Page 976 - The Toxicology of Fishes

956 The Toxicology of Fishes

What Are the Candidate Causative Chemicals Associated with the Effect?
This step involves techniques that confirm the proposed substances are in fact responsible for the observed
toxicity. This is usually accomplished through a weight-of-evidence assemblage of information that
collectively establishes the identity of the active compounds. It is also equally important to establish
that the cause of the effect is consistent over time so that amelioration efforts can adequately address
the effect. Some judgment must be exercised in terms of the extent to which confirmatory tests are
carried out, which reflects the authenticity of the results. As an example, if a suspected substance can
be removed by inexpensive pretreatment or process modification, a higher level of uncertainty may be
acceptable than if an expensive treatment plant is required.

Can the Candidate Chemicals Be Confirmed to Cause the Effect?
Complete confirmation of isolated chemicals proposed as causative agents is challenging in that it requires
procurement of authentic standards for chemical and toxicological verification. It is possible that authentic
standards of candidate structures will not be commercially available and custom synthesis may be
required. Custom synthesis can be expensive, time consuming, and, depending on the structure, difficult
to carry out. In the absence of complete confirmation it is important to recognize that valuable information
regarding the chemical characteristics of the active compounds will nevertheless be derived from all
previous work. With this information and tentative chemical structures it may be sufficient to tentatively
assign cause and proceed on confirmatory approaches that may include:

• Correlation approach—A strong consistent relationship between the concentrations of the
suspected agents and the bioassay response can be established.
• Symptom approach—Different active substances often produce different symptoms in response.
By comparing exposures of the effluent sample to those of pure suspected active substances,
one can obtain further evidence regarding whether or not the suspected agents are responsible.
Examples of symptoms include species sensitivities, shapes of dose–response curves, and time
for the effect to occur.
• Spiking approach—Suspected agents are added to the effluent to determine if a proportional
response in the bioassay is obtained.

Bioaccumulation Model
Applying TIEs to PMEs has proven to be problematic for the reasons outlined above. As a result, an
alternative strategy to investigating complex mixtures using PMEs as a model has been developed (Parrott
et al., 2000b; Hewitt et al., 2000, 2003b, 2004, 2005b). This approach utilizes controlled fish exposures
to final effluents and investigates tissue burdens of bioactive substances to determine what compounds
are bioavailable to fish and therefore of the most relevance. In the development of this model, it has
been shown that under high exposure conditions (50% v/v), multiple ligands for fish sex steroid receptors
and the aryl hydrocarbon (Ah) receptor are readily bioavailable to fish exposed to effluent from a bleached
kraft mill (Hewitt et al., 2000) and a bleached sulfite/groundwood mill (Hewitt et al., 2003b). Further
studies have validated the accumulation model to show that under spring conditions of high dilution at
a third bleached kraft mill, fish accumulate hormonally active substances and that there are gender-
specific differences in patterns of accumulation (Hewitt et al., 2004, 2005b). These studies were con-
ducted at sites where wild fish or fathead minnow tests have demonstrated clear effects on the reproductive
endocrine system, thereby providing a mechanistic linkage to the exposure of these compounds at these
sites. The investigation of active substances in tissues considers one of the basic axioms of toxicology:
The response of an organism is the result of a sufficient dose at the site of toxic action. Conducting
characterizations of unknowns in highly complex mixtures in this manner considers multiple exposure
pathways that would be ignored in direct investigations of final combined effluent or process stream
investigations: (1) the modification or creation of active compounds in secondary treatment, (2) the
modification or creation of active substances in the receiving environment, (3) the metabolic activation
of inactive precursors within the organism, and (4) the ability of organisms to excrete the compounds
by an inducible mechanism to reduce the effective dose at the target site.