Michael R. van den Heuvel

Factors impacting on pharmaceutical leaching following sewage application to land.

Sewage effluent application to land is a treatment technology that requires appropriate consideration of various design factors. Soil type, level of sewage pre-treatment and irrigation rate were assessed for their influence on the success of soil treatment in removing pharmaceuticals remaining after conventional sewage treatment. A large scale experimental site was built to assess treatment performance in a realistic environment. Of the factors investigated, soil type had the biggest impact on treatment performance. In particular, carbamazepine was very efficiently removed (>99%) when irrigated onto a volcanic sandy loam soil. This was in contrast to irrigation onto a sandy soil where no carbamazepine removal occurred after irrigation. Differences were likely caused by the presence of allophane in the volcanic soil which is able to accumulate a high level of organic matter. Carbamazepine apparent adsorption distribution coefficients (K(d)) for both soils when irrigated with treated sewage effluent were determined as 25 L kg(-1) for the volcanic soil and 0.08 L kg(-1) for the sandy soil. Overall, a volcanic soil was reasonably efficient in removing carbamazepine while soil type was not a major factor for caffeine removal. Removal of caffeine, however, was more efficient when a partially treated rather than fully treated effluent was applied. Based on the investigated pharmaceuticals and given an appropriate design, effluent irrigation onto land, in conjunction with conventional sewage treatment may be considered a beneficial treatment for pharmaceutical removal.

Relative sensitivities of common freshwater fish and invertebrates to acute hypoxia

Abstract The acute lethality of low dissolved oxygen (DO) was examined in laboratory studies using several New Zealand freshwater fish and two invertebrates at 15°C. The 48‐h LC50 value was used as the endpoint for acute DO sensitivity as, owing to rapid mortality, this was found to best approximate the threshold lethal concentration. Median lethal time to death did not provide a reliable endpoint for comparing sensitivities. Fish LC50 values varied from 0.54 to 2.65 mg litre‐1 , with inanga whitebait (Galaxias maculatus; 2.65 ± 0.19 mg litre‐1 , mean + SEM) being the most sensitive species tested. Common smelt (Retropinna retropinna; 1.83 ± 0.08 mg litre–1) and rainbow trout (Oncorhynchus mykiss; 1.61 ± 0.06 mg litre–1) were similar in their sensitivities, whereas common bully (Gobiomorphus cotidianus; 0.91 ± 0.06 mg litre–1) and shortfin eel elvers (Anguilla australis; 0.54 ± 0.03 mg litre–1) were the most tolerant fish. The shrimp (Paratya curvirostris; 0.82 ± 0.09 mg litre–1) and freshwater crayfish (Koura, Paranephrops planifrons; 0.77 ± 0.06 mg litre–1) were also tolerant to low DO. A subset of experiments to determine the relative sensitivities of larval and juvenile trout and bully indicated no significant differences between these life stages.

REPRODUCTIVE DEVELOPMENT OF YELLOW PERCH (PERCA FLAVESCENS) EXPOSED TO OIL SANDS-AFFECTED WATERS

In similar experiments conducted in 1996 and 2009, yellow perch (Perca flavescens) were stocked into two experimental systems: a demonstration lake where oil sands fine tailings were capped with natural water and a lake in a watershed containing bitumen-bearing sodic clays. In both experiments, yellow perch were captured in May from a nearby reservoir and released into the experimental ponds. Perch were recaptured in the experimental systems, the source lake, and two reference lakes in late September and lethally sampled to examine reproductive parameters. In the 1996 experiment, gonad size and steroid hormones were not affected in either pond environment. In the 2009 experiment, male perch in the water-capped tailings pond showed a significant reduction in the testicular development and reductions in circulating testosterone and 11-ketotestosterone, while no reductions were seen in the second experimental pond. No changes were observed in ovarian size or circulating steroid levels in female perch. In the pond containing tailings, the release of water from underlying tailings caused approximately a twofold increase in salinity, alkalinity, and naphthenic acids, and a pH increase from 8.4 to 9.4 over the 13-year period of the study. In the pond influenced by unextracted oil sands materials, total dissolved solids, major ions, and pH did not change substantially. However, naphthenic acids in this system dropped more than twofold post-watershed reclamation. Because the selective reproductive effect observed in male perch in the experimental end-pit lake were accompanied by increases in naphthenic acids, alkalinity, and pH, a specific cause cannot be determined. The present study adds to the evidence, suggesting the presence of endocrine-disrupting substances in oil sands.

