The mismeasure of biological efficacy in ballast water treatment testing

Transcription

1 The mismeasure of biological efficacy in ballast water treatment testing Nick Welschmeyer, Moss Landing Marine Laboratories, CA (CSU) and Golden Bear Facility, Cal Maritime (CSU) Presented: September 28, 2016; Miami, BWM-Tech 1

2 1. Let s see how organism counting fits with our expectations. 2. Let s see how ballast treatment success compares with other applications in pollution* and public health *Bloomberg Report 2013;

3 Conception 1: We can count live/active organisms accurately/precisely Coun ng Comparison: Microscope vs. Flow Cytometer Flow Cytometer log(beads/ml) y = x R² = Microscope log(beads/ml) Fantastic: Humans = Machines!! 3

4 Let s count live um organisms with the microscope and flow cytometer during full-scale ballast treatment testing What could possibly go wrong? 4

5 Real-world ballast tests: Cytometer vs. Microscope What a Mess!! Live Phytoplankton, um (Log (cells/ml) Epifluorescence microscope, FDA y = x R² = N = 142 y = x R² = Microscope log(beads/ml) Live Phytoplankton, um (Log (cells/ml): Flow cytometer, FDA Flow Cytometer log(beads/ml) Coun ng Comparison: Microscope vs. Flow Cytometer 5

6 Live Phytoplankton, um (Log (cells/ml) Epifluorescence microscope, FDA y = x R² = N = Live Phytoplankton, um (Log (cells/ml): N = 76 Flow cytometer, FDA 6

7 Logarithmic plot of the same data shown previously Live Phytoplankton, um (Log (cells/ml) Epifluorescence microscope, FDA y = x R² = Treatment Uptake Live Phytoplankton, um (Log (cells/ml): Flow cytometer, FDA 7

8 Live Phytoplankton, um (Log (cells/ml) Epifluorescence microscope, FDA Treatment No Man s Land Uptake Treatment Uptake Live Phytoplankton, um (Log (cells/ml): Flow cytometer, FDA 8

9 Do we provide an accurate assessment of Numerical CHALLENGE in um counting? um 9

10 Conception 2. Natural organism concentrations are not challenging enough Analyze beads of known diameters to generate a calibration curve Phytoplankton show red fluorescence due to chlorophyll content These particles exhibit low red fluorescence and are considered noise (detritus, inorganic particles)

11 10-50 um All active (live) phytoplantkon (10-50 um) N = 174,373 cells All active (live) phytoplantkon (1-50 um) N = 1,422,447 cells Count Ratio (1-50 um)/(10-50 um) = 8.2x 11

12 The outcome of 1) Whole-Water and 2) Size-fractionated (10-50 um) MPN assays (UV) Uptake Treatment Discharge 12

13 The outcome of 1) Whole-Water and 2) Size-fractionated (10-50 um) MPN assays (UV): Numeric Challenge and Biological Efficacy are significantly larger than we think Uptake Biological Efficacy (Uptake/Discharge) Treatment Discharge Whole Water MPN concentrations Whole Water MPN concentrations Sample Type Live cells/ml Sample Type Live cells/ml KLB1_UU KLB1_UU x KLB1_DT KLB1_DT KLB1_UU KLB2_UU KLB2_UU KLB2_UU X = 4267 KLB1_DT KLB2-DT KLB2-DT KLB2-DT X = Size-fractionated MPN concentrations Size-fractionated MPN concentrations Sample Type Live cells/ml KLB1_UU x Sample Type Live cells/ml KLB1_DT KLB1_UU2 800 KLB1_UU3 140 KLB2_UU1 600 KLB2_UU2 240 KLB2_UU3 140 X = 360 KLB1_DT KLB1_DT KLB2-DT KLB2-DT KLB2-DT X =

14 We just saw how two independent counting methods (microscope vs. cytometer) compared when using the same Living/Viable method (FDA). Now how about a comparison of two independent methods, operating under two Independent working-principles - but attempting to count the same thing: MPN vs. FDA

15 Fluorescein diacetate (FDA; colorless) Fluorescein O O O + H 2 0 Esterase-like enzymes (living metabolism) O OH O O O CH 3 HO O O H 3 C O Rotman and Papermaster (CH 3 COOH)

16 The FDA false posi ve : Abio c Produc on of Fluorescein (no organisms; no enzymes; no life ; 5 mg FDA in each tube) o ph 7 ph μm filtered Milli-Q H 2 0 ph 7; Δt=10 min 0.2 μm filtered Milli-Q H 2 0 ph 12; Δt=10 min (40 mm NaOH) Fig. 1: Abio c flur escein produc on resul ng from bulk FDA incuba ons (10 um FDA final conc.) using sterile, filtered seawater (FSW; 30 PSU) as the incuba on fluid (no organisms added). Non-acidified (ph 8) and acidified (ph 7 and ph 6) condi ons are shown. 30 min incuba on.! Fig. 2. Aqueous fluorescein standards (held constant at 5 ppb) a er ph adjustments with NaOH and/or HCl. Ex: 489 nm/em :515 nm.!

