Biological test method for determining toxicity of sediment using luminescent bacteria: chapter 2

Front Matter

• Readers' Comments
• Review Notice
• Abstract
• Foreword
• List of Abbreviations and Chemical Formulae
• Terminology
• Acknowledgements

Readers' Comments

Comments regarding the content of this report should be addressed to:

Richard Scroggins
Method Development and Applications Section
Environmental Technology Centre
Environment Canada
335 River Road
Ottawa, Ontario
K1A 0H3

Review Notice

This report has been reviewed by the staff of the Environmental Technology Advancement Directorate, Environment Canada, and approved for publication. Mention of trade names or commercial products does not constitute endorsement by Environment Canada. Other products of similar value are available.

Abstract

A reference method for measuring the toxicity of samples of whole sediment under controlled and defined laboratory conditions is described in this report. It uses luminescent bacteria (Vibrio fischeri) as the test organism and inhibition of light production by the bacteria in a solid-phase test as the biological endpoint. The test involves the preparation of a series of concentrations of the sample by serial dilution in water, their mixing with an inoculum of test organisms (V. fischeri) and incubation for 20 minutes in test tubes held in a water bath at 15 ± 0.5°C, the filtration of the contents of each test tube, the subsequent stabilization of the filtrate at 15 ± 0.5°C for 10 minutes in a series of glass cuvettes held within wells of a photometer, and thereafter the photometric reading of light produced by the luminescent bacteria remaining in the filtrate. The statistical endpoint of the test is the concentration of sample which is estimated to cause 50% inhibition of light production by the bacteria (i.e., the IC50).

This reference method follows and is built upon the generic (multipurpose) biological test method “Toxicity Test Using Luminescent Bacteria (Photobacterium phosphoreum)” published previously by Environment Canada (1992; EPS 1/RM/24). It is intended for use with samples of contaminated or potentially contaminated sediment.

Specific conditions and procedures are stipulated that include instructions on obtaining, shipping, holding, and storing test organisms (Bacterial Reagent); acceptable procedures and conditions for transporting, storing, and manipulating samples of sediment to be used in the test; required physicochemical analyses of sediment; procedures and conditions to be followed in preparing for and conducting the test; criteria for acceptable performance and valid test results; measurements and observations to be made; required or recommended data analyses; guidance for interpreting test results; and minimum reporting requirements. Instructions on the use of reference toxicity tests are also provided.

Foreword

This is one of a series of reference methods for measuring and assessing the toxic effect(s) on single species of aquatic or terrestrial organisms, caused by their exposure to samples of test materials or substances under controlled and defined laboratory conditions.

A reference method is defined herein as a specific biological test method for performing a toxicity test, i.e., a toxicity test method with an explicit set of test instructions and conditions which are described precisely in a written document. Unlike other multi-purpose (generic) biological test methods published by Environment Canada, the use of a reference method is frequently restricted to testing requirements associated with specific regulations (e.g., Disposal at Sea Regulations under the Canadian Environmental Protection Act; CEPA, 1999; Government of Canada, 2001).

Reference methods are those that have been developed and published by Environment Canada (EC), and are favoured:

• for regulatory use in the environmental toxicity laboratories of federal and provincial agencies;
• for regulatory testing which is contracted out by Environment Canada or requested from outside agencies or industry;
• for incorporation in federal, provincial, or municipal environmental regulations or permits, as a regulatory monitoring requirement; and
• as a foundation for the provision of very explicit instructions.

Appendix A lists those Reference Methods prepared for publication by Environment Canada’s Method Development and Applications Section in Ottawa, ON, along with other generic (more widely applicable) biological test methods and supporting guidance documents.

Words defined in the Terminology section of this document are italicized when first used in the body of the report according to the definition. Italics are also used as emphasis for these and other words, throughout the report.

