ecologic research and advising From 2012 onwards data will be processed in Excel See separate SOP Plankton viability T5 Incubation LITERATURE Koeman R P T Esslink K Fockens A L de Haan amp G L Verweij 2002 Biomonitoring van microzooplankton in de Nederlandse zoute wateren 2000 Rapport 2001 22 Bureau Koeman en Bijkerk Haren Koeman en Bijkerk intern rapport genoteerde soortenlijst 2000 2010 70 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 03 05 AUTHOR Louis Peperzak Plankton viability T5 Incubation 2012 1 Goal To measure the development of phyto and microzooplankton in treated ballast water samples that were discharged after
98. mples is accurately determined by flow cytometry using the DNA specific stain PicoGreen cf Gasol amp Del Giorgio 2000 Veldhuis et al 1997 Bacteria gt Viruses Figure 19 Epifluorescence microscopic picture showing bacteria and viruses in a natural water sample The yellow green fluorescence signal is DNA stained with a nucleic acid dye The dye PicoGreen is a green nucleic acid specific dye that only stains dsDNA with little or no cross over for ssDNA and RNA Veldhuis et al 1997 This makes the staining method ideal to for staining of DNA and therefore to determine bacterial abundance Flow cytometric analysis shows a clear signal with an excellent signal to noise ratio and bacteria are made visible easily and distinguishable from viruses and larger organisms This approach has extensively been compared with bacteria staining and counting using an epifluorescent microscope Figure 19 resulting in nearly identical results However because the flow cytometer method is much faster results are obtained within 100 seconds and over 100 samples can be analyzed per day and highly reproducible this counting method is to be preferred above the far more time consuming and labour intensive microscopic observations 39 NIOZ Land based Project Plan 5 2 4 Human pathogens S eurofins Cemark The samples for microbiological analysis are taken in special bottles of 300 or 600 mL and send to a special laboratory Eurofins C mark
99. n Mrs Eva Immler is responsible for the correct operation of the first NIOZ test installation on the Pelagia quay and for correct sampling sample handling and storage Mr Dennis Mosk is responsible for the correct operation of the NIOZ test installation on the Navicula quay and for correct sampling sample handling and storage The operation and maintenance of both NIOZ test installations is contracted to Mr A Smit of Smittech Den Hoorn The Netherlands and Mr J Witte Witte Klusbedrijf Den Burg The Netherlands The NIOZ ballast water team advisory committee consists of prof Dr Hein de Baar chemistry Dr J van Bleijswijck molecular biology Dr Corina Brussaard microbial ecology and Dr Klaas Timmermans head of the department of Biological Oceanography at NIOZ The Quality Management Plan QMP and Quality Assurance Project Plan QAPP will be reviewed annually by an external independent consultant In 2012 this was performed by dr S Kools of Grontmij The Netherlands In addition by request of the BSH a review on QA QC aspects was performed by dr A Cangelosi of GSI USA Comments of both parties have been used in the present project plan that includes both QMP and QAPP NIOZ Ballast Water Report 2012 9 QUALITY SYSTEM COMPONENTS The quality system of the NIOZ BWTS test facility contains several components of which this QMP is the main document The QMP is the joint responsibility of both project leaders of the NI
100. n Canto FCM 2012 1 59 61 64 organs 20 90 HE FCM Canto operation 2012 1 FCM Canto data processing 2012 3 Phytoplankton diversity Koeman amp Bijkerk b v quality Koeman amp assessments in www planktonforum eu Bijkerk outsourced Phytoplankton vitality Phytoplankton vitality PAM 2012 1 66 PAM fluorimetry Phytoplankton vitality Phytoplankton vitality SYTOX FCM 2012 1 68 SYTOX Green Phytoplankton viability Plankton viability T5 incubation 71 Microzooplankton Microzooplankton 2012 2 69 organisms 10 50 um including diversity Microzooplankton viability Plankton viability T5 incubation 71 Phytoplankton Phytoplankton Canto FCM 2012 1 59 61 64 organisms 20 00 FCM Canto operation 2012 1 FCM Canto data processing 2012 3 Heterotrophic bacteria Bacteria count PicoGreen 2012 1 73 E coli NEN EN ISO 9308 1 Eurofins C mark outsourced Enterococci NEN EN ISO 7899 2 Eurofins C mark outsourced 5 NIOZ Land based Project Plan 8 9 Section 1 Quality Management Plan Introduction Quality system management and organisation Quality system components Personnel qualifications and training Procurement of items and activities Documents and records Computer hardware and software Planning Implementation of work processes 10 Assessment and response 11 Quality improvement 6 NIOZ Land based Project Plan MP 10 10 10 10 11 11 11 1 Introduction The NIOZ Ballast Water Treatment
101. n are evaluated by research staff or Mr A Smit in collaboration with the project leader of Ballast Water Research or in his absence the quality manager Outsourced sample analyses are performed by commercial parties that have an established quality management program 6 Documents and records Documents related to the NIOZ test facility and BWTS testing include legal and financial contracts BWTS specific project plans including QAPP QMP SOPs North Sea Ballast Water Opportunity NSBWO documents notebooks data and custody forms student reports internal reports on for instance inter comparison workshops and scientific publications Legal and financial contracts are archived as required by NIOZ under the responsibility of the financial management assistant and the overall project manager North Sea Ballast Water Opportunity NSBWO documents are maintained by the general and financial management assistants or by the project leaders Notebooks data and custody forms are archived for at least five years by the project leader of ballast water research BWTS specific project plans including QAPP QMP SOPs student reports internal reports and scientific publications are also archived by the project leader of ballast water research Reports on new technologies as well as for bench scale tests to examine prototypes and components of BWTSs and other documents pertaining to the test facility are numbered sequentially as NIOZ Ballast
102. ncy tests for the enumeration of aquatic organisms will improve the quality of analyses and will enhance the comparison of results between test facilities The NIOZ test facility invites comments to improve the quality of its test and its test data for instance from class societies and through its membership of Global Testnet NIOZ Ballast Water Report 2012 9 Vani et Cathelco NIOZ QUALITY MANAGEMENT PLAN CATHELCO BWMS END PAGE Revision Date Description Author Checked Approved 01 13 09 13 Revision PH RF MD 00 13 09 13 Initial Issue PH Revision 01 Cathelco Page 16 of 16 10 10 2013 Marine House Dunston Road Chesterfield Derbyshire ENGLAND S41 8NY
103. ndard as a sample Repeat Calculate as a sample and record the data with those of the test samples 49 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 03 21 AUTHOR Josje Snoek Dissolved Oxygen 2012 1 Goal Determination of Total Dissolved Oxygen in seawater MATERIAL amp EQUIPMENT e Volume calibrated bottles 120 ml Dispenser bottles 3 500 ml or 1 liter Dispensers 3 max 2 or 5 ml per dose Chemicals MnCl2 NaOH KI H2S04 KIO3 Magnetic stirring bars many small some large Magnetic stirrer Balance g mg Hitachi U 1100 Spectrophotometer Introduction The chemical determination of oxygen concentrations in seawater is based on the method first proposed by Winkler 1888 In the Winkler method Oxygen is fixated by adding manganese chloride and alkaline iodide causing Oxygen to precipitate This precipitate is subsequently dissolved by adding acid causing a yellow coloured Iodine solution The yellow colour can be determined directly with a Spectrophotometer Preparation of the Chemicals Reagent A 2 liter MnCl2 4H20 600 g l e Weight 2 x 600 g MnCl2 in a 500 ml plastic measure cup e Dissolve bit by bit while mixing constantly with a spoon in 1000 ml MQ in a 2 liter plastic beaker e When dissolved fill up to 2 liter with MQ e Filter the solution through a coarse filter paper e Filla dispenser bottle with reagent A set
104. ndl 2005 Standards are prepared with potassium hydrogen phthalate Nacalao Tesque Inc Kioto Japan The mean concentration of triplicate injections of each sample three in total is calculated The average analytical precision of the instrument is lt 3 NIOZ Ballast Water Report 5 2 5 2 1 32 Table 4 List of variables measured in land based tests at NIOZ Variable unit IMO required Reference Salinity PSU Y SOP Temperature 2G Y SOP pH Y SOP TSS mg L Y SOP Particulate Organic Carbon mg L Y SOP Dissolved Oxygen saturation Y SOP Dissolved Organic Carbon mg L Y SOP Viable organisms 250 um number per m Y SOP including diversity number of phyla and species Organisms 10 50 um number per mL Y SOP phytoplankton Phytoplankton diversity number of phyla Y SOP and species Phytoplankton viability PAM Fv Fm Y SOP fluorimetry Phytoplankton viability number per mL Y SOP SYTOX Green Organisms 10 50 um number per mL Y SOP microzooplankton Microzooplankton diversity number of phyla Y SOP and species Microzooplankton viability or Y SOP Organisms lt 10 um number per mL N SOP phytoplankton Heterotrophic bacteria number per mL Y SOP E coli cfu per 100 mL Y NEN EN ISO 9308 1 Enterococci cfu per 100 mL Y NEN EN ISO 7899 2 Biological variables Counting of organisms 250 um For minimum dimension measurements the body of the organism should be
105. nducted according to standard procedures e g Shapiro 2003 Samples will be counted using standard protocols covering the particles in the size range of ca 2 to 50 um Total analysis time will be equal to an exact sampling volume of 1 mL or otherwise when relevant Of all particles present in the volume counted the cell size and the presence or absence of chlorophyll a fluorescence will be measured Only phytoplankton has chlorophyll a fluorescence Figure 18a b Absolute numbers cell sizes and chlorophyll a content of the particles will be analyzed using the software package FCS Express V3 or V4 DeNovo US Cell sizes will be estimated relative to 10um standard fluorescent beads Flow Check Fluorospheres Beckman Coulter 660539 or relative to 10 and 50 um beads on the Canto II For measuring viable phytoplankton three subsamples will be stained with SYTOX Green Veldhuis et al 2001 This nucleic acid specific dye only stains DNA of cells with a compromised cell membrane Of each phytoplankton cell present the green SYTOX fluorescence Figure 15b will be determined and compared with the green autofluorescent signal Veldhuis et al 2001 Cassoti et al 2005 Peperzak amp Brussaard 2011 NIOZ Ballast Water Report 5 2 3 35 Figure 17 Bench top flow cytometer Becton Dickinson Canto II an instrument to enumerate live and dead organisms lt 50 um nucleus Figure 18 a Epifluorescence microscopic picture of a live
106. nisms during staining or during sample analysis due to for instance warming of the sample This is because organisms that die after addition of the Neutral Red will still be clearly stained while those already dead prior to the addition will not be stained Neutral Red is pipetted in a ratio that yields an end concentration of approx 1 50 000 The Neutral Red stock solution is 1 2 000 i e approximately 4 mL of stock solution is needed to stain a sample of 100 mL The staining time is 2 hours Stained samples are filtered over a 30 um sieve and flushed into a Bogorov dish with filtered seawater Samples are analysed using a binocular with a 20x magnification for counting and up to 80x for species identification and measurements when necessary Neutral Red stains all major plankton groups including phytoplankton but it seems to have some limitations for bivalve larvae For the latter movement including that of hart and gill is used to verify viability This is dependent on the expertise of the person analysing the samples Therefore only persons with a dedicated training period will analyse samples Organisms that are able to swim are also considered alive In doubt the organism can be poked with a dissection needle The procedure is outlined in Figure 16 36 NIOZ Land based Project Plan Organisms larger 50um landbased tests Viability testing Filling emptying of test tanks Structural integrity undisturbed Struc
