Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells.

Alternative complement activity was determined in whole body homogenates (WBHs) and serum samples of different fish species, by measuring the amount of sample that induces 50% hemolysis of red blood cells using the ACH50 assay (Alternative Complement pathway Hemolytic activity). Values of ACH50 obtained for serum samples were about two-fold higher when using rabbit red blood cells (RRBC), as compared to sheep red blood cells (SRBC). The increase in ACH50 when using RRBCs for WBH samples was 28, 7 and 4 folds for guppy, molly and zebrafish, respectively. Large variability in complement activity was evident between fish species for both serum and WBHs. Evaluating the effect of freeze-thaw cycles on complement revealed significant reduction in complement activity in all tested samples. Loss of activity following three freeze-thaw cycles amounted to 48-59% when serum was tested and over 95% loss in activity for WBH. To our knowledge, this is the first study where fish WBHs were used for assaying complement activity. Our results support the suitability of this method in evaluating complement activity in small fish species or larvae, where blood cannot be obtained, as long as samples can be tested upon first thawing.

Genotoxic and mutagenic assessment of iron oxide (maghemite-γ-Fe2O3) nanoparticle in the guppy Poecilia reticulata.

The environmental risk of nanomaterials (NMs) designed and used in nanoremediation process is of emerging concern, but their ecotoxic effects to aquatic organism remains unclear. In this study, the citrate-coated (maghemite) nanoparticles (IONPs) were synthesized and its genotoxic and mutagenic effects were investigated in the female guppy Poecilia reticulata. Fish were exposed to IONPs at environmentally relevant iron concentration (0.3 mg L-1) during 21 days and the animals were collected at the beginning of the experiment and after 3, 7, 14 and 21 days of exposure. The genotoxicity and mutagenicity were evaluated in terms of DNA damage (comet assay), micronucleus (MN) test and erythrocyte nuclear abnormalities (ENA) frequency. Results showed differential genotoxic and mutagenic effects of IONPs in the P. reticulata according to exposure time. The IONP induced DNA damage in P. reticulata after acute (3 and 7 days) and long-term exposure (14 and 21 days), while the mutagenic effects were observed only after long-term exposure. The DNA damage and the total ENA frequency increase linearly over the exposure time, indicating a higher induction rate of clastogenic and aneugenic effects in P. reticulata erythrocytes after long-term exposure to IONPs. Results indicated that the P. reticulata erythrocytes are target of ecotoxicity of IONPs.

Single-cell gel electrophoresis assay in the ten spotted live-bearer fish, Cnesterodon decemmaculatus (Jenyns, 1842), as bioassay for agrochemical-induced genotoxicity.

The ability of two 48 percent chlorpyrifos-based insecticides (Lorsban* 48E® and CPF Zamba®), two 50 percent pirimicarb-based insecticides (Aficida® and Patton Flow®), and two 48 percent glyphosate-based herbicides (Panzer® and Credit®) to induce DNA single-strand breaks in peripheral blood erythrocytes of Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces, Poeciliidae) exposed under laboratory conditions was evaluated by the single-cell gel electrophoresis (SCGE) assay. In those fish exposed to Lorsban* 48E®, CPF Zamba®, Aficida®, Patton Flow®, Credit®, and Panzer®, a significant increase of the genetic damage was observed for all formulations regardless of the harvesting time. This genotoxic effect was achieved by an enhancement of Type II-IV comets and a concomitant decrease of Type 0-I comets over control values. A regression analysis revealed that the damage varied as a negative function of the exposure time in those Lorsban* 48E®- and Aficida®-treated fish. On the other hand, a positive correlation between damage increase and exposure time was achieved after Patton Flow® and Credit® treatment. Finally, no correlation was observed between increase in the genetic damage and exposure time after treatment with CPF Zamba® or Panzer®. These results highlight that all agrochemicals inflict primary genotoxic damage at the DNA level at sublethal concentrations, regardless of the exposure time of the aquatic organisms under study, at least within a period of 96 h of treatment.

Measuring water-borne cortisol in Poecilia latipinna:is the process stressful, can stress be minimized and is cortisol correlated with sex steroid release rates?

The stress of water-borne hormone collection process was examined in sailfin mollies Poecilia latipinna. Baseline release rates of the stress hormone cortisol were measured and minimum confinement time for water sampling was evaluated for a standard 60 min v. a 30 min protocol. A 30 min hormone collection period reflects release rates over 60 min. Potential stress response to confinement in the beaker for the water-borne collection process was tested over 4 days. There was no evidence of stress due to the collection methods, as cortisol release rates did not differ significantly across four sequential days of handling for P. latipinna. Males and females did not differ significantly in baseline cortisol release rates. Baseline cortisol release rates from fish immediately after being collected in the field were also not significantly different than those in the 4 day confinement experiment. After exposure to a novel environment, however, P. latipinna mounted a stress response. Stress may also affect sex steroids and behaviour but cortisol release rates were not significantly correlated with sex steroids [11-ketotestosterone (KT), testosterone, or oestradiol], or mating attempts. The correlation between water-borne release rates and plasma steroid levels was validated for both cortisol and KT. Finally, normalizing cortisol release rates using standard length in lieu of mass is viable and accurate. Water-borne hormone assays are a valuable tool for investigating questions concerning the role of hormones in mediating stress responses and reproductive behaviours in P. latipinna and other livebearing fishes.