List of Abbreviations and Chemical Formulae

cm

centimetre(s)

CV

coefficient of variation

g

gram(s)

h

hour(s)

HCl

hydrochloric acid

HNO₃

nitric acid

ICp

inhibiting concentration for a (specified) percent effect

IC50

50% inhibiting concentration

L

litre(s)

mg

milligram(s)

min

minute(s)

mL

millilitre(s)

mm

millimetre(s)

NaCl

sodium chloride

NaNO₃

sodium nitrate

nm

nanometre(s)

NRCC

National Research Council of Canada

p

probability

SD

standard deviation

SOP

standard operating procedure

SPT

solid-phase test

TM (^TM)

Trade Mark

v:v

volume-to-volume

°C

degree(s) Celsius

µL

microlitre(s)

µm

micrometre(s)

wt:v

weight-to-volume

>

greater than

<

less than

≥

greater than or equal to

≤

less than or equal to

±

plus or minus

%

percentage or percent

Terminology

Words defined herein are italicized when first used in the body of the report according to the definition. Italics are also used as emphasis for these and other words, throughout the report.

Note: all definitions are given in the context of the procedures in this report, and might not be appropriate in another context.

Grammatical Terms

Must is used to express an absolute requirement.

Should is used to state that the specified condition or procedure is recommended and ought to be met if possible.

May is used to mean “is (are) allowed to”.

Can is used to mean “is (are) able to”.

Might is used to express the possibility that something could exist or happen.

General Technical Terms

Compliance means in accordance with governmental regulations or requirements for issuing a permit.

Estuarine (water) is from a coastal body of ocean water that is measurably diluted with fresh water derived from land drainage.

Fines refers to (sediment) particles which are ≤0.063 mm in size. Measurements of % fines include all particles defined as silt (i.e., particles ≤0.063 mm but ≥0.004 mm) or clay (i.e., particles <0.004 mm).

Marine (water) is from or within the ocean, sea, or inshore location where there is no appreciable dilution of water by natural fresh water derived from land drainage.

Monitoring is the routine (e.g., daily, weekly, monthly, quarterly) checking of quality, or collection and reporting of information. In the context of this report, it means either the periodic (routine) checking and measurement of certain biological or water quality variables, or the collection and testing of samples of sediment for toxicity.

pH is the negative logarithm of the activity of hydrogen ions in gram equivalents per litre. The pH value expresses the degree or intensity of both acidic and alkaline reactions on a scale from 0 to 14, with 7 representing neutrality, numbers less than 7 indicating increasingly greater acidic reactions, and numbers greater than 7 indicating increasingly basic or alkaline reactions.

Reference method refers to a specific protocol for performing a toxicity test, i.e., a biological test method with an explicit set of test procedures and conditions, formally agreed upon by the parties involved and described precisely in a written document. Unlike other multi-purpose (generic) biological test methods published by Environment Canada, the use of a reference method is frequently restricted to testing requirements associated with specific regulations (e.g., Government of Canada, 2001).

Salinity is the total amount of solid substance, in grams, dissolved in 1 litre of (sea)water; and is traditionally expressed as parts per thousand (‰). It is determined after all carbonates have been converted to oxides, all bromide and iodide have been replaced by chloride, and all organic matter has been oxidized. Salinity can also be measured directly using a salinity/conductivity meter or other means (see APHA et al., 1995).

Terms for Test Materials or Substances

Artificial sediment refers to a synthetic sediment, prepared in the laboratory using a specific formulation of clay, silt, and/or sand intended to simulate a natural sediment. A mixture of appropriate quantities of clean clay, silt, or sand-sized particles with the desired percentages of fine and coarse-grained material is mixed in the laboratory to prepare (clean) artificial negative control sediment. A specific formulation of clay, silt, and/or sand is mixed (spiked) together with a toxic chemical (or, in some instances, a highly toxic contaminated sample of field-collected sediment) to prepare one or more concentrations of artificial positive control sediment. See also “clean sediment”, “negative control sediment”, and “positive control sediment”.