107. ns all organisms quantitatively retained on the 50 um net are considered as larger than 50 um in minimum dimension The organisms retained in the cod end of the Hydrobios net are flushed into a beaker using a squeeze bottle containing filtered seawater Organisms are kept in approximately 100 to 200 mL of filtered lt 0 2 um filter sterile water of the relevant salinity Samples are transferred to the lab directly after sampling treated for 2 hours with the viability stain Neutral Red and counted These samples are not stored Figure 14 Intermediate Bulk Containers IBCs of 1 m each A 50 Hm Hydrobios plankton net is fitted in the containers through the lid for easy sampling 4 5 2 Sampling and storage of 10 lt um lt 50 organisms Samples for the 10 lt um lt 50 fraction are taken as undisturbed unfiltered one litre samples This size fraction is not separated from the organisms lt 10 um or gt 50 um at sampling or during sample processing but during data analysis This approach reduces damage to more delicate organisms as ciliates and non armoured flagellates Whole intact samples of 1 L are stored in the dark prior to analysis in the laboratory The total concentration of 10 lt um lt 50 phytoplankton cells is measured in non fixed samples The concentration of dead phytoplankton cells is measured using SYTOX Green also in non fixed samples The difference between the concentrations of total and dead organisms is the conce
108. nths 69 l NIOZ Land based Project Plan ANALYSIS Samples need to be homogenized by mixing very carefully Use a pipette with a big opening to assure that ciliates will not be damaged For the control sampling point TO take 1ml out of all tree concentrated samples and mix carefully in a tube take a subsample of 1ml and analyze in the Uterm hl cuvette In general analyze at least 1 ml per control sampling point and at least 3 ml per treated sampling point Place the volume in a tube and include one or more droplets of sodium thiosulphate to neutralize the Lugol Then the sample is stained with a small droplet of Bengal Rose This sample is placed in a Uterm hl cuvette Fill this cuvette fully by filtrated 0 2 um and sterilized sea water Analysis will be done after 2 hours of staining and settling down of the sampling Use a 0 17 mm cover glass over the cuvette to assure no movement in the cuvette itself Analysis of abundance and species determination to the lowest possible taxonomic unit is done by using an inverted microscope 200x objective 400 and 600 for determination For more detailed information see micro zooplankton booklet in the microscope room F00 05 At the start of the BWTS test season subsamples of 3 or more old samples are counted by three analysts the individual counts are recorded the difference between analysts should become lt 10 A number of samples will be compared with counts made in the same sample by Koe
109. ntration of viable organisms As a reserve a 5 mL sample is fixed with formaldehyde and stored at 80 C for up to one year This fixed sample can no longer be used to determine the viability of the organisms The samples for 10 lt um lt 50 microzooplankton are fixed with Lugol s solution they can be stored in a refrigerator for up to one year before counting takes place The viability of the organisms is assessed from their intact morphology 4 5 3 Sampling and storage of total and viable heterotrophic bacteria To determine the total concentration of bacteria samples fixed with formaldehyde are frozen and stored at 80 C until counts are made after staining with PicoGreen Dead bacteria in non fixed samples are measured in fresh test water using SYTOX Green these samples cannot be stored The difference between the concentrations of total and dead bacteria is the concentration of viable bacteria 4 5 4 Sampling of human pathogens Live samples for microbiological analysis are taken in sterile bottles of 300 or 600 mL and sent to a special laboratory Eurofins C mark for further analysis The samples are transported immediately after sampling using a cooled transport container 4 C 32 NIOZ Land based Project Plan 4 5 5 Sampling for additional incubation viability experiments Additional incubation experiments which are not required by G8 serve to better assess the effect of the different ballast water treatment systems and
110. of the tube on the table to mix the SYTOX droplet with the sample Start a timer for 10 min after mixing the first sample Incubate for 10 15 min in the dark at room temperature e Put the tubes in the carousel of the FACS CANTO and start the analysis e Start a new series of 6 samples when the first 3 samples have been measured and repeat this until all samples are done Measurement e With SOPs Phytoplankton Canto FCM and FCM CANTO operation e Use Phyto SYTOX protocol measuring time is 200 sec CALCULATIONS e See SOP FCM Canto data processing e Use FCS Express Layout for Phyto SYTOX LITERATURE Peperzak L Brussaard CPD 2011 Flow cytometric applicability of fluorescent vitality probes on phytoplankton J Phycol 47 692 702 68 l NIOZ Land based Project Plan dr N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE 27 02 2012 Isabel van der Star Eva Immler Microzooplankton 2012 2 Goal Microzooplankton comprises by definition organisms in the size range of 20 200um This protocol refers to microzooplankton as defined by the IMO D2 Standards as a size class 10 lt um lt 50 um Maximum allowed numbers of organisms in this size class at discharge are 10 viable org ml This size class includes phytoplankton and zooplankton species Phytoplankton is quantitatively analyzed by flow cytometry Flagellates and dinoflagellates include autotrophic heterotrophic and mixotrophic species while ciliate
111. om the sample volume and the weight difference of the filter before and after sampling TSS is expressed in mg l e Open the petri dish and put the cover under the bottom of the dish with the filter and place them in the Dry oven at 60 C to dry overnight min 6 hrs and max 12 hrs e After 6 12 hrs remove the petri dishes from the oven and store in a desiccator to cool down under vacuum as long as needed e Weigh the GFC filters note the filter number weight and sample info in the lab journal e Calculate the difference from the weighing before and after in mg l To determine the POC concentration the same filter is combusted overnight at 500 C and allowed to cool in a dessicator and weighed again 48 NIOZ Land based Project Plan Fold the filters twice and place each filter in a numbered porcelain combustion dish Keep a record of the filter number sample info and combustion dish number Combust overnight at 500 C and cool down in a desiccator Weigh the filters and add the values to the record CALCULATIONS Calculate TSS weight after drying at 60 C weight pre dried empty filter at 60 C sample volume TSS is expressed in mg l Calculate POC weight after drying at 60 C weight after combustion at 500 C sample volume POC is expressed as mg C I CALIBRATION Accurately weigh approximately 50 mg reference dried mud Suspended in 1 L graduated cylinder 0 2 um filtered seawater Treat this laboratory sta
112. ook vermelden Filename datum projectnummer naam gebruiker informatie over kolom nieuw of niet soort monsters gebruik van ijklijn type aparaat gebruik van standaard en eventuele bijzonderheden Monsters kunnen overnacht worden geanalyseerd met de autosampler In carousel niet meer dan 50 tot 60 vials zetten Altijd beginnen met milliQ sampler heeft dubbele naald eerste flesje wordt doorborreld terwijl het tweede wordt geanalyseerd In verband met het behoorlijke verloop van de metingen relatief gezien op dit lage detectieniveau is het aan te bevelen om wel drie ijklijnen te meten per serie aan het begin halverwege en aan het eind Aan de hand van meetresultaten kan dan achteraf worden gecorrigeerd Na b v iedere zeven vials een schoonmaakvial met 0 1 m HCL zetten Het monsterschema voor 18 monsters ziet er dan b v als volgt uit 1 milliQ 21 12 Di 0 22 HCI 3 25 23 0 4 50 24 25 5 100 25 50 6 200 26 100 7 HCI 27 200 8 1 28 HCI 9 2 29 13 10 3 30 14 11 4 31 15 12 5 32 16 13 6 33 17 14 HCI 34 18 15 CRM 35 CRM 16 7 36 0 17 8 37 25 18 9 38 50 19 10 39 100 20 11 40 200 Ampullen met monsters openbreken gebruik een opgevouwen tissue ter voorkoming van snijwondjes door de top er met je duim af te breken zet je duim op de witte stip De inhoud van de ampul moet worden overgedaan in een 25 ml vial 5x gespoeld met m
113. ording to IMO regulations and NIOZ quality objectives can be performed Second as part of a research institute it may be of interest to the facility that the BWTS to be tested is of a different design as previously tested BWTSs The two project leaders of the test facility jointly decide on the admission of a BWTS for land based testing In case of requests for combined land based and ship board tests the admission will be discussed with the NIOZ partners IMARES and GoConsult Contracts on behalf of the NIOZ will be signed by one of the NIOZ directors The provisional project plan and planning for testing the BWTS is made by the project leader of ballast water research Next the provisional project plan and planning is discussed in the ballast water team where all practical issues including those related to quality management are addressed 9 Implementation of work processes Quality management objectives as specified in this QMP and in the QAPP and SOPs are implemented in the work process At the lowest level individual members of the facility s test team are responsible for carrying out quality checks as detailed in SOPs on a daily basis The quality manager is responsible for supervising QA QC activities during all test and laboratory activities The quality manager will give his feedback during each team meeting that is scheduled at least bi weekly during BWTS tests On advice of the quality manager the project leader of ballast water rese
114. ored at 4 C until analysis For TSS POC measurements pre weighed glass fibre filters GF C are used Each filter is coded and stored individually in a Petri dish The filtered water volume is not constant but depends on the particle concentration of the test water The higher the particle concentration in the sample the smaller the volume that can be filtered before clogging Practical volumes are 100 1000 mL per sample After filtration the filter is rinsed with demineralised water to remove sea salts The filter is then put back in its original Petri dish and stored temporarily in a freezer at 20 C or long term at 80 C until further analysis Glass Winkler bottles for oxygen DO are flushed at least three times their volume ca 120 mL with test or control water Great care is taken to avoid gas bubbles on the wall of the bottle or excessive mixing of air and water Next MnCl and NaOH solutions are added to the bottle just below the surface by using dispenser units to fix the oxygen concentration A stopper secured with a rubber band is put on the bottle and the bottle is gently mixed Bottles are stored in a dark container filled with water of the same temperature as the samples until further analysis in the laboratory Figure 12 Figure 12 Glass bottles for Dissolved Oxygen DO measurements are stored submerged prior to analysis 30 NIOZ Land based Project Plan Figure 13 Sampling point on a tank at the Navicula quay for s
