Table 1 presents the highest measured concentrations of nodularin in whole organisms and/or selected organs and tissues for each study cited. Error estimates are not considered, as not all reports specified what such estimates represented. Data are presented in increasing order of nodularin concentration. Publications that used ELISA as the sole analytical method generally reported their results in terms such as “total cyanobacterial hepatotoxins” or “microcystin-LR equivalents”, as the ELISA kits used in most studies detect and quantify the ADDA moiety common to microcystins and nodularin. However, the studies in which ELISA alone was used are included in this table because reported exposures were to nodularin-producing cyanobacteria (Nodularia spumigena), i.e., exposure to microcystins or microcystin-producing cyanobacteria would appear to be unlikely or minimal.

Nodularin concentrations measured in liver, muscle, peritoneal and stomach contents are presented in Table 2. Common fish names are as defined by the Australian Fish Names Standard [33]. Coefficients of variation for replicate analyses were calculated for nodularin-positive samples. Reported concentrations are not adjusted for recoveries. Microcystin-LR was not detected in any tissues. Figure 2 presents reconstructed partial chromatograms of nodularin product ions.

Table 3 presents recoveries from control fish tissues spiked with nodularin and MC-LR standards. Tissues were spiked in triplicate.

The livers of all five mullet captured from the study lake on 7 September 2009 were abnormal in appearance: gross hepatomegaly, orange-brown discolouration, and extremely friable. The liver of the single mullet captured at a nearby “control” lake on that day was a dark brown colour, which was obviously different to that of the livers of the study lake fish from which nodularin residue was subsequently detected (see Figure 3). Figure 3 and Figure 4 demonstrate the abnormal colour of mullet liver tissue seen on the 7 September capture.

Table 4 shows relative liver weights (a.k.a. hepatosomatic index, HSI, i.e., liver weight as a proportion of whole body weight).

Liver sections from study lake mullet caught on 7 September 2009, i.e., with hepatomegaly, orange discolouration and nodularin present (n = 3), and 29 September 2009 (no gross abnormality at necropsy but containing nodularin; n = 3) and from control lake sites (livers macroscopically normal, nodularin not detected; n = 5) were examined, but the results were inconclusive. Findings of the examination are presented in Table 5; because specimens were not fixed under ideal conditions, and other organs—particularly gills, kidney and spleen—were not available for inspection, we have not attempted to interpret these findings in the context of the main focus of this report, that of the chemical analysis of nodularin in mullet tissues.

In brief, the gross appearance of livers and histopathology examination suggests that the sampled fish had a variety of liver changes. Whether this is due to nodularin exposure and bioaccumulation cannot be ascertained from this study. A program of laboratory-based studies is indicated in order to investigate dose-response and time-series effects of exposure to nodularin, where environmental variables and age or class size can be controlled.

Figure 5 is a photomicrograph taken during examination of the stomach contents of a mullet recovered after the initial fish kill at the study lake. Remnants of Nodularia trichomes could not be identified microscopically in stomach contents of fish captured in September 2009, however nodularin was found in the stomach contents of mullet caught at the study lake on the second fish capture excursion, on 29 September 2009 (see Table 2).

Tissue dry weight equivalents presented in Table 2 were calculated from average moisture content of lyophilized tissues. Mullet liver contained 72.4% water (C.V. of duplicate measurements = 1.06%); muscle tissues of mullet recovered after the fish kill contained 69.1% water (C.V. 0.44%) and muscle tissue of captured mullet contained 75.8% water (C.V. 0.69%).

