In this paper data have been presented on the response of A. awamori to different toxicant concentrations by examining changes in Relative Light Units (RLU) and [Ca2+]c in the presence of an external CaCl2 concentration of 5 mM. This study differs from that of Torrecilla et al.  which examined changes in intracellular Ca2+ of the cyanobacterium Anabaena able to express apoaequorin constitutively when subjected to heat and cold shock. It is also the first study, as far as we know, examining responses of aequorin to toxicants in a filamentous fungus. The concentrations of Zn2+, Cr6+ and 3,5-DCP used in the experiments to assess toxicity were based on experience with V. fischeri bioassays, through range finding and interest in observing effects at low and high concentrations. Initially, the effect of each of these three chemicals on [Ca2+]c was examined. The [Ca2+]c response was not significantly different from that obtained with the control solution (data not shown). A second approach studied the effect of preincubation of the fungus with toxicants on [Ca2+]c response to external CaCl2.
There is no doubt that aequorin transformed A. awamori, using the protocol developed, responds to toxic chemical challenge in a reproducible way. The aequorin system has more parameters which can be assessed than other bioassays i.e. final [Ca2+]c resting level, recovery time and amplitude of [Ca2+]c/RLU following chemical challenge for 5 or 30 min.
A. awamori has been shown to respond to organic and inorganic compounds by a decrease in the amplitude of [Ca2+]c response to external CaCl2 with increasing toxicant concentration. Zn2+ and DCP also affected the final Ca2+ resting levels and recovery times. In the case of 30 min preincubation with either 112 mg l-1 DCP or 700 mg l-1 Zn2+, the final [Ca2+]c resting levels remained significantly higher than after 5 min preincubation with these toxicants indicating a greater toxicity to the fungus on longer contact with the toxicant. This is not an unusual response even in the V. fischeri bioassay where it is generally observed that toxicity increases (IC50 values decrease) with longer incubation times. IC50 values calculated using amplitude changes of [Ca2+]c showed decreased toxicity to Zn2+ at longer preincubation times. This needs further analytical consideration to determine toxicity values of relevance.
The proprietary Microtox bioassay and other bacterial bioluminescence methods, including that used in the present study, which do not involve genetically modified bacteria, utilise Vibrio (e.g. V. fischeri) and related Photobacterium species. Acute toxicity tests utilising such luminescent bacteria can underestimate the toxicity of chemicals and Backhaus et al.  showed that more reliable toxicity estimates can be obtained through the use of long-term toxicity testing with the same organisms. It may be prudent to test the response of A. awamori using longer preincubation times with toxicants e.g. > 24 h, prior to monitoring Ca2+ homeostasis. This may increase the sensitivity of the bioassay. It must be remembered however that for one of the toxicants (Zn2+), a decrease in sensitivity was evident at 30 min compared to the result at 5 min. There was also evidence of toxicity recovery through adaptation with increased incubation. This may be due to acquired resistance by the organism through synthesis of metal-binding proteins (metallothioneins) or constituents in the growth medium removing/immobilising the metal (e.g. EDTA, phosphate precipitation). This needs to be examined in the fungal bioassay.
It is interesting to compare toxicity data obtained with A. awamori with those obtained with the V. fischeri bioassay. The toxicity values for the fungus are higher than those of the bacterial test indicating lower bioassay sensitivity with the parameter used to calculate the IC50 values. For example, the IC50 values for the 30 min Zn2+ incubations were approximately 3 orders of magnitude lower for V. fischeri. Such insensitivity to Zn2+ has been observed with an ATP luminescence assay . The IC50 results with Cr6+ (30 min preincubation) were 400 mg l-1, 12 times higher than in the bacterial biosensor. This value is the same order of magnitude as the 15 min Microtox assay (339.6 mg l-1) carried out by Codina et al. . Indeed, the use of V. fischeri in various proprietary tests (LumisTox, Microtox, ToxAlert etc) have been shown to exhibit differences in sensitivity to different toxicants . It is, therefore, difficult to categorically state that V. fischeri is more sensitive to Cr6+ than A. awamori particularly when different incubation periods are used in bioassays affecting any ultimate IC50 value. It should be also noted that since the aequorin test is based on changes in [Ca2+]c, toxic chemicals often exert an increase in [Ca2+]c thus affecting IC50 calculations. The calculation of IC50 for this fungal bioassay may not be appropriate. Other parameters such as LT50, rise time, final Ca2+ resting levels and recovery time would provide ideal candidate parameters, but more tests and comparisons with other bioassays are needed. The aequorin bioassay can also generate up to 15 parameters to assess the effect of toxicants due to the complex pattern of [Ca2+]c changes . These parameters can be further used to create a profile for toxicants with specific modes of action . With different parameters available for analysis in the aequorin test it could be useful to assess the toxicity by calculating the NOEC (no observable effect concentration) or a nominal IC10 value .
Codina et al.  also used a yeast bioassay to test metal toxicity. The eukaryotic yeast was found to be less sensitive to metals than prokaryotic organisms including Vibrio and Pseudomonas species. An IC50 value of 549.1 mg l-1 was obtained for Zn2+ using the yeast assay  which is comparable to some of the values obtained with A. awamori in our study. The yeast was slightly more sensitive to Cr6+ (30.9 mg l-1) than the filamentous A awamori but this is not unusual among members of the same trophic level (see . Recent chronic toxicity studies have been using wild-type and genetically modified mutants of the yeast Saccharomyces cerevisiae . EC50 values of >1000 mg l-1 were determined for Zn2+ and 1.7-4.79 mg l-1 for Cr6+ indicating variability in assays that rely on conditions imposed including exposure time, species tested and criteria used in the final assessment.
An interesting observation is the enhanced stimulation of light output in some systems with a low concentration of toxicant. This is seen in the 5 min treated systems using 0.11 mg l-1 DCP (Figure 1a), 180 mg l-1 Zn2+ (Figure 1e) and also in the 30 min treated system with 180 mg l-1 Zn2+ (Figure 1f). This stimulation, referred to as hormesis, is a common occurrence in toxicity bioassays  and is often observed in our laboratory using the V. fischeri bioassay.
It is not clear based on these preliminary observations what causes Ca2+ and light retention. In the case of DCP it may be that this polar narcotic is affecting membrane permeability and the transport of Ca2+ out of the cytosol (Ca2+ ATPase and efflux of Ca2+ via e.g. Ca2+/H+ antiporters) and hence, a delay in RLU dissipation. The metals may be competing with the transport mechanisms in membranes. The metals thus act by interacting with physiological ions affecting transport and its concomitant effect on light output.
Most of the organic chemicals discharged to the environment exert a narcotic effect on biota. This is either Type I (non-polar narcosis) or Type II (polar narcosis). It would be interesting to utilise A. awamori to test whether non-polar and polar narcoses operate in the fungus and whether these can be predicted by QSAR (Quantitative Structure Activity Relationships). Cronin and Schulz  showed, using QSAR, that non-polar narcosis occurs in V. fischeri but they could not validate polar narcosis in the organism.
There is no doubt that a comprehensive testing of metals and organic compounds needs to be carried out to assess the value of A. awamori as a toxicity sensing tool both in systems using pure chemicals and those involving real samples (e.g. complex effluents and soil water matrices). Toxicant preincubation time and effect on fungus response would be an important factor to test fully.