Healthy marine water: Is dilution a solution?

1) The need or issue

2) How is innovation deployed?

3) What does the near future require?

Toxicology Factors

1. Chemistry (concentration)

2. Biology (harmful effects)

3. Environment (presence + severity)

Chemistry + Biology = Toxicity (most references are based on lab work)

Chemistry + Biology + Environment = Ecotoxicology (ambient situations)

Chemistry = anything at high enough levels is harmful (toxic)

Analytical Chemistry always required (to discriminate a specific compound)

Biological live species tests (bioassays):

1. Endpoint test (dilution, compliance for permitting)

2. Screening test (easiest, most common current need)

Tolerance and Toxicology

Larvae

Mammals

Birds

Fish

Amphibians

Invertebrates

Plants

Co

nce

ntr

atio

n

Time (Exposure)

Biological Communities

Tolerance and Toxicology

Larvae

Mammals

Birds

Fish

Amphibians

Invertebrates

Plants

Time (Exposure)

Biological Communities C

ost

Co

st

Anthropogenic intrusions on biota are forcing toxicity testing sooner, faster, less cost.

Challenges to healthy marine water: The “Carbon Footprint” of water is Chemicals

1) Naturally occurring sources (small adverse effects)

2) Anthropogenic (people are the source):

Effluents (pipes with mixtures)

Runoff (soluble or not, things flow downhill)

3) Maritime Industrial/Anthropogenic

Leaching

Dredging

Ballast water ( 7-9 billion gallons annually)

= TMDL (Total Maximum Daily Load)

The “Information Gap”- 7 Current Challenges

1) Complex solutions of water or mixtures

2) Need results in 24 hours or less

3) Many samples to evaluate

4) Is the sample “Normal” or meets expectations?

5) Operations are held up pending results or data

6) No laboratory is readily available

7) Unskilled personnel usually closest to the water

Innovative Technology: Biosensor

Bioluminescent glow from diver at night

Lig

ht

Contaminants

Inorganic concentration (+ or -)

Metals concentration (+ or -)

Organic concentration (+ or -)

organic

inorganic

metals

StressStressStressStress

• 270 million years of biological performance

• The plankton emit bright light when disturbed by wave action or other agitation

• Approximately 10 years of test-measurements by U.S. Navy

• > $2.5M in R&D

Contaminants

Bioluminescence

IC50%

Surface craft

Boat’s wake

This property is inhibited in a linear fashion when contamination (of all types) is at elevated concentrations.

Technology Description: Biosensor Advantages

Bioluminescent glow from diver at night

Surface craft

Inorganic concentration (+ or -)

Metals concentration (+ or -)

Organic concentration (+ or -)

Nitrates

Zinc

Selenium

CopperThallium

Calcium

Bromine

Gallium

Beryllium

Aluminum

Iron

Magnesium

Iodine

Nickel

Sulfur

Arsenic

Cadmium

Phosphates

Etc….

• The presence of ANYTHING at a level which is harmful to the environment.

Lig

ht

Contaminants

Contaminants

Bioluminescence

IC50%

• The SEVERITY of the situation, if unchanged.

• Cannot specify a contaminant, literally thousands of combinations.

• (Bacteria does not affect the organism)

Biosensor: Why it Works

Cellular concentrations

Inorganic concentration (+ or -)

Metals concentration (+ or -)

Organic concentration (+ or -)

organic

inorganic

metals+

+

+

++

+

+

+++ +

++

+ +

+

++++

+

+++++

+

CellCell EnvironmentEnvironment

Contaminants(elevated levels)

Contaminants

Bioluminescence

StressStressStressStress

Significance and Use

Protection of aquatic species requires prevention of unacceptable effects on populations in natural habitats. Toxicity tests are conducted to provide data to predict what changes in viable numbers of individual species might result from similar exposure in the natural habit. Information might also be obtained on the effects of the material on the health of other species. Bioluminescent dinoflagellates represent an important eucaryotic group which are widely distributed in the oceanic environment.

1. Scope1.1 This guide covers two distinct procedures, based on similar principles, for obtaining data concerning the adverse effects of a test material (added to dilution water) on oceanic bioluminescent dinoflagellates.

1.1.1 The endpoint for both procedures is based on a measurable reduction or inhibition in light output from the dinoflagellates . Both procedures are similar in that when bioluminescent dinoflagellates are exposed to toxicants, a measurable reduction in bioluminescence is observed from their cells following mechanical stimulation when compared to control cells. In the first procedure, cells of the bioluminescent dinoflagellate Gonyaulax polyedra can be tested over a range of up to seven days of exposure (or longer) to a toxicant. The second procedure uses another species, Pyrocystis lunula, for a 4 h test.

1.2 Both procedures can measure the toxic effects of many chemicals, various marine and freshwater effluents, antifouling coatings, leachates, and sediments to bioluminescent dinoflagellates (1-5). Compounds with low water solubility such as large organic molecules may be solubilized with methanol, ethanol, and acetone solvents for testing (4) (see Guide E 729).1.3 An IC50 in light output (bioluminescence) is the recommended endpoint (1). However, percent inhibition of bioluminescence is an appropriate endpoint in some cases (5).1.4 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute and chronic tests.1.5 The values stated in SI units are to be regarded as the standard.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

2. Referenced DocumentsD 1141 Practice for the Preparation of Substitute Ocean WaterD 5196 Guide for Biomedical Grade WaterE 1192 Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and AmphibiansE 1218 Guide for Conducting Static 96-h Toxicity Tests with MicroalgaeE 1733 Guide for Use of Lighting in Laboratory TestingE 178 Practice for Dealing with Outlying ObservationsE 729 Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians

ASTM E1924 - 97(2004) Standard Guide for Conducting Toxicity Tests with Bioluminescent Dinoflagellates

International Standard (ASTM E1924)

1010

11

55

OKOK

Concern

Toxic

OKOK OKOK

• $100 to $200 per test

Breakthrough in Rapid Water Quality Assessments

Current Process: Over 30 years old

• Expensive labor and facility

• $1,000 to $3,000 per test

• Desktop solution

Biosensor Testing:

5 to 7 days for results

Results in as little as 4 hours

How Hard is the Test to Do?

1. Mix small amount (35mL) of sample water with 5mL of our live culture that was shipped the prior day.

Dispense into the cartridges, six replicates.

Effort : 5-10 minutes each cartridge

1. Set aside in the dark for 3 to 4 hours (exposure time)

Effort: 0 minutes

1. Put cartridge in the Instrument

press “Test” button

Effort: not much, 2 minutes

for automated results

Ballast Water, Ship or Marine Discharge Testing

Challenges to healthy marine water: What does the near future require?

1) Filter it (fairly inexpensive, return to source, dispose, remediate)

2) Treat it (can be expensive, do it early, isolate and manage)

3) Measure water more pragmatically, and frequently

Indicative measurements

Faster actions save $

4) Get to the source (environmental “outfitting” and responsibility)

SOPs (Standard Operating Procedures)

BMPs (Best Management Practices)

= Urgent need to use sustainable practices and have managed industries in maritime business