Of the five mullet caught in the study lake on 7 September 2009, three specimens were identified as Mugil cephalus, whereas two were mullet species that could not be matched to the BOLD database. These two unidentified mullet belonged to the same species, but we were unable to determine exactly which species it was. Interestingly, the nodularin profile varied across these different mullet species. While the sample numbers here are small (n = 3 and n = 2), thus demanding caution in the interpretation of these findings, the three M. cephalus specimens had essentially identical nodularin concentrations in their livers (median 4.7 mg/kg wet weight) and nodularin was detected in the muscle of these fish (median 3.1 μg/kg). By contrast, the hepatic nodularin concentrations in the two unidentified mullet were substantially lower (0.96 mg/kg and 1.2 mg/kg) than those found in the sea mullet; nodularin was not detected in the muscle. This pattern was repeated on the second mullet capture excursion on 29 September. Of the four mullet caught that day from the study lake, DNA sequence analysis showed that two specimens were M. cephalus, and the other two were an unidentified mullet of the same species. Further examination of nucleotide sequences from the unidentified mullet caught from the study recreational lake on both days in September 2009 has shown that these four specimens belong to the same species. Thus we have evidence for two mullet species inhabiting this lake: M. cephalus and an unknown mugilid. Of these two species, M. cephalus consistently had higher hepatic nodularin levels than the unidentified mullet. Differences in wet weight liver nodularin concentration between both species were statistically significant with a low-power non-parametric test (Mann-Whitney U statistic: 0.0, two-tailed p = 0.016). So we appear to have found variation in tissue nodularin concentrations between different mullet species. While variable nodularin uptake and/or elimination kinetics across individual Mugilidae species may explain these results, we suggest that different feeding strategies and resource partitioning should be priority avenues for inquiry in this regard. The evolutionary diversity of the Mugilidae is centred on the mouth parts; different mullet species display preferences for specific inorganic particle size in the substrate [34,40]. Mullet species are reported to select specific particle sizes with a pharyngeal filter apparatus, though choice of substrate may also contribute to observed species-dependent variability in particle size in the stomach. Thus individual mullet species will partition the food resource on the basis of inorganic particle size preference [34,41]. A mosaic distribution of Nodularia across surface sediments of the study lake, and species-specific substrate preferences may be factors worth investigating in order to explain these provisional findings of inter-species variability in tissue nodularin levels within the Mugilidae.

Livers from mullet captured at the study lake on 7 September 2009 (n = 5) were an orange/tan colour, and appeared to be enlarged at necropsy. The relative liver weights presented in Table 4 support the visual assessment of hepatomegaly in study lake fish from 7 September 2009 when compared to relative weights of control lake mullet livers (p = 0.016). However, that observation did not hold for mullet caught at the study lake some three weeks later. Livers at that time were a more normal-looking brown colour, and did not appear enlarged. Again, relative liver weights of 29 September 2009 fish were not significantly larger as a group compared to those of fish from the nearby reference lakes (p = 0.23). Yet we found no obvious correlation between liver nodularin concentration and gross appearance of the livers; while there appeared to be a species relationship to liver nodularin concentration, as discussed above, nodularin concentrations measured in both species were higher on the second capture day (29 September 2009) when livers were macroscopically normal. Livers from the control lake fish did not contain detectable nodularin but controlled studies are required to ascertain whether liver discolouration in mullet is a reliable indicator of exposure to nodularin, and this will require a better understanding of the normal range of liver colouration in mugilids. We considered the possibility that our findings of hepatomegaly and orange/tan discolouration of the liver were related to exposure to Nodularia and/or nodularin, but we were unable to find reports that supported this hypothesis, as macroscopic changes in fish livers are not described in the literature on nodularin exposure in fish (Table 1). Kankaanpää et al. [21], in their oral dosing study of nodularin in trout, reported no gross pathological changes apart from inflammation of the intestine. From the larger body of literature on the effects of microcystins on fish, reviewed by Malbrouck and Kestemont [42], varying descriptions of gross pathological presentation of the liver are found. Kotak et al. [43] describe livers that were “wet, soft and pale tan or creamy white, with multiple, small ecchymoses visible beneath the capsule” in rainbow trout (Oncorhynchus mykiss) dosed with MC-LR at 1 ppm, and “red/tan mottled livers” seen at lower exposures. Best et al. [44], also working with O. mykiss, describe swollen, pale grey-coloured livers in fish exposed to MC-LR. Hepatomegaly, as reported by the hepatosomatic index (HSI), is also reported by these same authors. Kotak et al. [43] noted dose-related increases of 39% and 45% in HSI of MC-LR-dosed trout compared to controls; Best et al. [44] reported increased HSI of approximately 50%. In this study, the median HSI in mullet caught on 7 September 2009 was 1.66, a 52% increase over the median “control” group HSI of 0.8 (p = 0.016). A histological finding (Table 5) that correlated with the macroscopic examination of livers was that the three fish with gross hepatomegaly and orange discolouration seen at necropsy had a markedly reduced number of melanomacrophage centres (MMCs) in liver tissues; all other specimens, i.e., fish from control lakes and fish from the study lake caught on 29 September 2009—the latter with nodularin present but macroscopically normal livers—had clearly identifiable MMCs in liver tissue. Fish MMCs are macrophage aggregates, found in splenic and renal tissues and in the livers of some groups [45].