17 FDA Cytometer [log(cells/ml)] Fig. 4: This is Fig. 3C extrapoalated to x = 0 (e.g., 1 cell/ml) y = x R² = Assumed Predicted (Fig. 3C) MPN FDA cells/ml Cells/mL Figure 3. Summary histograms showing the comparison of FDA-based numeric concentra ons to MPN-based 6 numeric 30.4 concentra ons. Data from Welschmeyer and Maurer (2014). The ra o of the two procedural results was computed as: log[fda 8 (live cells/ ml) / MPN 3 (live 3.5 cells/ml)]. 4 This 9 ra o formed 40.3 the bin limits on the x-axis of the 10frequency 43.3 MPN [log (cells/ml)] histograms given in A and B. The sca erplot in C was taken from data in Figure 3(B) for the bins 0.5 to 0.5; this bin range marks the collec on of data showing the best rela onship for FDA and MPN determina ons of numeric live counts; marked by ver cal do ed line.

18 WHY? 1. MPN lower limit of detection 2. DOC POC Land-based augmentation Figure 3. Summary histograms showing the comparison of FDA-based numeric concentra ons to MPN-based numeric concentra ons. Data from Welschmeyer and Maurer (2014). The ra o of the two procedural results was computed as: log[fda (live cells/ ml) / MPN (live cells/ml)]. This ra o formed the bin limits on the x-axis of the frequency histograms given in A and B. The sca erplot in C was taken from data in Figure 3(B) for the bins 0.5 to 0.5; this bin range marks the collec on of data showing the best rela onship for FDA and MPN determina ons of numeric live counts; marked by ver cal do ed line.

19 2 Minutes of Biological and Chemical Oceanography

20 Refractory Semi-refractory Semi-labile 6000 years old 4000 years old 300 years old (= 0.8 mg C/L) Hansell, D. A., and C. A. Carlson (2015), Dissolved organic matter in the ocean carbon cycle, Eos, 96, doi: /2015eo

21 Effects of DOC augmentation in Land-based Testing The resultant bacterial production rate exceeds the highest measured values in Chesapeake Bay by up to 10x DOC consump on per 24h (augmenta on w/ lignin sulfonate) Dissolved Organic Carbon (mg/l) Uptake Control Discharge

22 Unaugmented: coccoid Augmented: rod-shaped

23 Examples of bacteria colonization of large particles 48 h after adding lignin sulfonate at nominal GBF land-based levels (6 L lignin sulfonate/300 m^3). No particle colonization was noticed at T-zero, before augmentation. Magnification was 500x in photos below; Sedgewick-Rafter counting of um protists is typically lower (e.g., 40x 100x). Size bar = 10 um.

24 100x 40x Magnification = 500x 500x magnification

25 Biological efficacy does not obey the CHALLENGE Concept in Ballast Water Testing Biological Efficacy as a Func on of CHALLENGE Concentra on Phytoplankton (10-50 um) Biological Efficacy (Challenge/Discharge) N = um Live Phytoplankton (FDA) CHALLENGE Concentra on (Cells/mL) Biological Efficacy as a Func on of CHALLENGE Concentra on Zooplankton (>50 um) Biological Efficacy (Challenge/Discharge) N = 110 >50 um Live Zooplankton Uptake Concentra on (organisms/m3) 25

26 1. Let s see how organism counting fits with our expectations. 2. Let s see how ballast treatment success compares with other applications in pollution* and public health *Bloomberg Report 2013;

27 Environmental Successes: 1. Visible reductions in Los Angeles smog Then Now

28 Environmental Successes: 1. Visible reductions in Los Angeles smog How? Regulatory emissions control Roughly 10x reduction in pollution emissions, even with modern 3-way converters

29 Environmental Successes: 2. Reduction in Acid Rain Death to acid-intolerant forests The Clean Air Act 1970 Stack-gas scrubbers: Roughly 5x 20x reductions in SO 2 and NO x

30 Environmental Successes: 3. Reduction of the Antarctic Ozone Hole The Montreal Agreement (1987) Ozone = 300 DU Ozone = 100 DU Roughly 10x reduction in Fluorocarbons, over 30 years!! Ozone = 93 DU Ozone = 126 DU

31 Successes in Disease Elimination/Eradication: Vaccination programs against organismic invaders Progress to date against diseases for which vaccines already exist and deaths from diseases for which vaccines might be developed Annual deaths (all ages) if no immunization Prevented Occurring % prevented Smallpox 5.0 million 5.0 million Diphtheria 260, ,000 37, Whooping cough 990, , , Measles 2.7 million 1.6 million 1.1 million 60 Neonatal tetanus 1.2 million 0.7 million 0.5 million 58 Hepatitis B 1.2 million 0.4 million 0.8 million 33 Tuberculosis 3.2 million 0.2 million 3.0 million 6 Polio (cases of lifelong paralysis) 640, ,000 90, Malaria/other parasitic infections 2.2 million million 0 HIV/sexually transmitted diseases 1.3 million million 0 Diarrhoea/enteric fevers* 3.0 million million 0 Acute respiratory infections 3.7 million million 0 Reduction: 7.0x 2.8x 2.5x 2.4x 1.5x 7.1x NOTE Figures for the number of deaths that would occur in the absence of immunization are generally calculated by taking the known mortality rate of each disease in the unvaccinated and applying it to the total