115. ory e Pipet 1 5ml e Pipet tip e Beaker glass 300ml e Sieve 30um diagonal mesh size e Squeeze bottle 0 2 um filtered seawater e Bogorov or Borgorov Gollasch dish e Microscope 20x magnification for determination 30 60x ZEISS V8 and V12 CHEMICALS e Neutral Red preparation of the solution 125mg standard Neutral Red stock in 250 ml demi water Neutral Red is added to the sample in a ratio to yield an end concentration of 1 50 000 SAMPLING Sampling of mesozooplankton for land based certification tests of a ballast water treatment system is done in triplicates Day 0 3x 1m after treatment 3x 20 liters control after the pump Day 5 3x 1m after treatment 3x 20 liters control after the pump Harbour control e Buckets are flushed 3 times with sample water e Fill both buckets with 10 liters sample water e 20 Liters are carefully filtered over a 50um sieve e Organisms are carefully flushed from the sieve with 0 2 um filtered seawater into a 2 Liter bottle mesozooplankton is sensitive to damage by nets and sieves Harbour treated samples e Plankton nets are flushed with fresh water The plankton net is placed in an empty 1000 liter IBC with a closed net beaker Fill the IBC with 1000 liters of sample water When during sampling the net is clogging When the IBC is full take the net slowly out of the IBC Organisms are flushed from the net into the net beaker by using a squeeze bottle filled with filtered seawater 0 2
116. p Harbour water samples are analysed regularly from February onwards in order to monitor the spring plankton bloom Immediately before the treatment equipment from the main pipeline but after the ballast pump that is used to pump up the test water from the harbour control TO Immediately after treatment from the main pipeline treated TO and During discharge from the main pipeline after the pump after 5 days control and treated T5 holding time 2 3 2 and 2 3 26 G8 guidelines and after completing a second passage through the BWTS when this step forms part of the treatment prescribed by the vendor of the BWTS i e in the case of the Cathelco BWTS treated only During ballast water tests samples will be taken sequentially covering the entire intake or discharge periods 2 8 NIOZ Land based Project Plan During the tests the following sample sizes will be used 1 Untreated water control To and Ts Sampling is conducted in line three times with sample volumes of 20 L gt 50 um 10 Land 2x 1 L The 10 L samples are used to subsample for abiotic variables The 1 L samples are used 1 to subsample for phytoplankton bacteria as well as for phytoplankton lt 10 um and 2 microzooplankton 10 lt um lt 50 An additional 10 L sample is taken for an incubation experiment To only 2 Treated water intake To Sampling is conducted in line three times with sample volumes of 1 m gt 50 um using 3 IBCs of
117. phytoplankton cell The red signal is due to the presence of chlorophyll a and b a dead phytoplankton cell with a yellow green fluorescence of the nucleus after staining with SYTOX Green Counting total heterotrophic bacteria The classical method for counting bacteria in many applications is based on plating on selective media Unfortunately for studies in the aquatic environment this approach is by far insufficient for various reasons Gasol amp Del Giorgio 2000 Therefore the total bacteria concentration in fixed samples is accurately determined by flow cytometry using the DNA specific stain PicoGreen cf Gasol amp Del Giorgio 2000 Veldhuis et al 1997 NIOZ Ballast Water Report 5 2 4 36 Bacteria Vi uses Figure 19 Epifluorescence microscopic picture showing bacteria and viruses in a natural water sample The yellow green fluorescence signal is DNA stained with a nucleic acid dye The dye PicoGreen is a green nucleic acid specific dye that only stains dsDNA with little or no cross over for ssDNA and RNA Veldhuis et al 1997 This makes the staining method ideal to for staining of DNA and therefore to determine bacterial abundance Flow cytometric analysis shows a clear signal with an excellent signal to noise ratio and bacteria are made visible easily and distinguishable from viruses and larger organisms This approach has extensively been compared with bacteria staining and counting using an epifluorescent mi
118. prove the quality of its tests and its test data for instance from class societies and through its membership in the Global Testnet 11 NIOZ Land based Project Plan 12 NIOZ Land based Project Plan Section 2 Quality Assurance Project Plan APP INTRODUCTION GENERAL DESCRIPTIONS 2 1 NIOZ profile 2 2 Profile of the CATHELCO group 2 3 Technical overview of CATHELCO s BWTS TEST FACILITY NIOZ test facility Freshwater intake at Den Helder and transport to NIOZ BWTS test set up treatment and control tanks Toolbox meetings Ballast water book Test season time planning WWW Ww WW AuURWNE TEST WATER QUALITY SAMPLING STORING Abiotic quality Biological quality General sampling strategy Abiotic variables sampling and storing Biological variables sampling and storing Sampling organisms 250 um Sampling and storage of 10 lt um lt 50 organisms Sampling and storage of total and viable heterotrophic bacteria Sampling of human pathogens Sampling for additional incubation viability experiments 33 nn een UU WUUuUrswNE URUNE MEASUREMENT OF VARIABLES Abiotic variables Salinity Temperature and pH TSS POC Total Suspended Solids Particulate Organic Carbon Dissolved Oxygen DO Dissolved Organic Carbon DOC Biological variables Counting of organisms gt 50 um Counting of organisms 10 lt um lt 50 1 Counting viable microzooplankton 2 Counting viable phytoplankton Counting total heterotrophic bacteria Human
119. quired which specialises in reverse osmosis water makers from a manufacturing facility in Bournemouth United Kingdom To serve the shipbuilding and repair industry in South East Asia Cathelco S E A based in Singapore became a subsidiary in 2011 In addition to its marine engineering activities the Cathelco Group encompasses Casting Repairs Ltd specialising in the repair of architectural cast ironwork It also has an active property division focusing on property development and rental via the Broomco Ltd subsidiary Cathelco GmbH was established in 2010 in Kiel Germany to research and develop ballast water treatment equipment for the worldwide market The aim is to develop a chemical free two step ballast water treatment system This system should be easy to retrofit for existing vessels and innovative for new builds Beside the ballast water issue the Cathelco R amp D Centre provides also services to the Cathelco Group in respect of testing and improving of existing products This includes but is not limited to antifouling systems for vessels and other marine structures NIOZ Ballast Water Report 2 3 13 Technical Overview of Cathelco s BWTS Cathelco has developed a 100 chemical free BWTS Figure 2 It is based on the combination of filtration UV treatment There are no active substances needed for the treatment of the ballast water or for the cleaning of the UV system The system was designed with emphasis on retrofit
120. r e 1 litre graduated cylinder e Filtration unit Filtration frame with vacuum pump and four vacuum bottles with a filter holder e Filter Forceps SS Millipore e Freshwater MilliQ or mQ e Beaker e Reference dried 60 C mud as used in BWTS testing stored in an exsicattor SAMPLING e During sampling in the harbour samples are taken in 1 litre square bottles in triplicate for each test e Insert a pre weighed filter into the Filter unit and write down the number of the filter in the harbour journal and add the code of the test onto the petri dish e To avoid precipitation of TSS mix gently but thorough and pore about 1 litre in one go into a 1 litre graduated cylinder e Read the volume and write it down in the harbour journal estimate the last ml e Filter the whole volume and rinse out the graduated cylinder with mQ also rinsing salt from the filter e When filtering is finished turn off the vacuum pump and release the vacuum before you take the filter from the holder by using a pair of tweezers and put it back in the numbered petri disk e Store temporary in the fridge in the harbour at the end of the day take all filters into the lab and store in a freezer 80 C 20 C is allowed for a short period or directly put them in the Dry oven at 60 C to dry overnight ANALYSIS For TSS analysis GF C filters are dried at 60 C for at least 8 hours and weighed again The concentration of TSS per litre can be calculated fr
121. r gt 50 um at sampling or during sample processing but during data analysis This approach reduces damage to more delicate organisms as ciliates and non armoured flagellates Whole intact samples of 1 L are stored in the dark prior to analysis in the laboratory The total concentration of 10 lt um lt 50 phytoplankton cells is measured in non fixed samples The concentration of dead phytoplankton cells is measured using SYTOX Green also in non fixed samples The difference between the concentrations of total and dead organisms is the concentration of viable organisms As a reserve a 5 mL sample is fixed with formaldehyde and stored at 80 C for up to one year This fixed sample can no longer be used to determine the viability of the organisms The samples for 10 lt um lt 50 microzooplankton are fixed with Lugol s solution they can be stored in a refrigerator for up to one year before counting takes place The viability of the organisms is assessed from their intact morphology Sampling and storage of total and viable heterotrophic bacteria To determine the total concentration of bacteria samples fixed with formaldehyde are frozen and stored at 80 C until counts are made after staining with PicoGreen Dead bacteria in non fixed samples are measured in fresh test water using SYTOX Green these samples cannot be stored The difference between the concentrations of total and dead bacteria is the concentration of viable bacteria Sampling of
122. r consumption of the system The UV dose at T10 70 will be about 135 J m at 200m3 h flow rate This dose is above the requirements for a 4 log reduction of many microorganisms if no photo repair mechanism occurs NIOZ Ballast Water Report As there is no light inside the ballast water tanks our system accepts this dose during ballasting only If the UV T decreases further the flow will be reduced automatically to ensure that the minimum dose 133 J m2 is maintained During de ballasting the water is much clearer i e UV T is higher and the Cathelco system will dose between 400 J m2 and 700 J m2 which is well above the dose requirements for a 4 log reduction for the majority of organisms even in the presence of photo repair mechanisms Cathelco s BWTS will be tested at a treatment related capacity TRC of 200 m3 h during the land based Type Approval Tests at the NIOZ test facility There will be two UV reactors and two different filters involved during the tests The general layout of the test system should be according to the P amp ID in Figure 4 Symbol Legend PS Pressure Sensor m Magnetic Flow Meter DK Actuated Butterfly Valve D Manual Butterfly Valve Filter By Pass vm UV Intensity Meter Temperature Sensor All equipment within boundary X Air Bleed 7 filter supply i LG Cath