Because fish liver specimens were not prepared under ideal conditions, i.e., they were not removed and formalin fixed in the field immediately after capture, some autolysis was evident in three of 11 slides examined. However, fish were placed on ice at the field site, which presumably slowed down autolytic changes sufficiently for some histological assessment to be made. Histological examination of liver tissues not affected by autolysis could not differentiate specimens that were exposed to nodularin from those captured from the reference lakes with no nodularin liver residues (both fish groups were independently allocated a HRS of 6.0).

In future exposure studies, organs in addition to the liver such as gills, kidney, spleen, central nervous system (brain, spine), gastrointestinal tract, gonads and muscle should be collected for histopathology and nodularin residues. Collectively, examination of these tissues may provide important information on the toxicokinetics of nodularin in the fish and will allow us to determine whether nodularin produces recognisably consistent pathological changes.

Future use of organ histopathology in the study of the pathogenic toxicology of nodularin in mullet would need to consider the exclusion of water quality factors not related to nodularin, e.g., other xenobiotic pollutants. The reason being that liver and organ responses to toxicants are generally non-specific, such that concurrent exposure to toxicants or poor water quality may obscure changes due to nodularin exposure alone. For this investigation, we measured only Nodularia and nodularin in water from the study lake and reference lakes, and in tissues of fish captured from these sites. Field studies in closed water bodies where a broader range of water quality parameters are measured for both reference and affected sites may aid in the interpretation of liver and organ changes where the risk of interest is nodularin or other cyanotoxins.

Our findings in this regard bear similarities to those reported by Kankaanpää et al. In that study, nodularin was measured in livers of flounders captured from the Gulf of Finland; histological examination of livers was also conducted. A range of pathological changes were seen that could not be correlated with nodularin concentration. Lesions seen in their wild-caught fish were attributed to a variety of probable causes such as parasite infestation and exposure to anthropogenic toxicants [28].

We considered the possibility of post-mortem redistribution of nodularin, given that fish sent to us after the fish kill in November 2008 were in poor condition; some specimens presented with obvious early decomposition of internal organs. The likely sequence of events following (presumed) death due to deoxygenation resulting from increased heterotrophic bacterial activity associated with the senescent Nodularia bloom is that dead fish would have sunk to the lake bottom, where they may have remained for a day or more before buoyancy due to gases produced during decomposition made the fish kill apparent. Post-mortem redistribution of pharmaceuticals is a concern for forensic toxicologists; lipophilic compounds are more prone to redistribution, which follows a concentration gradient as tissue breakdown occurs [46,47]. While nodularin is a water soluble toxin, we were nonetheless concerned about the possibility that undigested toxin present in the alimentary tract of the fish-kill mullet may have contributed to the high concentrations of nodularin we detected in hepatic tissues of these fish. We extracted peritoneal contents of these three specimens, but peritoneal nodularin concentrations were well below those found in liver tissues (median 0.2 mg/kg vs. 12 mg/kg wet weight; see Table 2). We conclude, therefore, that the toxin we detected in the livers of these mullet was concentrated in those tissues through dietary exposure and active uptake, as the toxicokinetics and organ-specific affinities are understood for the ADDA-containing cyanotoxins [48,49].

Nodularin concentrations in carnivorous fish were several orders of magnitude lower than those found in the two species of Mugilidae. This supports the general premise outlined by Ibelings and Havens [50] that for water-soluble cyanotoxins such as nodularin, biodilution rather than biomagnification can be expected at higher trophic levels. Our work also supports the findings and conclusions of Berry et al. [51] regarding bioaccumulation in fish of the microcystin (MC) group of cyanotoxins. Berry et al. found higher concentration of MCs in phytoplanktivorous fish compared to omnivores and zooplanktivores, with the implication that bioaccumulation of these water-soluble toxins is more a function of direct dietary exposure to cyanobacterial cells, not a biomagnification effect. The discovery that mullet are capable of concentrating extraordinarily high concentrations of nodularin in hepatic tissues may be explained both by a presumed congruity in uptake kinetics with the cyclic peptide microcystins, these utilising a process of active transport into the liver [52], and a high degree of exposure to Nodularia and hence nodularin through their feeding strategies.