Immunotoxic effects of oil sands-derived naphthenic acids to rainbow trout

Naphthenic acids are the major organic constituents in waters impacted by oil sands. To investigate their immunotoxicity, rainbow trout (Oncorhynchus mykiss) were injected with naphthenic acids extracted from aged oil sands tailings water. In two experiments, rainbow trout were injected intraperitoneally with 0, 10, or 100 mg/kg of naphthenic acids, and sampled after 5 or 21 d. Half of the fish from the 21 d exposure were co-exposed to inactivated Aeromonas salmonicida (A.s.) to induce an immune response. A positive control experiment was conducted using an intraperitoneal injection of 100 mg/kg of benzo[a]pyrene, a known immune suppressing compound. T-lymphocytes, B-lymphocytes, thrombocytes, and myeloid cells were counted in blood and lymphatic tissue using flow cytometry. In the 5d exposure, there was a reduction in blood leucocytes and spleen thrombocytes at the 100 mg/kg dose. However, at 21 d, leucocyte populations showed no effects of exposure with the exception that spleen thrombocyte populations increase at the 100 mg/kg dose. In the 21 d exposure, B- and T-lymphocytes in blood showed a significant Dose × A.s. interaction, indicating stimulated blood cell proliferation due to naphthenic acids alone as well as due to A.s. Naphthenic acid injections did not result in elevated bile fluorescent metabolites or elevated hepatic EROD activity. In contrast to naphthenic acids exposures, as similar dose of benzo[a]pyrene caused a significant decrease in B- and T-lymphocyte absolute counts in blood and relative B-lymphocyte counts in spleen. Results suggest that the naphthenic acids may act via a generally toxic mechanism rather than by specific toxic effects on immune cells.

Immunological impacts of oil sands-affected waters on rainbow trout evaluated using an in situ exposure

Rainbow trout were exposed in situ to oil sands-affected waters for 21 d, either with or without an immune stimulation using inactivated Aeromonas salmonicida. Three aquatic systems were utilized for the experiment: a pond containing oil sands tailings capped with approximately 3 m of natural surface water, a second pond where unextracted oil sands materials were deposited in the watershed, and a reservoir receiving Athabasca River water as a reference caging location. The three systems showed a gradient of oil sands-related compounds, most notably, total naphthenic acids were highest in the system containing tailings (13 mg/L), followed by the system influenced by unextracted oil sands (4 mg/L), followed by the reference cage location (1 mg/L). Biochemical and chemical measures of exposure in rainbow trout showed the same trend, with the tailings-influenced system having the highest hepatic EROD activity and elevated bile fluorescence measured at phenanthrene wavelengths. Trout caged in the tailings-influenced location had significantly fewer leukocytes and smaller spleens as compared to the reference fish, though liver size and condition factor were unaffected. Fish in the tailings-influenced waters also demonstrated increased fin erosion, indicative of opportunistic infection. The trout exposed to tailing-influenced waters also showed a significantly decreased ability to produce antibodies to the inactivated A. salmonicida. Given the complexity of the exposure conditions, exact causative agents could not be determined, however, naphthenic acids, polycyclic aromatic hydrocarbons and pH correlate with the immunotoxic effects while elevated salinity or metals seem unlikely causes.