123. riables sampling and storing 4 5 1 Sampling organisms 250 um 4 5 2 Sampling and storage of 10 lt um lt 50 organisms 4 5 3 Sampling and storage of total and viable heterotrophic bacteria 4 5 4 Sampling of human pathogens 4 5 5 Sampling for additional incubation viability experiments MEASUREMENT OF VARIABLES Abiotic variables 1 Salinity Temperature and pH 2 TSS POC Total Suspended Solids Particulate Organic Carbon 3 Dissolved Oxygen DO 4 Dissolved Organic Carbon DOC Biological variables Counting of organisms 50 um Counting of organisms 10 lt um lt 50 1 Counting viable microzooplankton 2 Counting viable phytoplankton Counting total heterotrophic bacteria Human pathogens PAM measurement for total phytoplankton viability Counting phytoplankton lt 10 um UA AUW UU a UU UW UUNWNNNEHE WN Quality assurance Quality control QA QC 6 1 Ballast water tests 6 2 Laboratory analyses 6 3 Data analysis REFERENCES 4 NIOZ Land based Project Plan 14 15 16 16 42 42 44 Section 3 Standard Operating Procedures SOPs Variable Reference SOP Page Salinity and Temperature Salinity and Temperature 2012 2 46 pH pH 2012 1 47 TSS and Particulate Organic TSS POC 2012 2 48 Carbon Dissolved Oxygen Dissolved Oxygen 2012 1 50 Dissolved Organic Carbon DOC 2012 1 53 Viable organisms 250 um Mesozooplankton 2012 1 57 including diversity Phytoplankton Phytoplankto
124. rol To and Ts Sampling is conducted in line three times with sample volumes of 20 L gt 50 um 10 L and 2x 1 L The 10 L samples are used to subsample for abiotic variables The 1 L samples are used 1 to subsample for phytoplankton bacteria as well as for phytoplankton lt 10 um and 2 microzooplankton 10 lt yum lt 50 An additional 10 L sample is taken for an incubation experiment To only Treated water intake To Sampling is conducted in line three times with sample volumes of 1 m gt 50 um using 3 IBCs of 1000 L each Furthermore in line sampling is conducted three times with a sample volume of 10 L and 2x 1 L The 10 L samples are used to subsample for abiotic variables The 1 L samples are used 1 to subsample for phytoplankton bacteria as well as for phytoplankton lt 10 um and 2 microzooplankton 10 lt um lt 50 Additional samples of 10 L are taken for incubation experiments Treated discharge Ts Sampling is conducted in line three times with sample volumes of 1 m gt 50 um using 3 IBCs of 1000 L each Furthermore in line sampling is conducted three times with a sample volume of 10 L and 2x 1 L The 10 L samples are used to subsample for abiotic variables The 1 L samples are used 1 to subsample for phytoplankton bacteria as well as for phytoplankton lt 10 um and 2 microzooplankton 10 lt um lt 50 Additional samples of 10 L each are taken for incubation experiments The sub sample volumes taken fro
125. s as well as the identification and enumeration of gt 50 u organisms All forms are stored on paper in the NIOZ Ballast Water archive or in electronic format on the Ballast Water project server Specific forms for sample custody are also used in case of the analyses of pathogenic bacteria or specific chemical toxicological analyses which are carried out in third parties commercial laboratories Standard Operating Procedures or SOPs are available for each IMO variable that the test facility is obliged to measure For abiotic variables the analysis methods stem from decades of NIOZ experience as an oceanographic institute New methods in particular those for biological variables are or will be published in international peer reviewed scientific journals In addition SOPs are present for specific actions during the BWTS testing such as for adjusting the salinity of the test water flow cytometer data analysis or the cleaning of ballast water tanks SOPs are numbered by year and version Paper copies are kept in the NIOZ test facility archive Electronic copies are stored on a dedicated server that is accessible to Ballast Water project personnel only SOPs will be critically reviewed during the operation of the test facility as well in general annual reviews by the quality manager SOPs relevant to the QAPP can be found in section 3 of this project plan The test protocol QAPP QMP and SOPs are submitted before the start of the BWTS tests to
126. s a special strain of E coli also incubated like a normal sample to confirm that this species can grow and form colonies on the used media NIOZ Ballast Water Report Enterococci group Analysis for this group is carried out according NEN EN ISO 7899 2 For this the samples are filtered through membrane filters pore size 0 45 um and these filters are incubated on a selective agar plate Incubation is 44 4 hours at 36 2 C on Slanetz amp Bartley medium After that red and pink colonies are counted If the presence of enterococcus bacteria can be suspected after the colour of the colonies the filter is transferred to a pre heated selective agar plate and incubated for another 2 hours at 44 0 5 C After that the medium is examined whether or not a brown to black colour can be found in it Results are confirmed via a positive and a negative control For the latter sterilized water is incubated like a regular sample and to confirm the results it may only yield less than 1 colony forming unit cfu per 100 mL The positive control uses a strain of Enterococcus faecium 5 2 5 2 PAM measurement for total phytoplankton viability The photochemical efficiency of photosystem II is an indicator of the physiological health condition of phytoplankton cells It is a bulk variable that is measured using a Pulse Amplitude Modulated PAM fluorimeter Schreiber et al 1993 Figure 20 The simple fluorescence ratio Fv Fm gives a qualitative indica
127. s are able to perform a specific sampling or analysis This should prevent personal bias in sample and data analysis In addition this procedure ensures that in case of unexpected absence sampling or sample analysis can still be carried out by trained personnel In case of non automated sample analysis the microscopic identification and enumeration of planktonic organisms the research personnel is trained in groups of three At the beginning of each year NIOZ test water is analysed by all three staff until the difference in individual plankton concentrations is lt 10 Furthermore in the case of gt 50 um organisms the correct execution of sample and analysis procedures and identification and enumeration of the organisms is audited by annually by an external independent consultant In 2012 this was performed by Mr F Fuhr of KiTe Aquatic Resources Consulting The Netherlands Additional training of staff members takes place on the job during inter comparison workshops of the ballast water test team in cooperation with the planktonlab of the department of biological oceanography of NIOZ One such workshop was the comparison of flow cytometers held in January 2012 Furthermore a statistics course is planned for autumn winter 2012 PROCUREMENT OF ITEMS AND ACTIVITIES The BWTS test facility is part of the NIOZ research institute and is required to use the NIOZ facilities for procuring items and services that include all sampling and laboratory eq
128. s are heterotrophic or mixotrophic Heterotrophic species miss chlorophyll fluorescence and cannot be analysed by flow cytometry MATERIAL amp EQUIPMENT Sampling 1 L bottle e 4ml Lugol L Laboratory e 50ml bottles e Low pressure vacuum pump lt 0 1 bar e Weighing scale e Tube e Pipettes 0 5 5 ml 100 1000pl e Pipette tips 0 5 5 ml 100 1000ul e Uterm hl cuvette of 5 6 ml 0 17mm bottom thickness e Inverted microscope CHEMICALS e Lugol 100 KI 50g I and 100ml HAc in 1 liter demi water see SOP Lugol plankton fixative e Sodium thiosulphate 1 dissolved in demiwater e Bengal rose SAMPLING Harbour e iL bottles with 4 ml lugol are prepared keep in the dark prior to filling e Per sampling point 3 samples of 1 liter each are taken 3 controls after pump and 3 after treatment Bottles are filled without filtering ciliates are highly sensitive to damage by filters Laboratory e Samples need to settle down for at least 24 hours They are placed in a dark solid closet to avoid light and vibrations e After settling sample bottles are weighted without lid o Full sample o Concentrated sample o Empty bottle e Sample is concentrated by sucking the non concentrated part of the sample by using a vacuum pump A volume of max 50 ml of the homogenized concentrated sample will be saved in a dark bottle e The concentrate is saved in a fridge for max one year At room temperature the sample can be stored for 6 mo
129. s feedback during each team meeting that is scheduled at least bi weekly during BWTS tests On advice of the quality manager the project leader of ballast water research gives permission in cases that quality documents need to be revised or new ones have to be made NIOZ Ballast Water Report 2012 9 10 11 12 ASSESSMENT AND RESPONSE During BWTS tests inspections may be made by inspectors of Lloyds Register or the BSH depending on the national authority that will apply for type approval at IMO All ballast water team members are obliged to report deviations from quality standards or procedures to the quality manager At the end of each BWTS test the quality manager will report on all deviations from the QAPP or from QA QC measures in SOPs The quality manager also checks the BWTS test data that are compiled by the project leader of ballast water research The project leader of ballast water research will report any deviations in harbour test water monitoring data and intake test water data from the QAPP QUALITY IMPROVEMENT All team members are motivated to enhance specific QA QC measures and methods Regular feedback between team members and the quality manager will enhance self improvement In addition the NIOZ quality objectives are submitted to independent external review NIOZ test facility methods are compared and discussed in NSBWO workshops with other test facilities and experts in the field of ballast water research Proficie
130. s is contracted to Mr A Smit of Smittech Den Hoorn The Netherlands and Mr J Witte Witte Klusbedrijf Den Burg The Netherlands The NIOZ ballast water team advisory committee consists of Prof Dr Hein de Baar chemistry Dr J van Bleijswijk molecular biology Dr Corina Brussaard microbial ecology and Dr Klaas Timmermans head of the Department of Biological Oceanography at NIOZ The Quality Management Plan QMP and Quality Assurance Project Plan QAPP are reviewed annually by an external independent consultant In 2012 this was performed by Dr S Kools of Grontmij The Netherlands In addition at the request of the BSH a review on QA QC aspects was performed by Dr A Cangelosi of GSI USA Comments of both parties have been used in the present project plan that includes both QMP and QAPP 3 Quality system components The quality system of the NIOZ BWTS test facility contains several components of which this QMP is the main document The QMP is the joint responsibility of both project leaders of the NIOZ Ballast Water Project The QMP forms the basis of the project s quality assurance and quality control QA QC The Quality Assurance Project Plan QAPP is the project specific technical document reflecting the specifics of the NIOZ test facility the BWTS tested and other conditions affecting the actual design and implementation of the required experiments such as test water quality sampling and sample storing the me
131. scope at 200x magnification method by Uterm hl The fixed samples or sub samples thereof are transferred into settling chambers and neutralized using sodium thiosulfate After this the sample is stained using Bengal rose stain This stain specifically stains organic material and helps to identify organisms between sediment particles After staining the samples are left undisturbed in the dark to settle The time of settling depends on the settling chamber used but should be at least 1 hour per cm height of the settling chamber The iodide component of the Lugol s solution increases the weight of particles Therefore they settle on the bottom glass and can be counted Live dead separation in these samples is mainly based on the structural integrity of organisms This method can be applied for both zoo and phytoplankton Nevertheless certain groups are known to be affected by this standard method of fixation and therefore they will be systematically underestimated if present 5 2 2 2 Counting viable phytoplankton Organisms in the 10 lt um lt 50 size class will be analyzed via flow cytometry Figure 17 a semi automated method used in the NIOZ for the counting of phytoplankton bacteria and viruses In principle flow cytometry allows to assess a complete view of the effectiveness of the applied treatment technique for all organisms irrespective of their size Veldhuis amp Kraay 2000 By applying special staining techniques the numerical ab