Public health authorities and researchers first became aware of a dense Nodularia bloom at the study lake in September 2008. As discussed above, the fish kill some two months later followed treatment of the lake with Phoslock^®. We thought that a deoxygenation event triggered by collapse of the bloom was a more likely explanation for the fish kill than a direct toxicity effect due to nodularin, in part because fish other than mullet involved in the kill had much lower concentrations of nodularin in their livers at the time. On two occasions three weeks apart in September 2009, we again found high concentrations of nodularin in the livers of mullet captured from the study lake. Lake water at the time of the two fish capture surveys in September 2009 was free of detectable nodularin, and Nodularia populations were low. We did, however, receive anecdotal reports of a green discolouration in the lake some two weeks before our first capture survey on 7 September 2009. We assumed that the findings of high nodularin levels, enlarged and discoloured livers in early September, followed by high nodularin levels and normal-looking livers three weeks later could be explained by re-exposure to and partial recovery from nodularin produced by a transient Nodularia bloom in late August 2009. We suspect that this lake is intermittently affected by phytoplankton blooms; a review of satellite images of the site since 2003 using the historical imagery function in Google Earth suggests a dynamic process in this regard. However, we cannot rule out the possibility that nodularin may have remained intact in the lake sediment since the 2008 bloom, and may have been available to mullet—again, through their unusual feeding strategy involving uptake of sediment material—for subsequent exposure through the following year. Some organic compounds produced by cyanobacteria and other phytoplankton are extremely stable in sediments. Indeed, chlorophyll, carotenoids and other compounds can be reliably detected in prehistoric sediments and are used as the basis for determining phytoplankton assemblages in ancient lakes [53,54]. Nodularin is reportedly capable of persisting for “several months” in sediments [12], and the related cyclic peptide toxins, the microcystins, have been recovered from lake sediments at a depth of 30cm, which implies a capacity for longevity in some circumstances [55]. Experimental work has shown that nodularin can adsorb to sediment particles [56].

The discovery of nodularin in liver and muscle tissues of mullet has implications for public health, with the potential for dietary transfer of the toxin should these fish be caught and eaten. Nodularin in marine food products clearly concerns researchers in Baltic Sea states, as seen in the literature cited in Table 1. Other cyanotoxins are also coming under scrutiny from a food safety perspective, with a growing body of literature reporting the presence of cyanobacterial toxins in foods of marine and freshwater origin. The majority of reports pertain to microcystins; for recent examples see [57,58,59,60]. Microcystins are probably the most frequently encountered and widely distributed group of freshwater cyanobacterial toxins. Saxitoxins (of cyanobacterial origin) and cylindrospermopsin have been found in edible marine and aquatic animals after both natural and experimental exposures [61,62].

The emerging public health issue of cyanobacterial toxins in fish and shellfish was discussed by Ibelings and Chorus [63], who present a three-tier guideline value proposal for microcystin-LR in aquatic foods. These authors have calculated tolerable exposure levels for both children and adults, through acute, sub-acute and chronic exposures. The Australian state of Victoria has produced guideline concentrations for a range of cyanotoxins in seafood and shellfish harvested from the Gippsland Lakes [64]. This estuarine lake system has long been adversely affected by toxigenic cyanobacterial blooms, principally Nodularia. To our knowledge, Victoria is the first government agency anywhere in the world to move beyond ad hoc decision-making to direct fisheries closures for protection of public health from toxic cyanobacteria by adopting and publishing guideline values developed from a human health risk assessment approach. However, governments across the world are recognising the risks these potent toxins present, and have sanctioned guidelines for exposure from drinking water and recreational waters [65]. Many national and trans-national agencies (the European Union, for example) monitor and regulate concentrations of eukaryotic marine algal toxins such as saxitoxins, okadaic acid and domoic acid in seafood and shellfish [66]. Cyanobacterial toxins are considered along with eukaryotic HAB toxins in Denmark, where commercial harvesting of mussels is subject to closure by regulatory authorities in the event that toxins are detected through their monitoring program [65]. Victoria has assumed a food safety leadership role with regard to monitoring and regulation of cyanotoxins in foods harvested from a lake system frequently subject to toxic cyanobacteria blooms. Their guidelines now form the basis for specific public health advice by the Victorian Department of Health regarding consumption of seafood and shellfish from the Gippsland Lakes; results of their monitoring program are posted on a publicly accessible website [64,67].

