Page 975 - The Toxicology of Fishes

Case Study: Pulp and Paper Mill Impacts 955

field trials, and MFO induction peaked within 2 days in warm water; (2) the inducers at some mills
could be excreted rapidly by fish, but excretion involved an inducible mechanism which required greater
than 4 days of exposure; and (3) at Jackfish Bay in Ontario, it seemed likely that the inducers were not
accumulated from the food chain, although this historically may have been a component.
Various methods and approaches have been used to isolate bioactive substances from PMEs. Despite
the efforts of several investigators, limited information is available on the identities of compounds present
in final effluents that are associated with reproductive abnormalities in wild fish (Munkittrick et al.,
2002). This is due to a number of factors, such as the chemical complexity of effluent discharges, a lack
of understanding of the mechanisms involved, and differences in the response patterns of wild fish and
laboratory species used to conduct investigations (Van Der Kraak et al., 1998).
The last tiers of the framework reduce this seemingly overwhelming task into achievable components
by asking a progressive, more detailed set of questions regarding the chemical identities of the bioactive
substances. In the case of EEM, this is done primarily to provide investigators with discrete steps of
information such that causal investigations may be halted when sufficient information is gained (Hewitt
et al., 2005a). A primary means by which to achieve this goal is through implementation of toxicity
identification evaluations (TIEs), which provide guidance in the identification of substances causing
toxicity in complex mixtures (USEPA, 1991, 1993a,b, 1997).

Toxicity Identification Evaluation Procedures
The TIE approach uses the responses of organisms or appropriate bioassay surrogate to detect the
presence of active chemicals in a complex mixture. TIEs characterize the active substances of interest
in three phases, and they were developed for municipal sewage in concert with toxicity reduction
evaluations (TREs) to ameliorate effluent acute and chronic toxicity. Modifications of this approach
have been used to investigate hormonally active substances associated with metabolic disruption in
fish exposed to PMEs, and it is possible to investigate any activity of interest by selecting a different
endpoint. The first phase of a TIE involves: (1) determining the characteristics of the active agents
and (2) establishing whether or not the effect is caused by the same substances. Failure to establish
effect variability related to the active substances could lead to erroneous conclusions and control
measures that do not eliminate the effect. The physicochemical properties of the active substances
can be described using effluent manipulations coupled to a bioassay that either duplicates the field
effects or is mechanistically linked to them. Each test is designed to alter the substances themselves
or change their bioavailability so that information on the nature of the substances can be obtained.
Repeating these tests over time on the same sample will provide information on the consistency of
the substances to cause the effect. Examples of effluent manipulations include filtration, pH adjust-
ments, addition of oxidizing agents and chelating agents, temperature adjustments, aeration, and solid-
phase extraction. If relatively simple modifications of this stage remove the effect during testing, it
may be possible that the investigation can be halted at this juncture and these manipulations employed
on an industrial scale.

What Are the Chemical Classes Involved in the Effect?
The first phase of a TIE involves specific methods to isolate the active chemicals and propose structures
for their identification. In this step, further separation of active components from inactive substances is
likely necessary for their identification and confirmation. These methods are specific to the classes of
chemicals outlined above and utilize bioassay responses to evaluate the success or failure of extraction,
separation, and concentration steps in isolating the bioactive substances. The question of whether one
or more bioactive substances are involved complicates this process, and the solution to this problem is
to focus on the active component that is easiest to identify. Examples of isolation techniques include
solid-phase extraction, high-performance liquid chromatography (HPLC), and solvent extraction. Chem-
ical isolation steps proceed in an iterative fashion, directed by bioassay responses until either further
isolations are not possible or candidate chemicals are identified. Once there is strong evidence that one
or more candidate chemicals are associated with the response, the last phase can be initiated.