132. se your grip When open add 1 ml reagent C with a dispenser do not place the dispenser tube in the sample Watch out for spilling reagent C it is a strong acid Drop a stirring bar in the bottle and mix using a magnetic stirrer until precipitate is fully dissolved The color of the sample should be brownish orange in different intensities depending on the amount of oxygen trapped Take the left side of the tubing attached to the flow through cuvette of the spectrophotometer quickly out of the MQ dry the tubing on the outside and place it in the sample Let it run through the cuvette until the transmission reading is stable Calculation To be able calculate the Oxygen concentration you need the slope and the intercept of a calibration curve with a Potassium Iodate Standard solution For standard use slope 0 0021 intercept 0 0312 or determine them from your own calibration curve Use the following equation for calculation of the Oxygen concentration of your sample O2 umol l E456 sample intercept slope bottle volume ml 1 bottle volume ml 3 1 05 02 mg l 02 umol l 32 1000 For making a calibration curve prepare a Potassium Iodate Standard solution Dry KIO3 for 6 hours at 180 C cool down in an excicator Weigh 2 5 KIO3g and dissolve in 250 ml MQ Calculate the oxygen equivalent of the KIO3 solution 02 umol ul 1 5 ug KIO3 1000 1 00016 ml MQ 214 2 5 g KIO3 250 ml 0 07 umol O2 ul Prepare a blank
133. sea systems Climate variability and the sea and Biodiversity and ecosystem functioning Together with a number of oceanographic partners NIOZ also maintains the popular marine website www seaonscreen org For more information please contact our Communication amp PR department at cpr nioz nl or visit our website at www nioz nl NIOZ has extensive experiences in the field of ballast water and ballast water treatment technologies at its harbour on the island of Texel During the past seven years several pilot tests for ballast water treatment were conducted in the NIOZ harbour and so far between 2007 and 2010 seven full scale land based tests were carried out for Final and Type Approval Profile of the Cathelco Group Cathelco Ltd was formed in 1956 and has become a world leading supplier of cathodic protection equipment to the shipping and offshore marine markets The parent company based in Chesterfield United Kingdom manufactures marine pipe work anti fouling AF and impressed current cathodic protection ICCP systems The Cathelco Group has grown through a series of acquisitions beginning with Corrintec Ltd in 1995 Today Corrintec Marine continues to operate as a wholly owned subsidiary serving the military sector worldwide In 2005 the company established Cathelco Korea a subsidiary engaged in manufacturing and distribution serving the Korean shipbuilding market More recently in 2010 Seafresh Desalinators Ltd was ac
134. sms even in the presence of photo repair mechanisms Cathelco s BWTS will be tested at a treatment related capacity TRC of 200 m3 h during the land based Type Approval Tests at the NIOZ test facility There will be two UV reactors and two different filters involved during the tests The general layout of the test system should be according to the P amp ID in Figure 4 Symbol Legend PS Pressure Sensor f m Magnetic Flow Meter 3 Da Actuated Butterfly Valve DK Manual Butterfly Valve Filter By Pass Cw UV Intensity Meter A Temperature Sensor cei All equipment within boundary 4 Air Bleed ifilter supply X TE ilter supply Cal Cathelco S Sample Point i Pre Filtration i amp Turbidity UV Meter eS Filter Drain required Overboard Figure 4 Piping and Instrumentation diagram of Cathelco s BWTS for a TRC treatment related capacity of 200 m3 h 19 NIOZ Land based Project Plan 3 TEST FACILITY 3 1 NIOZ test facility The land based tests will be carried out on the island of Texel NIOZ harbour NL from March to July spring and early summer season The NIOZ test site is equipped with two times Navicula and Pelagia quay three silos or underground storage tanks of 300 m3 each to simulate ship s ballast water tanks Figure 5 The NIOZ harbour is located at the Marsdiep tidal inlet betwe
135. t OK Haal de cap van de carrousel pak de carrousel eraf en zet de vials er goed in in de uitsparingen en in juiste volgorde File haal oude file op b v 8 oktober 08 ballastwater t32 Edit Delete All alle meetresultaten worden weggegooid connect even wachten carousel gaat draaien en plunjer gaat op en neer gt stoplicht start In scherm de vial nummers invullen b v 1 t m 40 bij iedere regel OK start Calibration Per serie twee of drie keer een CRM standaard meten Certificate Reference Material b v Deep Seawater Reference REMARKS e See also TOC V user s manual and for data processing TOC V Administrator s manual 56 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE Author Frank Fuhr Dennis Mosk Eva Immler 2012 02 27 SOP Mesozooplankton 2012 1 Goal This protocol describes sampling and analyzing of mesozooplankton in a ballast water context for land based testing It refers to mesozooplankton as defined by the IMO D2 Standards as a size class gt 50 um After treatment the allowed number of organisms in this size class at discharge is lt 10 viable org m3 MATERIAL amp EQUIPMENT Harbour e 2 buckets 10 liter Bottle 2 liter Sieve 50um diagonal mesh size Squeeze bottle 0 2 um filtered seawater Plankton net 50um diagonal mesh size net beker Hydrobios 1000 liter tank IBC Laborat
136. t 50 um gt 10 mL different phyla divisions heterotrophic bacteria not further defined The natural waters of the test area include a large range of organisms varying in sensitivity to mechanical stress UV radiation or various active substances Besides fragile organisms also plankton that is highly adapted to harsh environmental conditions mostly hard shell organisms are present in the test water For completeness the plankton fraction lt 10 um is also included in the NIOZ analyses although this is not required by the G8 guideline NIOZ Ballast Water Report 4 3 25 Table 3 Biodiversity as number of species in NIOZ test water according to phylum and to size class based on data from the 2011 spring and early summer test season The test water contains at least 18 phyla with a total of 16 phyla in the 10 50 and gt 50 um size classes The total number of species in each G8 size class is 42 8 phyla for the 10 lt um lt 50 and 31 10 phyla for the 250um size classes Organisms lt 10um that are not bacteria are not part of the D 2 regulation The high test water biodiversity largely exceeds the G8 guideline 2 3 20 Phylum Number lt 10 um Number 10 50 um Number gt 50 um Amoebozoa 1 Annelida 5 Arthropoda 13 ess __ Chlorophyta 1 1 Choanozoa Ciliophora Cnidaria Cryptophyta Ctenophora Echinodermata Euglenozoa Haptophyta Mollusca Myzozoa Nematoda Ochrophyta Rotifera Unknown
137. t ca 10 minutes e Cytometer gt CST e Page Cytometer Setup and Tracking appears e Right top screen Characterize Check Performance gt Run gt OK e Filla flow cytometer tube with 0 35 ml mQ water e Mix the bead vial by gentle vortexing e Add one drop of BD Cytometer Setup amp Tracking Beads e Vortex the solution e Store the solution in the dark and make this solution daily fresh e Vink on Load Tube Manually you find this directly under Run in Setup Control e Window Cytometer Setup and Tracking appears gt OK e Unload the tube e After the calibration the report appears e Cytometer Setup and Tracking gt View report e Please watch the variance bright beads CV lt 6 when not ok the values are in red e File print e After you ve run the CST beads click on Performance Tracking You ll find an overview of the last CST beads files e Close window CST e Check performance e CST mismatch window appears e Click on middle option Use CST Settings 61 NIOZ Land based Project Plan Trucount beads Get a tube of Trucount beads from the drawer left of the Canto flow cytometer Read the number of beads from the bag and write it on the tube Add 1 ml of filtered seawater to the tubes Click on the book Trucount beads Right mouse button gt Duplicate without data Rename the experiment with the correct date Open specimen Run first the tube with Trucount beads on Low flow speed for 180s Continue with me
138. t te worden moet wel dagelijks het geklonterde Platinised Alumina uit de glasbuis worden gehaald na afkoelen m b v een ijzerenstaaf en vervolgens moet er een beetje Platinised Alumina aan worden toegevoegd dan ook het inj blok schoonspoelen en droogblazen tevens bovenplaatje van inj blok schoon spuiten met milliQ dagelijks moet wel de brug f met Anhydrone worden ververst trek de koppelingen g los van de brug en trek vervolgens de dop er af aan Anhydrone kant schud leeg haal de pluk glaswol eruit en laat de Tin korrels zitten vervolgens weer een nieuwe pluk Silica Wool goed aandrukken Anhydrone erop gieten schudden en de dop er weer op doen en de brug weer terugplaatsen Buis h bijvullen met milliQ indien niveau te laag is zie min max markering controleer meterstanden van carri rgassnelheid i moet 110 130 zijn manometers in apparaat en bij de zuurstofcylinder j 200 kPa Indien het glazen buisje 1 voor opvang van condenswater vol zit dan twee van de drie koppelingen losmaken het water in een bekerglas gieten en vervolgens de slangen weer aankoppelen Als de buis m met halogen scrubber soort koperwol donker van kleur is geworden dan deze vervangen niet te snel hij is vrij prijzig Zet de TOC V aan knop voorop deur rechtsonder Kolom wordt 680 C Detector zo n 0 6 C Acclimatisation Voor je kunt beginnen met de analyse van de monsters moet het apparaat eerst geacclimatiseerd
139. tep ballast water treatment system This system should be easy to retrofit for existing vessels and innovative for new builds Beside the ballast water issue the Cathelco R amp D Centre provides also services to the Cathelco Group in respect of testing and improving of existing products This includes but is not limited to antifouling systems for vessels and other marine structures 2 3 Technical Overview of Cathelco s BWTS Cathelco has developed a 100 chemical free BWTS Figure 2 It is based on the combination of filtration UV treatment There are no active substances needed for the treatment of the ballast water or for the cleaning of the UV system The system was designed with emphasis on retrofitting e g installation of the different components as required by different engine room designs However skid mounted systems will also be available which are optimized for foot print 16 NIOZ Land based Project Plan Cathelco s BWTS is a modular ballast water management system The system is installed in bypass to the main ballast line and provides a safe flexible and economical process for the treatment of ballast water and eradication of aquatic invasive species Treatment of ballast water is achieved through a simple and efficient two step process 1 Filtration The ballast water passes through an automatic back flushing filter capable of removing particulates and organisms zooplankton and phytoplankton using a 40 um super mes