Bacterial Reagent is a standard culture of a specific strain of freeze-dried (lyophilized) Vibrio fischeri, stored in small, sealed vials which each contain about 100 million organisms.

Chemical is, in this report, any element, compound, formulation, or mixture of a substance that might be mixed with, deposited in, or found in association with sediment or water.

Clean sediment is sediment that does not contain concentrations of any contaminant(s) which would reduce the light produced by V. fischeri during the test.

Contaminated sediment is sediment containing chemical substances at concentrations that pose a known or potential threat to environmental or human health.

Control is a treatment in an investigation or study that duplicates all the conditions and factors that might affect the results of the investigation, except the specific condition that is being studied. In an aquatic toxicity test, the control must duplicate all the conditions of the exposure treatment(s), but must contain no added test material or substance. The control is used to determine the absence of measurable toxicity due to basic test conditions (e.g., temperature, health of test organisms, or effects due to their handling or manipulation).

Deionized water is water that has been purified by passing it through resin columns or a reverse osmosis system, for the purpose of removing ions such as Ca⁺⁺, Mg⁺⁺, etc.

Diluent is a solution of 3.5% sodium chloride in distilled or deionized water, which is prepared using reagent-grade salt. Diluent comprised of 3.5% NaCl may be used with samples of marine, estuarine, or freshwater sediment. See also “distilled water” and “deionized water”.

Distilled water is water that has been passed through a distillation apparatus of borosilicate glass or other material, to remove impurities.

Liquid-phase (toxicity test) means that which does not include any added sediment particulate (i.e., the test material and concentrations thereof represent an aqueous solution). See also “solid-phase (toxicity test)”.

Material is the substance or substances from which a thing is made. A material might have heterogeneous characteristics, even after mixing. Soil, sediment, or surface water are considered herein as materials. Usually, the material would contain several or many substances. See also “substance”.

Negative control sediment means uncontaminated (clean) sediment which does not contain concentrations of one or more contaminants that could affect the performance (in this instance, light production) of the test organisms. This sediment may be natural, field-collected sediment from an uncontaminated site, or artificial sediment formulated in the laboratory using an appropriate mixture of uncontaminated (“clean”) sand, silt, and/or clay. This sediment must contain no added test material or substance, and must enable an acceptable rate of light production by V. fischeri according to the test conditions and procedures. The use of negative control sediment provides a basis for judging the toxicity of coarse-grained (<20% fines) test sediment. See also “artificial control sediment” and “clean sediment”.

Pore water (also called “interstitial” water) is the water occupying space between sediment particles.

Positive control sediment means sediment which is known to be contaminated with one or more toxic chemicals, and which causes a predictable toxic response (in this instance, inhibition of light production) with the test organisms according to the procedures and conditions of the test. This sediment might be one of the following: a standard contaminated sediment; artificial sediment or reference sediment that has been spiked experimentally with a toxic chemical; or a highly contaminated sample of field-collected sediment, shown previously to be toxic to V. fischeri and for which its physicochemical characteristics are known. The use of positive control sediment assists in interpreting data derived from toxicity tests using test sediment. For this reference method, positive control sediment must be used as a reference toxicant when appraising the sensitivity of the test organisms and the precision and reliability of results obtained by the laboratory for that material. See also “standard contaminated sediment”, “artificial sediment”, “reference sediment”, and “reference toxicant”.

Product is a commercial formulation of one or more chemicals. See also “chemical”.

Reconstitution Solution is non-toxic distilled or deionized water that is used to activate a vial of Bacterial Reagent.