A direct application of the Victorian Government guidelines to the data generated for this report should be interpreted cautiously, as the guidelines ascribe recommended safety values to cyanotoxin concentrations in whole fish [64], which we did not measure (we analysed muscle and liver tissues separately). Nodularin concentrations in mullet muscle were all below the guideline values of 39 ppb for adults and 24 ppb for children. But concentrations in liver were noteworthy: the highest wet weight liver concentration we found in fresh mullet, 7.2 ppm, represents a 185-fold exceedance for adults and a 300-fold compliance failure for a child.

In Australia, toxic Nodularia blooms have affected large estuarine systems in the southern and south-west regions of the continent. The Murray River’s estuarine lakes in South Australia, Western Australia’s Peel-Harvey and Vasse-Wonnerup estuaries and Victoria’s Gippsland Lakes all suffer massive intermittent Nodularia blooms; shellfish and prawn fisheries in the Gippsland Lakes have been subject to temporary closure [13,68,69,70,71,72]. Nodularia blooms have declined in the Harvey estuary since the construction in 1994 of an artificial channel to flush the system with seawater, though blooms still occur in tidal reaches of inflowing rivers. Nodularia blooms have also been reported from a coastal lake system in Tasmania, Australia’s most southerly State [73]. The Eastern seaboard (and lower latitude) state of New South Wales has not, to our knowledge, experienced any significant Nodularia blooms in its estuaries, and in Queensland this species has only been found in the recreational lake that is the focus of this report and its companion paper [35].

Various mullet species, principally M. cephalus, are harvested commercially and recreationally from estuaries in all Australian mainland states; the Nodularia-prone Peel-Harvey, Vasse-Wonnerup, Gippsland Lakes and Murray Lakes systems support valuable commercial mullet fisheries [72,74,75,76]. Australia’s wild-caught mullet fisheries are valued at more than A$14 million annually, with production of 5,500 tonnes in 2009–2010 [77]. Globally, various mullet species support commercial, recreational and subsistence fisheries. Mullet aquaculture and larviculture are very substantial industries worldwide [78,79]. Egypt produced over 150,000 tonnes in 2005, which then represented 29 per cent of that nation’s aquaculture production [80]. M. cephalus are being investigated for their potential as a mariculture bioremediator; benthic enclosures anchored beneath finfish net cages are stocked with mullet, which feed on the sediments with accumulated organic waste that falls out of the main enclosures [81,82].

We suggest that Mugilidae raised in polyculture systems or as bioremediators in nutrient-enriched aquaculture or mariculture processes that may facilitate proliferation of cyanobacterial blooms, should such stock enter the human food supply, should be subject to research scrutiny for their ability to accumulate cyanotoxins such as nodularin, microcystins and cylindrospermopsin. This study presents preliminary evidence that mullet have an unusual capacity to concentrate such toxins in their livers, and that the toxins may also be present in muscle tissues. Concentrations of cyanotoxins in mullet tissues that may pose a food safety risk may not manifest in obvious behavioural changes in the fish.

Viewing the high concentration of nodularin in mullet livers from a food safety perspective, the risk of acute intoxication arising from this particular event would appear to be somewhat remote, given that fish liver is generally not eaten by the dominant Caucasian population in Australia, and to capture mullet from this small recreational lake would have required a fairly concerted effort to trawl with nets during the night, as the proprietor of the cable ski business does not allow fishing activities when the park is operating in daytime hours. However, the potential for acute poisoning due to cyanotoxins in mullet liver should be considered; dietary practices within some communities, particularly those of island cultures for which seafood is of paramount significance, suggest fish liver and other viscera are consumed. Fish liver is a delicacy in Micronesia; indeed, nutritional research in these island states seeks to encourage consumption of fish liver as an excellent source of vitamin A [83]. As for mullet, we have anecdotal reports that sea mullet liver is a constituent of certain dishes prepared by particular ethnic communities in Australia. A paper from a Spanish group notes that the mugilid Liza ramada “…may be consumed with or without the liver, depending on eating habits” [84]. The internet is generously supplied with recipes for mullet liver, but these appear to refer to “red mullet”, a common name that encompasses several Mediterranean species not belonging to the Mugilidae. Liver of European flounder is regarded as a delicacy by certain cultural groups; bioaccumulation of nodularin by flounder is the focus of attention by researchers in Baltic Sea states, and recommendations have been made in this regard to avoid consumption of flounder liver [28].