140. termine the POC concentration the same filter is combusted overnight at 500 C and allowed to cool in a dessicator and weighed again The POC is calculated from the weight difference between this measurement and the dry TSS weight POC is expressed as mg C L 5 1 3 Dissolved Oxygen DO Fixed samples in Winkler bottles are acidified with H2SO prior to measuring the optical density OD at 456 nm with a spectrophotometer The oxygen concentration is calculated using standards and expressed as uM O L or mg O2 L uM O 0 032 Since both salinity and temperature change over the season the oxygen concentrations is expressed as percentage relative to the natural saturation value for the given temperature and salinity 5 1 4 Dissolved Organic Carbon DOC The DOC concentration is determined in the laboratory by a high temperature combustion method using a Shimadzu TOC Vcpn analyser according to Reinthaler amp Herndl 2005 Standards are prepared with potassium hydrogen phthalate Nacalao Tesque Inc Kioto Japan The mean concentration of triplicate injections of each sample three in total is calculated The average analytical precision of the instrument is lt 3 34 NIOZ Land based Project Plan Table 4 List of variables measured in land based tests at NIOZ Variable unit IMO required Reference Salinity PSU Y SOP Temperature C Y SOP pH i Y SOP TSS mg L Y SOP Particulate Or
141. the five day tank storage on cell viability and concentration In case the treatment is insufficient and residual viable organisms remain present or resting stages or cysts germinate growth of the plankton will be stimulated under favourable conditions This incubation method also allows studies on the effect of the treatment over a period longer than 5 days up to 20 days The experiments usually involve control and treated water that were sampled at T5 the day of discharge They are performed in 10 L flasks in a climate room under optimal growth conditions for the plankton community including irradiance temperature and turbulence A sufficiently high level of nutrients is ensured through the addition of nitrate phosphate and silicate favouring phytoplankton growth and stimulating cyst germination throughout these experiments The standard variables measured include phytoplankton and microzooplankton abundance lt 10um and 10 lt um lt 50 the viability of phytoplankton PAM fluorimetry and the abundance of bacteria Usually the incubation time is seven days In more elaborate experiments samples are taken daily and may include multiple analyses of phytoplankton and heterotrophic bacteria 33 NIOZ Land based Project Plan 5 MEASUREMENT OF VARIABLES A list of all abiotic and biotic variables that are required by G8 is provided in Table 5 1 All variables are measured in samples of test water taken during intake and discharge as required
142. the particles will be analyzed using the software package FCS Express V3 or V4 DeNovo US Cell sizes will be estimated relative to 10 um standard fluorescent beads Flow Check Fluorospheres Beckman Coulter 660539 or relative to 10 and 50 um beads on the Canto II For measuring viable phytoplankton three subsamples will be stained with SYTOX Green Veldhuis et al 2001 This nucleic acid specific dye only stains DNA of cells with a compromised cell membrane Of each phytoplankton cell present the green SYTOX fluorescence Figure 15b will be determined and compared with the green autofluorescent signal Veldhuis et al 2001 Cassoti et al 2005 Peperzak amp Brussaard 2011 Figure 17 Bench top flow cytometer Becton Dickinson Canto II an instrument to enumerate live and dead organisms lt 50 um 38 NIOZ Land based Project Plan nucleus Figure 18 a Epifluorescence microscopic picture of a live phytoplankton cell The red signal is due to the presence of chlorophyll a and b a dead phytoplankton cell with a yellow green fluorescence of the nucleus after staining with SYTOX Green 5 2 3 Counting total heterotrophic bacteria The classical method for counting bacteria in many applications is based on plating on selective media Unfortunately for studies in the aquatic environment this approach is by far insufficient for various reasons Gasol amp Del Giorgio 2000 Therefore the total bacteria concentration in fixed sa
143. the test facility is obliged to measure For abiotic variables the analysis methods stem from decades of NIOZ experience as an oceanographic institute New methods in particular those for biological variables are or will be published in international peer reviewed scientific journals i e subjected to anonymous scientific quality control In addition SOPs are present for specific actions during the BWTS testing such as for adjusting the salinity of the test water flow cytometer data analysis or the cleaning of ballast water tanks SOPs are numbered by year and version Paper copies are kept in the NIOZ test facility archive Electronic copies are stored on a dedicated server that is accessible to Ballast Water Project personnel only SOPs are critically reviewed during the operation of the test facility as well as during annual reviews by the quality manager SOPs relevant to the QAPP can be found in section 3 of this project plan The test protocol QAPP QMP and SOPs are submitted before the start of the BWTS tests to the class societies that were designated by the national authorities These are the BSH Bundesamt f r Seeschifffahrt und Hydrographie or Federal Maritime and Hydrographical Agency Hamburg for Germany and Lloyds Register London Rotterdam for The Netherlands Inspectie Leefomgeving en Transport ILT United Kingdom Maritime and Coastguard Agency MCA and Greece The BSH and Lloyd s Register are notified in advance of the BWT
144. the viable phytoplankton fraction 40 NIOZ Land based Project Plan Figure 20 PAM fluorimetry a fast method to determine bulk phytoplankton biomass and the physiological condition of the photosynthetic apparatus of the cells 5 2 5 3 Counting phytoplankton lt 10 pm Organisms in this size class will be analyzed via flow cytometry as explained for the 10 lt um lt 50 phytoplankton size class 41 NIOZ Land based Project Plan 6 Quality Assurance Quality Control QA QC The Quality Management Program QMP in section 1 of this project plan addresses the quality control management structure and policies of the test facility Sampling and analysis standard operating protocols SOPs contain QA QC measures where applicable The SOPs are listed in section 3 of this project plan 6 1 Ballast water tests For all ballast water test scenarios piping and instrumentation diagrams are available Prior to each test a tool box meeting is held to ensure that the proper procedures are followed during intake and discharge During the ballast water project all samples are taken stored and analysed according to the dedicated SOPs Prior to all tests sample codes are assigned following the rules explained in Table 4 Because the test site is within very short distance of the main NIOZ building all samples containing fresh and live material are immediately transported to the laboratory for direct analysis The sample storage flasks as well as cr
145. the volume at 1 ml e Store the left over at room temperature in a plastic container Reagent B 2 liter NaOH 250 g l KI 350 g l e Weight 500 g NaOH in a 500 ml plastic measure cup e Dissolve bit by bit while mixing constantly with a spoon in 1000 ml MQ in a 2 liter plastic beaker e Add 2 x 350 g KI bit by bit while mixing until dissolved e Fill up to 2 liter with MQ e Filla dispenser bottle with reagent B set the volume at 2 ml e Store the left over at room temperature in a plastic container Reagent C 1 8 liter H2SO4 20 N 10M e Filla 2 liter Erlenmeyer with 800 ml MQ e Place it in a bucket with ice water e Slowly add 1000 ml H2S04 96 end volume 1800 ml e Mix after each addition e Filla dispenser bottle with reagent C set the volume at 1 ml e Store the left over at room temperature in a 2 5 liter brown glass flask Sampling e Remove all air bubbles from the tubing of the dispensers e Each BW tank has a sampling point for small volumes Rinse the tubing of the sample point by opening the valve for some time e Sample in triplicate in 120 ml volume calibrated bottles e Place the sampling tube at the bottom of a 120 ml volume calibrated bottle e Fill the bottle smoothly without air bubbles and overflow for 2 3 volumes 50 NIOZ Land based Project Plan While the water is running remove air bubbles by tapping the side of the bottle with the back of the glass stopper Remove the tube from the bottle while
146. ting e g installation of the different components as required by different engine room designs However skid mounted systems will also be available which are optimized for foot print Cathelco s BWTS is a modular ballast water management system The system is installed in bypass to the main ballast line and provides a safe flexible and economical process for the treatment of ballast water and eradication of aquatic invasive species Treatment of ballast water is achieved through a simple and efficient two step process 1 Filtration The ballast water passes through an automatic back flushing filter capable of removing particulates and organisms zooplankton and phytoplankton using a 40 um super mesh screen The screen is arranged in cone shaped filter candles where the number of filter candles varies with the treatment related capacity TRC of the BWTS During the NIOZ G8 tests to be performed at 200 m h the number of filter candles will be nine The automatic cleaning cycle of the filter is activated by an increased pressure drop across the filter The filter candles are cleaned one after the other without interruption of the filtration process The frequency of the filter back flushes depends on the quality of the water e g the TSS content The concentrate is discharged over board This ensures that the screen is kept clean and the filtration process maintained at maximum efficiency at all times 2 UV disinfection The filtered
147. tion of the photosynthetic efficiency of the phytoplankton community In addition the maximum fluorescence value Fm is an indication of phytoplankton biomass Prior to the measurement the sample is kept in the dark for at least 30 minutes 3 mL of unfiltered sample water control and treated each in triplicate are filled into a glass cuvette and analysed within 2 minutes In the case of a high photosynthetic efficiency of the bulk phytoplankton community samples can be filtered using 50 and 10 um Hydrobios gauze to determine the exact size class of the viable phytoplankton fraction Figure 20 PAM fluorimetry a fast method to determine bulk phytoplankton biomass and the physiological condition of the photosynthetic apparatus of the cells 5 2 5 3 Counting phytoplankton lt 10 um 37 Organisms in this size class will be analyzed via flow cytometry as explained for the 10 lt um lt 50 phytoplankton size class NIOZ Ballast Water Report 38 NIOZ Ballast Water Report 6 1 6 2 6 3 39 QUALITY ASSURANCE QUALITY CONTROL QA QC The Quality Management Program QMP addresses the quality control management structure and policies of the test facility Sampling and analysis standard operating protocols SOPs contain QA QC measures where applicable Ballast water tests For all ballast water test scenarios piping and instrumentation diagrams are available Prior to each test a tool box meeting is held to ensure that the