Reference sediment is a field-collected sample of presumably clean (uncontaminated) sediment, selected for properties (e.g., particle size, compactness, total organic content) representing sediment conditions that closely match those of the sample(s) of test sediment except for the degree of chemical contaminants. It is often selected from a site that is uninfluenced or minimally influenced by the source(s) of anthropogenic contamination but within the general vicinity of the site(s) where samples of test sediment are collected. A sample of reference sediment should be included in each series of toxicity tests with test sediment(s). This reference sediment might or might not prove toxic due to the presence of naturally occurring chemicals such as hydrogen sulphide or ammonia, or the unanticipated presence of contaminants from human influence at harmful-effect concentrations. The use of such (toxic) sediment as reference sediment in future toxicity tests should be avoided, unless the experimental design is cognizant of this and the investigator(s) wish to compare test results for this material with those for one or more samples of test sediment. See also “clean sediment” and “test sediment”.

Reference toxicant is a standard positive control sediment used to measure the sensitivity of the test organisms to establish confidence in the toxicity data obtained for a test material or substance. In most instances, a toxicity test with a reference toxicant is performed to assess the sensitivity of the organisms at the time the test material or substance is evaluated, and the precision and reliability of results obtained by the laboratory for that reference toxicant. See also “positive control sediment”.

Reference toxicity test is a test conducted using a reference toxicant in conjunction with a sediment toxicity test, to appraise the sensitivity of the organisms and the precision and reliability of results obtained by the laboratory at the time the test material is evaluated. Deviations outside an established normal range indicate that the sensitivity of the test organisms, and the performance and precision of the test, are suspect. For this reference method, a reference toxicity test should be performed using a positive control sediment which can be either a subsample of a reference material comprised of standard contaminated sediment with known levels of contaminants (such as is available from the National Research Council of Canada), or a spiked control sediment test. See also “reference toxicant”, “positive control sediment”, “standard contaminated sediment”, and “spiked control sediment”.

Sampling station means a specific location, within a site or sampling unit (depending on the study design), where the sample(s) of field-collected sediment are obtained for toxicity tests and associated physicochemical analyses. See also “site”.

Sediment is natural particulate material, which has been transported and deposited in water, and which usually lies below water. The term can also describe a material that has been experimentally prepared (formulated) using selected particulate material (e.g., sand of particular grain size, bentonite clay, etc.).

Site means a delineated tract of sediment that is being used or considered as a study area, usually from the perspective of it being contaminated or potentially contaminated by human activity.

Soil is whole, intact material representative of the terrestrial environment, that has had minimal manipulation following collection. It is formed by the physical and chemical disintegration of rocks and the deposition of leaf litter and/or decomposition and recycling of nutrients from plant and animal life. Its physicochemical characteristics are influenced by microbial and invertebrate activities therein, and by anthropogenic activities.

Solid-phase (toxicity test) means that which includes a series of test concentrations prepared using an aliquot of the whole sediment (i.e., sample particulate plus pore water, added as a homogeneous mixture). See also “liquid-phase (toxicity test)”.

Spiked control sediment is clean artificial sediment or clean field-collected reference sediment to which a test substance or material such as a chemical, a mixture of chemicals, drilling mud, contaminated dredge spoil, sludge, or contaminated sediment has been added experimentally, and mixed thoroughly to evenly distribute the substance or material throughout the control sediment. See also “clean sediment”, “artificial sediment”, “reference sediment”, “contaminated sediment”, and “positive control sediment”.

Standard contaminated sediment is a field-collected sediment for which contaminant concentrations are known, documented, and available (e.g., from the National Research Council of Canada); and one which has proven to be toxic to V. fischeri using the reference method described herein.

Substance is a particular kind of material having more or less uniform properties. This term includes any distinguishable kind of organic or inorganic matter, whether inanimate or animate.

Test sediment is a field-collected sample of whole sediment, taken from a marine, estuarine, or freshwater site thought to be contaminated (or potentially so) with one or more chemicals, and intended for use in the solid-phase toxicity test with luminescent bacteria. In some instances, the term also applies to any solid-phase sample (including reference sediment, artificial sediment, negative control sediment, positive control sediment, or dredged material) used in the test. See also “solid-phase toxicity test”, “reference sediment”, “artificial sediment”, “negative control sediment”, and “positive control sediment”.