Distinct migratory and non-migratory ecotypes of an endemic New Zealand eleotrid (Gobiomorphus cotidianus) – implications for incipient speciation in island freshwater fish species

BackgroundMany postglacial lakes contain fish species with distinct ecomorphs. Similar evolutionary scenarios might be acting on evolutionarily young fish communities in lakes of remote islands. One process that drives diversification in island freshwater fish species is the colonization of depauperate freshwater environments by diadromous (migratory) taxa, which secondarily lose their migratory behaviour. The loss of migration limits dispersal and gene flow between distant populations, and, therefore, is expected to facilitate local morphological and genetic differentiation. To date, most studies have focused on interspecific relationships among migratory species and their non-migratory sister taxa. We hypothesize that the loss of migration facilitates intraspecific morphological, behavioural, and genetic differentiation between migratory and non-migratory populations of facultatively diadromous taxa, and, hence, incipient speciation of island freshwater fish species.ResultsMicrochemical analyses of otolith isotopes (88Sr, 137Ba and 43Ca) differentiated migratory and non-migratory stocks of the New Zealand endemic Gobiomorphus cotidianus McDowall (Eleotridae). Samples were taken from two rivers, one lake and two geographically-separated outgroup locations. Meristic analyses of oculoscapular lateral line canals documented a gradual reduction of these structures in the non-migratory populations. Amplified fragment length polymorphism (AFLP) fingerprints revealed considerable genetic isolation between migratory and non-migratory populations. Temporal differences in reproductive timing (migratory = winter spawners, non-migratory = summer spawners; as inferred from gonadosomatic indices) provide a prezygotic reproductive isolation mechanism between the two ecotypes.ConclusionThis study provides a holistic look at the role of diadromy in incipient speciation of island freshwater fish species. All four analytical approaches (otolith microchemistry, morphology, spawning timing, population genetics) yield congruent results, and provide clear and independent evidence for the existence of distinct migratory and non-migratory ecotypes within a river in a geographically confined range. The morphological changes within the non-migratory populations parallel interspecific patterns observed in all non-migratory New Zealand endemic Gobiomorphus species and other derived gobiid taxa, a pattern suggesting parallel evolution. This study indicates, for the first time, that distinct ecotypes of island freshwater fish species may be formed as a consequence of loss of migration and subsequent diversification. Therefore, if reproductive isolation persists, these processes may provide a mechanism to facilitate speciation.

The immunological effects of oil sands surface waters and naphthenic acids on rainbow trout (Oncorhynchus mykiss)

There is concern surrounding the immunotoxic potential of naphthenic acids (NAs), a major organic constituent in waters influenced by oil sands contamination. To assess the immunological response to NAs, rainbow trout (Oncorhynchus mykiss) waterborne exposures were conducted with oil sands-influenced waters, NAs extracted and purified from oil sands tailings waters, and benzo[a]pyrene (BaP) as a positive control. After a 7d exposure, blood, spleen, head kidney, and gill samples were removed from a subset of fish in order to evaluate the distribution of thrombocytes, B-lymphocytes, myeloid cells, and T-lymphocytes using fluorescent antibodies specific for those cell types coupled with flow cytometry. The remaining trout in each experimental tank were injected with inactivated Aeromonas salmonicida and held in laboratory water for 21 d and subjected to similar lymphatic cell evaluation in addition to evaluation of antibody production. Fluorescent metabolites in bile as well as liver CYP1A induction were also determined after the 7 and 21 d exposure. Oil sands waters and extracted NAs exposures resulted in an increase in bile fluorescence at phenanthrene wavelengths, though liver CYP1A was not induced in those treatments as it was with the BaP positive control. Trout in the oil sands-influenced water exposure showed a decrease in B- and T-lymphocytes in blood as well as B-lymphocytes and myeloid cells in spleen and an increase in B-lymphocytes in head kidney. The extracted NAs exposure showed a decrease in thrombocytes in spleen at 8 mg/L and an increase in T-lymphocytes at 1mg/L in head kidney after 7d. There was a significant decrease in antibody production against A. salmonicida in both oil sands-influenced water exposures. Because oil sands-influenced waters affected multiple immune parameters, while extracted NAs impacts were limited, the NAs tested here are likely not the cause of immunotoxicity found in the oil sands-influenced water.