148. tions are also archived by the project leader of ballast water research Reports on new technologies as well as for bench scale tests to examine prototypes and components of BWTSs are numbered sequentially as NIOZ Ballast Water Report yyyy SE The generic BWTS specific project plan including QAPP QMP SOPs as well as the G8 and G9 reports will also be numbered sequentially as NIOZ Ballast Water Reports These documents will be made publically available on the NIOZ web site COMPUTER HARDWARE AND SOFTWARE The computer hardware that is used in the NIOZ test facility consists of lap tops and personal computers that use Windows based software Both hard and software are maintained by the NIOZ Information and Presentation Centre IPC IPC is also responsible for the security of data and other information for instance by up to date virus protection and daily back ups The NIOZ network can only be accessed by authorised personnel and students In addition all BWTS test data are stored on a dedicated network share that is accessible only to authorised ballast water team members This authorisation needs to be granted by both IPC and one of the project leaders of the ballast water project All software used by the NIOZ test facility including Microsoft Office and dedicated software for data analysis is legally obtained PLANNING Manufacturers of BWTSs that approach the NIOZ facility for testing of their equipment should be awar
149. titute also carries out research based on societal issues The senior scientists at NIOZ all participate in international research projects Several of them also hold a professorship at Dutch or foreign Universities Education The institute educates PhD students and master students of universities and schools for professional education Together with several universities NIOZ also organises courses for PhD students and master students in the marine sciences A number of our senior scientists is also appointed as professor at Dutch and foreign universities NIOZ Ballast Water Report 2 2 12 Marine Technology NIOZ has its own workshops for mechanical instrumental en electronic engineering Here marine research equipment is being designed and built according to the wishes of our individual scientists Facilities NIOZ invites marine scientists from Dutch and foreign institutes and universities to write scientific proposals involving the institute s research vessels laboratories and large research equipment Our ocean going research vessel Pelagia is shared on a European level in the Ocean Facilities Exchange Group www ofeg org The basic scientific disciplines at NIOZ are physics chemistry biology and geology Multidisciplinary sea research is regarded as one of the main strengths of the institute Therefore the research is organised in 5 multi disciplinary themes Open ocean processes Sea floor dynamics Wadden and shelf
150. ton e Store the Greiner tube 30 minutes at room temperature PAM ANALYSIS e Measure phytoplankton viability with the PAM separate SOP e Measure bacteria and phytoplankton after thawing of samples with the flow cytometer separate SOP e Measure the microzooplankton concentration with an inverted microscope separate SOP 71 NIOZ Land based Project Plan REMARKS e see separate SOPs for PAM and the measurement of bacteria phytoplankton and microzooplankton e not all samples will be analysed this will depend on the microzooplankton concentration on discharge 72 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 09 06 AUTHOR Josje Snoek Bacteria PicoGreen count FCM 2012 1 Goal Determination the concentration of heterotrophic bacteria in ballast water samples MATERIAL amp EQUIPMENT e Nalgene Square bottles with 1 litre ballast water sample e Flowcytometer BD Canto II e FCM tubes for BD CANTO e PicoGreen 100 uL commercial stock in 400 uL TE buffer SAMPLING e Samples are fixed with formaldehyde and stored at 80 C e After thawing add 1 mL sample to 10 ul diluted PicoGreen Incubate for 10 30 minutes in the dark at room temperature e Put the tubes in the carousel of the FACS CANTO and start the analysis e Start a new series of 9 samples when the first 3 samples have been measured and repeat this until all samples have be
151. top Write down p peak x flat line uncertainty Write down the FO Fm and Fv Fm ratio Closing down the PAM Rinse the PAM cuvette with milli Q water Close the window WinControl Report The following message appears WinControl You will loose report chart and kinetics data by quitting WinControl unless they were saved in csv txt pcf and pkf files Really leave Press OK Switch off the laptop Switch off the PAM Control REMARKS The PHYTO ED system is best suited for field and on deck work The system is particularly recommended for work with open ocean water at chlorophyll contents below 0 5 ug l from Brochure Walz 04 2004 The Universal Control Unit of the PAM has serial number UKEA0117 The Water ED Emitter Detector Unit Red version has EDEE0196 67 NIOZ Land based Project Plan dp N 10Z Royal Netherlands Institute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 09 06 AUTHOR Josje Snoek Phytoplankton vitality SYTOX FCM 2012 1 Goal Determination of the vitality of phytoplankton in Ballast Water samples MATERIAL amp EQUIPMENT e Nalgene Square bottles with 1 liter BW sample e Flowcytometer BD Canto II e FCM tubes for BD CANTO e Sytox green SAMPLING e After Phytoplankton FCM measurements samples are taken from the same bottles Mix carefully and pipet 1 ml into a FCM tube add 10pl Sytox green to the wall of the tube When 6 tubes are filled tap with the bottom
152. tural integrity disturbed Sampling with 50um net Stained and Movement or Transfer of organisms into filtered water amp transport to lab ae Adjustment of volume to 100 200 ml and addition of stain in a 1 50 000 ratio Staining time 1 hrs Unstained or light colour and or no movement Physical manipulation with dissection needle or observation at higher magnificaton Filter stained sample over 30 um sieve No movement Transfer of organisms into Bogorov dish with filtered Alive Viable unstained water 2 ificati Gount at 20x magnification Retain filtered water for further analysis of organisms lt 50um Figure 16 Sampling and viability assessment for organisms larger than 50 um during land based tests 5 2 2 Counting organisms 10 lt um lt 50 5 2 2 1 Counting viable microzooplankton The sample bottles are transferred to the laboratory and left undisturbed in the dark for all organisms and material to settle After at least 24 hours the full bottle weight is measured Using a water jet pump and specialized tubing the supernatant is aspirated The bottle is weighed once more The concentrate is transferred to small storage bottles that can be stored refrigerated for one year The bottle is weighed once more to determine the sample and concentrate volumes The samples are analyzed with an inverted micro
153. ty Wadden Sea water flows towards the North Sea The second set of high salinity tests is performed in late spring or early summer Test water is pumped from the harbour at high tide when relatively saline North Sea water flows towards the Wadden Sea After consultation with the BSH the Cathelco BWTS tests carried out in spring and early summer of 2012 were combined to one salinity saline range This meant that an additional series of freshwater tests had to be performed The first appropriate months for these freshwater tests were September and October NIOZ Ballast Water Report 22 NIOZ Ballast Water Report 4 1 23 TEST WATER QUALITY SAMPLING STORING Abiotic quality The land based test cycles have to be carried out at specific water qualities as defined in the G8 guidelines The NIOZ harbour represents a brackish water environment with a varying salinity 20 35 PSU High salinity water originating from the North Sea is taken in around high tide Low salinity water from the Wadden Sea is taken in around low tide The salinity of the Wadden Sea water is dependent on the discharge of fresh water from Lake IJssel which itself depends on the amount of rainfall and on the flow rates in the rivers Rhine and IJssel In an effort to maintain a minimum 10 PSU salinity difference as requested under 2 3 17 of G8 per tank 15m fresh water is added in the pipelines to the natural water prior to the pump to reduce the ambient salinity ca
154. uipment and chemicals The financial management assistant to the test facility ensures that all procedures are as required by internal NIOZ rules and by Dutch law Material and equipment for the test installations is procured by Mr A Smit of Smittech Den Hoorn The Netherlands In all cases of procurement the quality costs and supplier reputation are evaluated by research staff or Mr A Smit in collaboration with the project leader of Ballast Water research or in his absence the quality manager DOCUMENTS AND RECORDS Documents related to the NIOZ test facility and BWTS testing include legal and financial contracts BWTS specific project plans including QAPP QMP SOPs North Sea Ballast Water Opportunity NSBWO documents notebooks data and custody forms student reports internal reports on for instance inter comparison workshops and scientific publications Legal and financial contracts are archived as required by NIOZ under the responsibility of the financial management assistant and the overall project manager North Sea Ballast Water Opportunity NSBWO documents are maintained by the general and financial management assistants or by the project leaders Notebooks data and custody forms will be archived for at least five years by the project leader of ballast water research BWTS specific NIOZ Ballast Water Report 2012 9 19 project plans including QAPP QMP SOPs student reports internal reports and scientific publica
155. um and are carefully flushed from the net beaker 57 NIOZ Land based Project Plan into a 2 Liter bottle Until further transport store the bottles away from direct sunlight at a temperature comparable to the sample water Laboratory The bottles are transferred back to the lab as soon as practical Samples are flushed into beaker glasses and the volume is adjusted to any convenient manifold of 50ml and at least 150 ml to assure that the sample is not too concentrated for the organisms 2ml Neutral Red is added per 50 ml sample Stain for a minimum of 2 hours without direct sunlight and at a comparable temperature as the sample temperature After staining the sample is filtered over a 30 um mesh net and carefully flushed with 0 2um filtered seawater into a Bogorov or Bogorov Gollasch counting dish Analysis Samples are counted complete no subsampling Subsamples are counted by at least two analysts and the individual counts are recorded Total amount of living organisms is counted and recorded on the plankton form Live dead determination is resulting from staining structure and movement of the organism At minimum the phyla and abundance of the organisms present in the Bogorov counting dish is determined and recorded on the plankton form For more information see the NIOZ mesozooplankton booklet in laboratory F00 09 At the start of the BWTS test season subsamples of 3 or more old samples are counted by three analysts the
156. undance of heterotrophic as well as autotrophic bacteria can also be estimated The vitality of the 37 NIOZ Land based Project Plan different organisms present will be addressed by using a specific fluorescent dye method Veldhuis et al 2001 Cassoti et al 2005 Veldhuis et al 2006 Peperzak amp Brussaard 2011 In addition the viability will be assessed by incubating discharge water see 8 4 5 5 The diversity of the phytoplankton is measured by microscopy of Lugol fixed samples of the NIOZ test water by Koeman amp Bijkerk The Netherlands This company is actively involved in international quality assessments http www planktonforum eu Three replicate samples from both control and treated water are placed in the carousel of a bench top flow cytometer Beckman Coulter XL MCL or Becton Dickson Canto II of which the Canto flow cytometer has an elaborate quality control system All procedures and handling are conducted according to standard procedures e g Shapiro 2003 Samples will be counted using standard protocols covering the particles in the size range of ca 2 to 50 um Total analysis time will be equal to an exact sampling volume of 1 mL or otherwise when relevant Of all particles present in the volume counted the cell size and the presence or absence of chlorophyll a fluorescence will be measured Only phytoplankton has chlorophyll a fluorescence Figure 18a b Absolute numbers cell sizes and chlorophyll a content of