32 Deaths Malaria Deaths by Region (WHO: Africa) x Year Deaths Malaria Deaths by Region (WHO: Americas ) x Year Deaths Malaria Deaths by Region (WHO: SE Asia) 1.6x Year Deaths Malaria Deaths by Region (WHO: West Pacific) x Year

33 60,000 Polio 50,000 Cases per year 40,000 30,000 20,000 10, x Recall the Challenge of Land-based Ballast Water Tests: >50 um Challenge = 100,000/m 3 Cases per Year Year Guinea Worm Cases per Year x Year Ballast Water Discharge Std. = 10/m3 A 10,000x reduction (in one day!) A 0.01% contamination will cause a FAIL

34 Three of the greatest environmental successes in modern history 1. Reduction in smog derived from automobiles 2. Reduction of acid rain 3. Shrinkage of the ozone hole were accomplished with reductions in the respective putative pollutants that were approximately 10x.

35 Biological efficacy does not obey the CHALLENGE Concept in Ballast Water Testing Biological Efficacy as a Func on of CHALLENGE Concentra on Phytoplankton (10-50 um) Biological Efficacy (Challenge/Discharge) um Live Phytoplankton (FDA) CHALLENGE Concentra on (Cells/mL) Biological Efficacy as a Func on of CHALLENGE Concentra on Zooplankton (>50 um) Biological Efficacy (Challenge/Discharge) >50 um Live Zooplankton Uptake Concentra on (organisms/m3) 35

36 Done! 36

37 Done! 37

38 Conception 1: We can count live/active organisms accurately/precisely Let s start simply with plastic calibration beads (15 um dia.) - no growth - no death - no shape variation (uniform spheres) Numeric Calibra on: Standards vs. Microscope Microscope Concentra on (beads/ml) y = x R² = Beautiful! Standard Concentra on (beads/ml) 38

39 Conception 1: We can count live/active organisms accurately/precisely Same data Expressed as log concentration Two independent counters WE (HUMANS) CAN COUNT!! 39

40 Comparing 10-50um size class organisms across locations Location Mean Volume (um 3 ) Equivalent Diameter (um) n Seattle, WA 2, Vallejo, CA 3, San Francisco, CA 3, Denmark 3, Moss Landing, CA 7, Port Angeles, WA 8,

41 Public Health Successes: Vaccination programs Only one successful eradication Small Pox 7 diseases under control for elimination

42 Conclusions: CONCEPTION 1: We can count accurately/precisely? Well yes we can, for perfectly shaped, inert plastic beads but real organisms present a significant incresase in variabilty CONCEPTION 2: Natural organism concentrations are not challenging enough? Actually, for phytoplankton, the true numerical challenge concentration is about 10x higher than for the um regulated size class. CONCEPTION 3: The concept of challlenge is a well-substantiated principle in ballast water treatment testing? Actually, we have no data to substantiate that conclusion. Our results are opposite to common logic. CONCEPTION 4: Ballast water treatment efficacy is NOT stringent enough. Actually, the current biological efficacy of ballast water treatment outpaces the well-documented environmental success stories by 2-4 orders of magnitude. A 1,000,000x reduction in zooplankton concentrations is not unusual? WE MIGHT BE DOING A LOT BETTER THEN YOU THINK!! 42

43 CHALLENGE in Ballast Water Treatment Testing: Conceptions and Misconceptions Nick Welschmeyer, Moss Landing Marine Laboratories, CA (CSU) Presented: Feb 2, 2016; ETV Tech Panel, Baltimore MD?? 43

44 Microscope Issues: Counting um What could possibly go wrong? Hey Dad. What is the minimum dimension of an organism shaped like the Eiffel Tower?? 44

45 Flow Cytometer issues: Individuals vs. entities Will it see 5 cells in a chain, or just one entity? Thalassiosira Cylinodrotheca Cerataulina Asterionellopsis Will one cell produce two pulses? Scan profiles from G. Dubelaar 45

46 Treatment Discharge Live Cells/mL 3. The CHALLENGE Concept: Higher uptake concentrations yield a more Challenging test ?? A Misconception?? Flow Cytometry (FDA): Phytoplankton um Uptake vs.treatment Discharge N = um Live Phytoplankton (FDA) Uptake Live Cells/mL Zooplankton Uptake vs. Discharge 35 Discharge Concentra on (organisms/m3) N = Uptake Concentra on (organisms/m3) >50 um Live Zooplankton 46

47 Conception 3: Is the concept of CHALLENGE meaningful in Ballast Water Treatment Testing? The CHALLENGE concept: as the concentration of challenge organisms increases, the biological efficacy of ballast water treatment systems will decrease. 47