Mullet roe is a profitable export commodity [85], including from Australia where roe is the most valuable component of the fish [86,87]. Future toxicokinetics studies of nodularin and microcystins in mullet will need to assess the capacity of these toxins to bioaccumulate in the roe.

A systematic review of the literature was conducted to identify observational and experimental measurements of nodularin in aquatic and marine animals that are food items for human consumption. The search string “nodularin AND (fish OR seafood OR shellfish)” was used in both PubMed and Web of Science bibliographic databases. Manuscript titles and abstracts were perused to identify those reports discussing measurement of nodularin in tissues of seafood products. The bibliographies of papers thus identified were reviewed in order to identify other published reports that appeared to meet inclusion criteria for Table 1. Only English-language papers were consulted; the grey literature was not searched. Several papers describing nodularin concentrations in the three-spined stickleback (Gasterosteus aculeatus), a species often used for laboratory-based experiments, were not included as this fish is not a common food item, though it has been commercially harvested in previous decades and processed into fishmeal and animal fodder [88].

Liver and muscle tissues were removed from seven specimens collected after a fish kill at the lake under investigation in November 2008. Tissues from three sea mullet (Mugil cephalus), two yellowfin bream (Acanthopagrus australis), one longfin eel (Anguilla reinhardtii) and a greasy rockcod (Epinephelus tauvina) were examined. The fish kill occurred 12 days after commencement of a treatment regime comprising Phoslock^® (a phosphorus sequestrant) and polyaluminium chloride (a flocculant) adopted in an attempt to mitigate a toxic bloom of Nodularia that occurred initially in September of 2008. Another eel was captured in April 2009, i.e., several months after the bloom had dissipated. Tissues for control purposes were provided by two seafood processors: sea mullet livers and fillets from A.J. Raptis & Sons, Colmslie, Queensland (sourced from a fishery offshore from Ballina, New South Wales) and a whole longfin eel was purchased from Manchester Eels, Coopers Plains, Queensland.

Specimens retrieved from the study lake in November 2008 after the fish kill were undergoing early decomposition, so macroscopic and microscopic assessment of the condition of internal organs was not possible. Liver tissue was identifiable and able to be removed in sufficient amounts for extraction and quantification of nodularin in all specimens except for the two bream; muscle tissue only was examined in those fish.

Subsequent field investigations focused on mullet, as analysis of tissues from the fish kill showed that these species accumulated much higher concentrations of nodularin compared to other fish collected at the time. Field excursions to capture mullet from the cable ski park and nearby lakes were undertaken on two days in September 2009. On 7 September, a combined approach using shore seine netting and vertical gill netting was used. A subsequent excursion to capture fish from the study lake, and in particular to acquire more specimens from the nearby control lakes, was conducted on 29 September. The seine method proved to be more successful for catching mullet, so was employed exclusively on the follow-up capture excursion on 29 September. Captured mullet were immobilised by placing in ice slurry, after which they were killed by rapidly severing the spinal cord with a sharp knife. Intact carcases were kept on ice at 4 °C until necropsies were conducted the following day.

The recreational lake that is the focus of this investigation—a cable ski park in Logan Shire, southeast Queensland—is referred to as the “study lake,” or “recreational lake” throughout this report. Properties located approximately 1.5 km NW of the cable ski park, occupied by a golf course and a sand processing facility, contain a series of artificial freshwater and brackish water lakes. Two of these lakes—“west pond” and “model yacht pond”—were fished for mullet to serve as reference specimens. These sites are referred to collectively as “control lakes” or “reference lakes” in this report.

Of the fish received after the 2008 fish kill, the longfin eel was in the best condition; the liver of this specimen was used to develop the most suitable extraction method. We initially used a methanol extract with hexane wash and solid-phase extraction clean-up, but a simple methanol extraction (in duplicate) followed by HPLC-MS/MS in multiple reaction mode was found to be a more effective and reproducible technique, with minimal matrix effects observed in the electrospray mode. 2.0 mL of 80% v/v methanol in chromatography-grade water was added to 1 g tissue (liver and muscle tissues, analysed separately), homogenised with an Ultra Turrax T25 macerator, centrifuged at 3,500 rpm for 20 minutes and the supernatant was decanted. A second extraction was conducted, supernatants were combined and syringe filtered (0.45 μm). Samples were initially analysed in triplicate when sufficient tissue was available; after satisfying ourselves that our method was reproducible, subsequent analyses were conducted in duplicate.