157. urement and processing of samples for the basic variables is ensured by the NIOZ harbour laboratory on the Navicula and Pelagia quays of the test facility Figure 11 A number of measurements is performed after sampling e g temperature salinity and pH while the other samples DOC POC TSS DO is processed for later analysis DOC samples are filtered through GF C filters and sealed in pre combusted glass ampoules or special screw cap bottles after adding hydrochloric acid HCI Sealed ampoules or bottles are stored at 4 C until analysis For TSS POC measurements pre weighed glass fibre filters GF C are used Each filter is coded and stored individually in a Petri dish The filtered water volume is not constant but depends on the particle concentration of the test water The higher the particle concentration in the sample the smaller the volume that can be filtered before clogging Practical volumes are 100 1000 mL per sample After filtration the filter is rinsed with demineralised water to remove sea salts The filter is then put back in its original Petri dish and stored temporarily in a freezer at 20 C or long term at 80 C until further analysis Glass Winkler bottles for oxygen DO are flushed at least three times their volume ca 120 mL with test or control water Great care is taken to avoid gas bubbles on the wall of the bottle or excessive mixing of air and water Next MnClz and NaOH solutions are added to the bottle just be
158. uring Light Actinic light Clock Freq 5 Int 6 Time 00 20 Width 00 10 Itemi SAT Pulse System Parameters F Off 4 Saturation Pulse Set PM Gain 10 15 20 Int 10 x Beeper Out Gain 2 Width 0 8 Mark A ETR F 0 84 LED Amplitudes Far Red Light Meas 8 FR Int 6 Actinic 12 FR Width 00 10 Status X Meas Light X Photo Multipl Blanc e Fill the PAM cuvette with filtered seawater e Place the cover over the PAM cuvette e Wait 1 min for the Ft signal to become stable e Check whether the Ft signal is zero If this is not the case press the button left from the Ft signal top of the screen right Write down the F Off s values for the three gains 10 15 and 20 e If you change the gain you have to change the F Off in the corresponding value e Sample e Fill the cuvette with sample e Wait 1 min before measuring the Ft signal Note The measurement is not reliable when the Ft value is below the 100 r e and above 1000 r e In this case you should increase respectively decrease the PM Gain versterkingsfactor photomultiplier Write the PM Gain down 10 15 or 20 66 NIOZ Land based Project Plan e Press the FO button e Check for a straight horizontal line on the small graph on the bottom of the screen right If not fill the cuvette with fresh sample and start again e Press the Fm button as soon as you see the FO value written on the computer screen e Check for a signal in the small graph on the bottom of the screen with a flat
159. ute for Sea Research b STANDARD OPERATING PROCEDURE DATE 2012 02 24 AUTHOR Anna Noordeloos FCM Canto operation 2012 1 Goal To operate and calibrate the Canto flow cytometer MATERIAL amp EQUIPMENT e BD Canto II Flowcytometer Starting up Switch the machine on with the green button on the left side of the flowcytometer It takes the machine 5 7 minutes to warm up e Switch on the computer The window Log On to Windows appears Username Administrator The password is BDIS gt OK e Click twice on the icon BD FACSDiva Software e Wait until the text The system is ready appears in the middle of the screen in the Cytometer window e Cytometer gt Fluidics Startup gt OK e During Fluidics Startup you need to get the air out of all the fluidic filters on the wet car You do this by turning the white knob on top of the filter anticlockwise while the liquid is flowing through Keep the white knob open till liquid is coming out e Window Startup Status appears Fluidics Startup is complete The system is ready gt OK e Open the instrument and check the flowcel for air bubbles In case of air bubbles Cytometer gt Cleaning Modes gt De gas flow cell e Window De gas Flow Cell is complete gt OK Cytometer Setup amp Tracking beads CST Beads BD Cytometer Setup amp Tracking Beads e Mixture of 2 and 3 um beads with different intensities e Type Lot ID e Check met beads dagelijks uitvoeren duur
160. veral books may be issued during the entire test period These books remain at NIOZ in the appropriate dossier Test season time planning The intermediate and high salinity range test season at the NIOZ harbour is restricted to spring and summer In this period of the year sufficiently high numbers of organisms are naturally present in the North and Wadden Sea At NIOZ the test water is not enriched with organisms neither artificially cultured nor collected at sea In general early spring sea water has a lower salinity then in summer due a decrease in river discharge A decrease in wind speed during spring will lead to diminishing concentrations of total suspended solids TSS and therefore sediment from the nearby Mokbaai Figure 1 is added to increase TSS to the required value of 50 mg l for brackish water The freshwater test season according to IMARES data begins in March and probably ends in November December In February sampling and measurements in the harbour start in order to monitor the start of the spring plankton bloom In March the first G8 tests may be performed depending on natural circumstances such as water temperature and underwater light climate that affect the plankton development The first set of tests is carried out at the intermediate salinity range of G8 2 3 17 because in early spring the freshwater content of the Wadden Sea is relatively high Test water is pumped from the harbour at low tide when low salini
161. water for BWTS testing at NIOZ will be collected The freshwater test water is transported over a relatively short distance across the Marsdiep tidal inlet of the Wadden Sea within 12 hours by ship to the NIOZ harbour Figure 8 The day following intake at Den Helder i e within 24 hours the test water is available in the NIOZ harbour and ready to be pumped into the NIOZ harbour installation 22 NIOZ Land based Project Plan NIOZ harbour Freshwater Intake site Figure 8 The test water will be transported to the NIOZ harbour over a distance of 6 5 km in less than 12 hours The pump to be used is an Amarex KRTK 100 401 354WG S that will be suspended by a crane from the NIOZ Pelagia quay Figure 9 The crane will be able to steadily position the pump because the freshwater surface level will change according to deballasting and tide This pump will be connected to the NIOZ installation Figure 6 by a flexible hose 23 NIOZ Land based Project Plan Figure 9 The Amarex pump to be used in pumping freshwater from a boat into the NIOZ installation 3 3 BWTS test set up treatment and control tanks A typical test of a treatment system is performed with two treatment tanks and one control tank that are filled in rapid succession i e on the same day at approximately the same time in the tidal cycle Figure 6 The control tank with untreated water serves as reference to examine the effect of the treatment including holding for
162. y to obtain a 10 unit salinity difference either brine or fresh water may be added natural mud is added to increase TSS as needed this also increases POC Biological quality In order to establish the biological efficacy of the BWTS it should be tested with water containing a high concentration of organisms as well as a sufficient biodiversity 2 3 20 of G8 This is required by G8 to guarantee the effectiveness of the BWTS in different ecosystems across the globe The variety of organisms in the influent test water should be documented according to the size classes mentioned in Table 2 Natural water originating from the coastal zone of the North Sea high tide and the inner Western Wadden Sea low tide will be used The test period will cover the whole spring and early summer of the plankton growth season and therefore includes the natural occurring biodiversity and species succession The ambient plankton content in terms of species diversity in the relevant size classes is very high For instance in 2011 16 phyla and more than 70 species were detected during the test season Table 3 Only 5 species and 3 phyla are required 2 3 20 of G8 Table 2 Minimal numbers and species diversity required at intake for different size classes and groups of organisms 1 um 1 micron 0 001 mm Intake test water Variety at least 5 species from at least 3 250 um different phyla divisions at least 5 species from at least 3 210 and l
163. yovial boxes are labelled with the same coloured labels and codes Samples that are fixed for long term storage are stored in specifically designated refrigerators 4 C and freezers 20 C 80 C The samples for microbiological analysis of the presence and number of human pathogens will be taken in special bottles of 300 and 600 mL and sent to Eurofins C mark in Heerenveen accreditation certificate RvA lab no L043 The samples will be transported immediately after sampling using a cooled transport container 4 C 6 2 Laboratory analyses The analyses of abiotic and biological variables are described in general in other parts of this project plan Detailed descriptions of each analysis are available These Standard Operating Procedures SOPs of sampling sample storage sample analyses data analyses and data management are part of the NIOZ Ballast Water QMP Specific quality assurance and quality control measures are contained in each SOP 6 3 Data analysis The sample codes assigned a priori to the harbour tests Table 4 are also used in data handling i e the transfer of data from laboratory instruments to Excel files dedicated to specific analyses All data files are collected on a NIOZ network disk that is backed up at least once a day The separate data files are combined in one Excel file in which all appropriate calculations for D 2 and G8 will be conducted The data on the NIOZ network disk are accessible to aut
164. ysis 50 gm BN 176263 e Silica Quarz wool fine Elemental Microanalysis 50 gm 502 177 B1102 e Deep Seawater Reference CRM 44 46 uM C amp 30 ml Hansell Laboratory University of Miami Batch 11 lot 12 11 e Stock solution 1000 ppm C 2 125 g of reagent grade potassium hydrogen phthalate that was previously dried at 105 120 C for about 1 hour and cooled in a desiccators before transferred in a 1 L volumetric flask and dissolved in milliQ water 1000 ppm C 100 mg C L This stock solution is necessary for making reference concentrations of 0 25 50 100 and 200 uM C e Hydrochloric Acid 37 38 2 5 max 5 ppb Hg Baker Analyzed J T Baker UN1789 e 0 1 M HCI solution e Anhydrone Magnesium perchlorate desiccant For Drying Baker Analyzed L T Baker Cas No 10034 81 8 Lot B27416 e MilliQ water Procedure DOC The TOC V instrument measures the amount of total carbon TC total inorganic carbon IC and total organic carbon TOC in water Oxidative combustion infrared analyses is a widely used TOC measurement method The procedures related to TOC Control V software system ANALYSIS Dutch Ongeveer wekelijks na 120 ml aan injecties 100 ul per injectie ca 5 tot 7 inj per monster moet de glazen kolom met katalysator vervangen worden onder aan de kolom het T stuk losdraaien a bovenaan de twee metalen schroeven links van inj poort losdraaien b rechts van inj poort de schroefverbinding

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