Water-only (toxicity test) refers to a (toxicity) test which does not include any sediment or other solid-phase material (e.g., a test using an aqueous solution of a reference toxicant). The term water-only (toxicity test) is synonymous with liquid-phase (toxicity test). This test is used to confirm that the Reconstitution Solution does not decrease light production by V. fischeri (see Section 3.2.4). See also “liquid-phase (toxicity test)” and “solid-phase (toxicity test)”.

Statistical and Toxicological Terms

Acute means within a short period in relation to the life span of the test organism (of the order of some minutes for bacteria).

Acute toxicity is an adverse effect (lethal or sublethal) induced in the test organisms within a short period (for purposes of this document, within a few minutes) of exposure to test sediment(s).

Battery of toxicity tests is a combination of several toxicity tests, normally using different species of test organisms (e.g., a series of sediment toxicity tests using V. fischeri, one or more species of marine or estuarine amphipods, and a polychaete worm).

Coefficient of Variation (CV) is the standard deviation (SD) of a set of data divided by the mean, expressed as a percentage. It is calculated as: CV (%) = 100 SD ÷ mean.

Endpoint means the measurement(s) or value(s) that characterize the results of a test (e.g., ICp). This term might also mean the reaction of the test organisms to show the effect which is measured upon completion of the test (e.g., inhibition of light production).

Gamma is the measure of light loss used in calculating the ICp. It is calculated individually for each cuvette containing a filtrate of a particular test concentration. Gamma(Γ) is calculated based on the ratio between the amount of light emitted by a test filtrate and that emitted by the control solutions, as follows: Γ = (I_c/I_t) - 1, where I_c = the average light reading of filtrates of the control solutions, and I_t = the light reading of a filtrate of a particular concentration of the test material (Section 6.1). When Gamma equals unity (Γ = 1), half of the light production has been lost as a result of toxicity or other confounding factors (Section 6.2).

Geometric mean is the mean of repeated measurements, calculated logarithmically. It is advantageous, in that extreme values do not influence the mean as is the case for an arithmetic mean. The geometric mean can be calculated as the n^th root of the product of the “n” values, and it can also be calculated as the antilogarithm of the mean of the logarithms of the “n” values.

ICp is the inhibiting concentration for a (specified) percentage effect. It represents a point estimate of the concentration of test substance or material in sediment that causes a designated percent inhibition (p) compared to the control, in a quantitative biological function such as light production by bacteria or growth of fish, relative to the control. For example, an IC50 could be the concentration estimated to cause a 50% reduction in the quantity of light emitted at the end of the test by the test organisms, relative to that in the control. This term should be used for any toxicological test which measures a continuously variable effect, such as light production, reproduction, respiration, or dry weight at test end.

Precision refers to the closeness of repeated measurements of the same quantity to each other, i.e., the degree to which data generated from replicate measurements differ. It describes the degree of certainty around a result, or the tightness of a statistically derived endpoint such as an ICp.

Replicate refers to a single test chamber containing a prescribed inoculum of organisms in either one concentration of the test material or substance, or in the control or reference treatment(s). A replicate is an independent test unit; therefore, any transfer of organisms or test material from one replicate to another would invalidate the test. The term is also used to refer to more than one sample of test material taken at one time from a particular location and depth (i.e., field replicates), or for subsamples of a particular test material taken for multiple (duplicate or more) toxicity tests using identical procedures and conditions (i.e., laboratory replicates).

Replicate samples are field-replicated samples of sediment collected from the same sampling station, to provide an estimate of the sampling error or to improve the precision of estimation. A single sediment sample from a sampling station is treated as one replicate. Additional samples are considered to be additional replicate samples when they are treated identically but stored in separate sample containers (i.e., not composited).

Toxic means poisonous. A toxic chemical or material can cause adverse effects on living organisms, if present in sufficient amount at the right location. Toxic is an adjective or adverb, and should not be used as a noun; whereas toxicant is a legitimate noun.