Nodularin was determined by HPLC-MS/MS using an AB/Sciex API4000Q mass spectrometer (AB/Sciex, Concord, ON, Canada) equipped with an electrospray (TurboV) interface coupled to a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan). Separation was achieved using a 5 micron 150 × 4.6 mm Alltima C₁₈ column (Alltech®, Deerfield, IL, USA) run at 40 °C, and a flow rate of 0.9 mL/min with a linear gradient starting at 100% A, ramped to 90% B in 12 minutes then to 100% B in 1 minute and held for 3 minutes followed by equilibration at 100% A for 5 minutes (A = 1% methanol in HPLC grade water, B = 95% methanol in HPLC grade water, both containing 0.1% formic acid). The mass spectrometer was operated in the positive ion, multiple reaction-monitoring mode using nitrogen as the collision gas. Transitions monitored for nodularin were 825.5 (M + H)⁺ to 135 and 163 (relative intensity 28%) using a declustering potential of 125 and collision energies of 75 and 65 electron volts. The transitions monitored for microcystin-LR were 995.6 (M + H)⁺ to 135 and 213.2 (relative intensity 52%) using a declustering potential of 130 and collision energies of 110 and 85 electron volts. The retention times for nodularin and MC-LR were 9.2 and 9.6 minutes respectively.

Positive samples were confirmed by retention time and by comparing transition intensity ratios between the sample and an appropriate concentration standard from the same run. Samples were reported as positive if the two transitions were present, retention time was within 0.15 minutes of the standard and the relative intensity of the confirmation transition was within 20% of the expected value. The value reported was that for the quantitation transition. Using a 10 μL injection volume the limit of detection for this method was typically less than 1 μg/L, with a reporting limit of 2 μg/L in the final aqueous methanol extract. Response was linear to at least 500 μg/L.

Control sea mullet liver and muscle samples were spiked in triplicate with 750 pg nodularin standard and separately with 145 pg microcystin-LR. Standards were dissolved in methanol. Extracts were prepared as described above, and spiked with toxin prior to maceration. Nodularin and MC-LR standards were purchased from Abraxis LLC (Warminster, PA, USA). Nodularin in-house standard was prepared from N. spumigena bloom material. Lyophilised, sonicated cells were extracted with aqueous methanol, further concentrated by C₁₈ solid-phase extraction, and fractionated by reverse-phase HPLC. The nodularin-containing fraction was quantified by reference to the purchased standard and by UV spectroscopy; this in-house standard was used for spiking and routine analytical quantification.

Fish specimens captured from the study and control lakes were identified by DNA barcoding and comparison with the BOLD database, as described in [89]. Specimens that were unable to be reliably identified to species level by this procedure are reported at the Family taxon.

Livers were removed in the laboratory the day after specimens were captured. Livers that were able to be removed intact from mullet caught on the two fish netting excursions in September 2009 (n = 12) were weighed. Liver sub-samples were fixed in 10% buffered formalin, sectioned, stained with haematoxylin & eosin and mounted on glass microscope slides. Slides were labelled with an alpha-numeric code in order to blind pathologists (authors RC and AAS) to the identity of control and study lake fish.

Muscle tissues of four mullet specimens from the study lake were weighed in duplicate (9–14 g), frozen and lyophilised (Martin Christ Alpha 2–4 LD, Osterode, Germany). Dry tissues were then reweighed for estimation of dry weight yields. Liver tissues from study fish were not available for lyophilisation; sea mullet livers were donated by A.J. Raptis & Sons, Brisbane, Australia. As for muscle, livers were weighed and lyophilised in duplicate.

The stomach of one of the mullet retrieved from the study lake after the initial fish kill in November 2008 was intact and able to be opened. There was insufficient material inside the stomach to perform a solvent extraction for analysis of nodularin, but microscopic examination of the stomach contents revealed numerous fragments of Nodularia (see Figure 5). Stomach contents of mullet captured from study and control lakes in September 2009 were examined microscopically, and solvent extraction and HPLC-MS/MS for nodularin detection and quantification was conducted.

Distribution-free Mann-Whitney tests were used to compare:

A non-parametric test was adopted because small sample sizes did not permit normal distribution assessments. Analyses were conducted with GraphPad Prism v5.03; a significance level of 5% (p < 0.05) was adopted.