Murray cod (Maccullochella peelii peelii) is the largest and best-known Australian freshwater species. Its distribution and abundance has declined in the past 50 years because of the construction of dams, changes to river flows and temperatures, and pollution of their habitat from various sources (; http://www.deh.gov.au/water/basins/murray-cod/). In contrast, Murray cod aquaculture is an emerging industry with a large potential for rapid growth. The industry now produces both fingerlings for the stocking of waterways and table-sized fish (500–800 g) for human consumption (; http://www.abareconomics.com/publications_html/fisheries/fisheries_03/er03_aquaculture.pdf). Murray cod was identified by our group as a native Australian freshwater fish that is well suited to immunoecotoxicology testing of xenobiotics under laboratory conditions. They are readily available from fish farmers, adapt extremely well to indoor tanks, are robust to handling and stress, and feed on a standard commercial pellet diet, and a large amount of immune tissue can be obtained from one fish. Furthermore, they also have a high ecological, economic, recreational, and cultural value. Our research group has successfully adapted functional immune assays for Murray cod (i.e., lysozyme, mitogenesis, and phagocytosis), which have been previously used in mammals and other fish species to assess the immunotoxicity of environmental pollutants .
Lysozyme is one of three hydrolyase enzymes that have a defensive role in the circulatory system. In fish, it is found in the blood, mucus, and lymphomyeloid tissue, highlighting its role in fish innate immune systems, which are increasingly important, as their specific immune system is slower and less developed in comparison to mammals . Lysozyme was chosen as an indicator of immunomodulation because of the easy and rapid assay method and its importance in the innate defense of fish. The lymphoproliferative response to mitogens is similar to the adaptive lymphocytic response to antigens that are presented by macrophages; however, it does not require the action of antigen-presenting cells and occurs rapidly in response to natural agents conserved in many foreign organisms, such as bacteria (e.g., lipopolysaccharide) and plants (e.g., phytohemagglutinin [PHA]). The mitogen-stimulated lymphoproliferation assay assesses the function of lymphocytes and has often been used in immunotoxicity testing protocols . Phagocytosis is a primitive defense mechanism, conserved in both vertebrates and invertebrates. It has been used in a tiered system for the immunotoxicological assessment of environmental pollutants and immunostimulants used in aquaculture . Our group has recently reported the use of flow cytometry to measure the phagocytic activity of head kidney cells from three native Australian freshwater fish .
Numerous studies from abroad have demonstrated that many aquatic pollutants are immunotoxic in exotic species of fish. However, only limited studies have applied standardized immune functional assays to assess the immunotoxicity of environmental pollutants in native Australian freshwater fish [8–10]. Tributyltin (TBT) and dibutyltin (DBT) contamination of harbors and marinas has been a significant environmental concern because of their disruption of endocrine and reproductive functions and immunotoxic properties at very low concentrations . The majority of environmental organotin pollution is from biocidal antifouling paints that leach TBT to protect ship hulls from algal and mollusk growth. Although an international treaty effectively banned the use of TBT-based marine paints in 2003, organotins are expected to persist in the environment for many years . Furthermore, TBT and DBT also enter both freshwater and marine environments through treated woods, runoff from landfill, sewage, and industrial discharges . Once in the aquatic environment, they are bioaccumulated by invertebrates, fish, and aquatic mammals and can reach extremely high levels in the tissues of these organisms [14,15].
Environmental organotin residues in freshwater ecosystems are reported less frequently than levels in marine ecosystems. Nevertheless, international studies have shown that TBT and DBT contamination of freshwater ecosystems is significant. Residues of the organotins have been reported up to 54 and 220 ng/L in water columns , 143 and 520 ng/g in sediments [17,18], and 0.7 and 2.5 mg/kg in fish tissues [17,19] for DBT and TBT, respectively. Very few studies have investigated the contamination of TBT and DBT in Australian waters, especially in recent years. A study of TBT contamination along the Western Australia coast reported high levels of TBT in marine sediment (0.001–1.35 μg/g TBT) and mussel (Mytilus edulis) tissue (0.003–0.32 μg/g TBT), which correlated with areas of high boating activity . In New South Wales, Sydney Harbour had levels of 0.220 μg/L TBT and 0.051 μg/L DBT, and Georges River had lower levels of 0.1 μg/L TBT and 0.04 μg/L DBT , while oysters in the Georges River had bioaccumulated levels of 0.019 mg/kg DBT and 0.234 mg/kg TBT . No studies have been reported in the literature concerning organotin contamination of the Murray-Darling basin despite the expectation of some contamination in this freshwater environment.
In mammals, TBT and DBT have been known for their specific reduction of T-lymphocytes through apoptotic mechanisms , and their immunotoxic effects in aquatic organisms appear to be similar to those observed in mammals (i.e., thymus atrophy and a reduction in lymphocyte numbers ). Numerous studies have reported TBT and DBT as immunotoxins in fish, and our group has previously reported organotin-induced immunotoxicity in rainbow trout and in the Australian freshwater native silver perch (Bidyanus bidyanus) . In vitro studies using isolated spleen and head kidney cells from juvenile rainbow trout showed that TBT and DBT suppressed mitogen-stimulated lymphoproliferation at concentrations ⩾ 50 μg/L. The results also indicated that DBT was the more potent immunosuppressant . Furthermore, extensive in vitro studies have reported that TBT can either suppress or increase the phagocytic activity (i.e., chemiluminescence) of oyster toadfish (Opsanus tau) macrophages, depending on the dose given [24,25].
The immunotoxicity of in vivo TBT exposure in channel catfish (Ictalurus punctatus) and rainbow trout have been previously reported. Catfish exposed to 1.0 mg/kg TBT displayed decreased natural killer cell activity (at 3 and 7 d) and suppressed phagocyte oxidative burst (3 d after exposure) . Hematological examination of the fish showed an increase in circulating monocytes and neutrophils but a decrease in hematocrit values and lymphocyte numbers. Additionally, humoral responses were suppressed in all groups exposed to 0.01, 0.1, or 1.0 mg/kg TBT . In vivo studies using rainbow trout bathed in 4.0 μg/L TBT for 28 d have reported a doserelated lymphocytic depletion, a marked proliferation of reticuloendothelial cells, an increased erythrophagia in the spleen, severe lesions within epithelia of the gills, and pseudobranch epithelial cells . Extensive field experiments involving a number of different chemicals linked TBT as the causative agent of an increased prevalence of lymphocystis virus infections in the flounder (Platichthys flesus) along the Dutch coastline .
Preliminary immunotoxicity studies by our group demonstrated that in vitro exposures to organotins resulted in a reduction of lymphocyte subpopulations in three large native Australian freshwater fish: silver perch, golden perch (Macquaria ambigua), and Murray cod. However, in the crimson-spotted rainbowfish (Melanotaenia fluviatilis), granulocytes were the most sensitive subpopulation. The phagocytic activity of head kidney granulocytes was suppressed in golden perch, Murray cod, and rainbowfish, and of the native fish species tested, silver perch head kidney cells were the least sensitive to in vitro exposures of TBT and DBT . In the present study, TBT and DBT were used as exemplar immunotoxins to assess the validity of immune function assays (i.e., mitogen-stimulated lymphoproliferation and phagocytosis in head kidney tissue and serum lysozyme activity) and to compare the sensitivity of Murray cod to other fish species.