Biological test method for determining toxicity of sediment using luminescent bacteria: chapter 3

Section 1: Introduction

This reference method specifies the procedures and conditions to be used when preparing for and undertaking a solid-phase test for measuring the toxicity of samples of test sediment, using luminescent bacteria (Vibrio fischeri). The reference method herein represents one of the biological test methods to be used as part of sediment assessments consistent with the Federal regulations on disposal at sea under the Canadian Environmental Protection Act (EC, 1997a; CEPA, 1999; Government of Canada, 2001). It can also be used to measure the toxicity of samples of sediment being considered for disposal on land or at any freshwater, estuarine, or marine sites where regulatory appraisals or stringent testing procedures apply. Another reference method, intended for applications with sediment samples, has been published by Environment Canada (EC, 1998) and other Federal (Environment Canada) biological test methods for measuring sediment toxicity are available (see Appendix A).

Solid-phase tests for measuring sediment toxicity, using luminescent bacteria (V. fischeri, formerly identified as  Photobacterium phosphoreum) have been used by Canadian, the US, and other researchers and regulators since the introduction of this test procedure by Canadian researchers for evaluating the toxicity of test sediments from Hamilton Harbour (Brouwer et al., 1990). An acute solid-phase test for sediment (or soil) toxicity was subsequently adopted and standardized by Microbics Corporation (Carlsbad, CA), as one of several Microtox^TM test methods (Microbics, 1992). At that time, Environment Canada (1992) recommended the use of the Microtox^TM solid-phase test method for evaluating the toxicity of samples of sediment or similar solids, while recognizing that the standardization of the test method was in its infancy. Notwithstanding, Environment Canada (1992) continues to serve as a useful reference source and companion to the present document, when performing acute solid-phase (or, using EC 1992 guidance, liquid-phase) toxicity tests with luminescent bacteria.

Many components of the procedures and conditions specified herein are consistent with the guidelines and approaches for measuring sediment toxicity using the solid-phase test option and V. fischeri as test organisms, which are described in various methodology documents or laboratory standard operating procedures (SOPs) including: EC (1992), Microbics (1992, 1995a), ASTM (1995), EC (1996a), AZUR (1997), AZUR (1998a, b), EC (1999a), and NICMM (1999). Appendix E provides a review of the similarities and differences associated with various procedures and conditions specified in those documents. The contribution of those methods and SOPs to all parts of this reference method is acknowledged, and they are recommended as sources of supporting rationale. Procedures and conditions stipulated in this report should, however, be taken as the definitive ones when planning and undertaking solid-phase sediment toxicity tests with luminescent bacteria (V. fischeri) for regulatory purposes in Canada.

Besides the existing methodology guideline documents or SOPs for which pre-test and test conditions and procedures are summarized in Appendix E, a number of reports are now available in the scientific literature that provide further useful details regarding the performance of solid-phase sediment toxicity tests using luminescent bacteria. These include but are not restricted to the following: studies of the influence of sediment composition on apparent toxicity (Benton et al., 1995; Ringwood et al., 1997; Tay et al., 1998); an investigation into the role of sulphide toxicity in reduced sediments as a factor in (freshwater) sediment toxicity (Brouwer and Murphy, 1995); a study of correlations between a number of solid-phase sediment toxicity tests and in-situ benthic community structure (Day et al., 1995); input to test design and data interpretation using the V. fischeri solid-phase test for sediment toxicity (Ross and Leitman, 1995); a survey of the toxicity of Halifax Harbour sediments (Cook and Wells, 1996); the role of this and other sediment assays in assessing the toxicity of test sediments (Ross, 1998; Bombardier and Birmingham, 1999); and the interlaboratory precision of a solid-phase test for sediment toxicity using V. fischeri (Ross et al., 1999).

Before finalizing this reference method, two series of interlaboratory studies with samples of reference and contaminated sediment were performed to determine intralaboratory and interlaboratory precision, and to validate the test method. Each series involved the same six testing laboratories, with varied (≤ 1 to 8 years) experience in performing sediment toxicity tests using luminescent bacteria in a solid-phase test. The first series of tests was conducted by each laboratory using an identical set of aliquots (subsamples) of eight dry test materials identified only as samples numbered 1 to 8. Seven of those aliquots were dry, certified standard sediments (reference or contaminated) provided by the National Research Council of Canada (NRCC), and the eighth aliquot was 100% dried kaolin clay. Although not identified as such, four of the eight aliquots submitted to each laboratory during the first series of tests were subsamples taken from the same batch of a single composite sample of a standard NRCC contaminated sediment. For the second series of tests, an identical set of eleven subsamples of field-collected “wet” sediment, obtained from a number of contaminated or reference sampling stations within Canadian coastal waters, was sent to each laboratory for testing. Although not identified, three of those eleven test materials were from the same batch of a composite sample of a single reference sediment. Findings from these studies, which are detailed in a technical report available from Environment Canada’s Method Development and Applications Section (McLeay et al., 2001), indicated very good intralaboratory and interlaboratory precision for each series of tests performed using the pre-test and test conditions detailed in this reference method.