Kraft mill effluent survey: Progress toward best management practices for reducing effects on fish reproduction

Pulp and paper mill effluents have been linked to effects on fish reproduction for more than 25 years. To date, the causes of these effects and remedial strategies have eluded investigators. Recent work has shown that the degree of reproductive effect caused by a mill effluent is related to the overall organic content. If verified, this could lead to breakthroughs for best management practices (BMPs). For this study, the effluents from seven kraft mills were assessed for their ability to reduce egg production in fathead minnow (Pimephales promelas) in the laboratory. At the same time, the effluents were analyzed for three parameters thought to be good indicators of organic losses: biochemical oxygen demand (BOD), methyl-substituted 2-cyclopentenones (cellulose degradation products), and a gas chromatographic (GC) profile index, which integrates the total area of the chromatographic peaks of solvent-extracted effluents using low-resolution mass spectrometry. The results showed that the degree to which the effluents reduced egg production increased with increased organic losses as characterized by BOD and the GC profile index. Therefore, these parameters could be used to guide BMPs at kraft mills according to specific targets: BOD < 20 mg/L and GC profile index equivalent to effluent with BOD of 20 mg/L. Such targets should be achievable by good in-plant control of organic losses and optimized effluent biotreatment systems.

The Role of Submerged Aquatic Vegetation in Structuring the Nearshore Fish Community Within an Estuary of the Southern Gulf of St. Lawrence

Artificial fertilizers are contributing to the replacement of eelgrass (Zostera marina) by sea lettuce (Ulva lactuca) in estuaries of Prince Edward Island (PEI), Canada. In this study, we found that the nearshore fish community differed between areas dominated by these two vegetations within an estuary in every month sampled (April–August). Adult northern pipefish (Syngnathus fuscus), threespine stickleback (Gasterosteus aculeatus), blackspotted stickleback (Gasterosteus wheatlandi), and Atlantic silverside (Menidia menidia) were most strongly associated with eelgrass, while mummichog (Fundulus heteroclitus), ninespine stickleback (Pungitius pungitius), and American eel (Anguilla rostrata) were often more numerous in sea lettuce. Sea lettuce stations tended to have more young-of-the-year mummichog, fourspine stickleback (Apeltes quadracus), and Gasterosteus sp. than eelgrass stations but fewer young-of-the-year northern pipefish and Atlantic silverside. Fish richness and abundance were significantly lower in the sea lettuce than eelgrass habitat during August when benthic hypoxia occurred. We conclude that the loss of eelgrass from PEI estuaries will result in significant declines in fish biodiversity.

Simultaneous determination of androgenic and estrogenic endpoints in the threespine stickleback (Gasterosteus aculeatus) using quantitative RT-PCR

A method to evaluate the expression of three hormone responsive genes, vitellogenin (estrogens), spiggin (androgens), and an androgen receptor (ARbeta) using real-time PCR in threespine stickleback is presented. Primers were designed from previously characterised spiggin and ARbeta sequences, while a homology cloning strategy was used to isolate a partial gene sequence for stickleback vitellogenin (Vtg). Spiggin mRNA was significantly higher in kidneys of field-caught males compared to females by greater than five orders of magnitude while ARbeta levels were only 1.4-fold higher in males. Female fish had four order of magnitude higher liver Vtg expression than wild-captured males. To determine the sensitivity of these genes to induction by hormones, male and female sticklebacks were exposed to 1, 10 and 100 ng/L of methyltestosterone (MT) or estradiol (E2) in a flow-through exposure system for 7 days. Spiggin induction in females, and Vtg induction in males were both detectable at 10 ng/L of MT and E2, respectively. MT exposure did not induce ARbeta expression in the kidneys of female stickleback. In vitro gonadal steroid hormones production was measured in testes and ovaries of exposed stickleback to compare gene expression endpoints to an endpoint of hormonal reproductive alteration. Reduction in testosterone production in ovaries at all three MT exposure concentrations, and ovarian estradiol synthesis at the 100 ng/L exposure were the only effects observed in the in vitro steroidogenesis for either hormone exposure. Application of these methods to assess both androgenic, estrogenic, and anti-steroidogenic properties of environmental contaminants in a single fish species will be a valuable tool for identifying compounds causing reproductive dysfunction in fishes.