Toxicant is a toxic substance or material.

Toxicity is the inherent potential or capacity of a material or substance to cause adverse effect(s) on living organisms. The effect(s) could be lethal or sublethal.

Toxicity test is a determination of the effect of a material or substance on the test organism (e.g., Vibrio fischeri), under defined conditions. An aquatic toxicity test usually measures either (a) the proportions of organisms affected (quantal); or (b) the degree of effect shown (quantitative or graded), after exposure to a specific test material (e.g., a sample of sediment) or mixture (e.g., a chemical/sediment mixture). A solid-phase toxicity test with luminescent bacteria must be considered a graded (quantitative) toxicity test since there is no measurement of the proportions of individual bacteria that are directly affected, but instead, overall measurements of the degrees of reduction in a physiological function (i.e., light emission), shown by groups of bacteria.

Treatment refers to a specific test sediment (e.g., site sediment or reference sediment from a particular sampling station and depth), or a concentration thereof. Samples or subsamples of test sediment representing a particular treatment are typically replicated in a toxicity test. See also “replicate”.

Warning chart is a graph used to follow changes over time, in the endpoints for a reference toxicant. Date of the test is on the horizontal axis and the effect-concentration is plotted on the vertical logarithmic scale.

Warning limit is plus or minus two standard deviations, calculated logarithmically, from a historic geometric mean of the endpoints from tests with a reference toxicant.

Acknowledgements

This reference method was written by D. McLeay (McLeay Environmental Ltd., Victoria, BC) and G. Wohlgeschaffen (Dartmouth, NS). The undertaking was initiated and funded by Environment Canada’s Marine Environment Branch, with the support and encouragement of L. Porebski and J. Osborne (Environment Canada, Hull, PQ). R. Scroggins (Method Development and Applications Section, Environmental Technology Centre, Environment Canada, Ottawa, ON) acted as Scientific Authority and provided technical input and direction throughout the work. The technical advice and review comments provided by K. Doe (Atlantic Environmental Science Centre, Environment Canada, Moncton, NB) during the development, validation, and writing of this biological test method were particularly helpful and are gratefully acknowledged. Technical data and comments provided by D. Lee (Environment Canada, North Vancouver, BC) and D. St. Laurent (Environment Canada, Montreal, PQ) also assisted in the preparation of this reference method. Mr. G. Schroeder (Environment Canada, North Vancouver, BC) is thanked for developing the electronic spreadsheet recommended herein for comparing two IC50s.

Members of the Inter-Governmental Environmental Toxicity Group (IGETG, Appendix B) participated in the development and review of this report and are thanked accordingly. Members of Environment Canada’s regional and headquarters offices (Appendix C) are also thanked for their support.

Special acknowledgement is made of the many useful comments provided by each member of the Scientific Advisory Group responsible for scientific input and advice during the development and review phases related to the preparation of this report. This team of advisors included: Mr. C. Buday (Environment Canada, North Vancouver, BC), Dr. A. Burton, Jr. (Wright State University, Dayton, OH), Mr. K. Doe (Environment Canada, Moncton, NB), Dr. K. Ho (U.S. Environmental Protection Agency, Narragansett, RI), Ms. P. Jackman (Environment Canada, Moncton, NB), Mr. J. Osborne (Environment Canada, Hull, PQ), Ms. L. Porebski (Environment Canada, Hull, PQ), Dr. P. Ross (Colorado School of Mines, Golden, CO), Mr. R. Scroggins (Environment Canada, Ottawa, ON), Mr. P. Topping (Environment Canada, Hull, PQ), and Mr. S. Trottier (Environment Canada, Montreal, PQ). Appendix D provides complete affiliations and points of contact for each member of this Scientific Advisory Group, and for the Scientific Authority and Consultants.

Photos for the cover were supplied by Paula Jackman, Troy Steeves, and Dale Hughes (Environmental Science Centre, Environment Canada, Moncton, NB).