Louis A. Tremblay

The use of biomarkers in ecological risk assessment: recommendations from the Christchurch conference on Biomarkers in Ecotoxicology

S. MARSHALL ADAMS1, JOHN P. GIESY 2, LOUIS A. TREMBLAY3 and CHARLES T. EASON3* 1 Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 USA 2 Aquatic Toxicology Laboratory, Department of Zoology, Institute of Environmental Toxicology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824 USA 3 CENTOX (Centre for Environmental Toxicology), Landcare Research, PO Box 69, Lincoln, New Zealand.

Steroid estrogens, conjugated estrogens and estrogenic activity in farm dairy shed effluents.

Agricultural wastes are a source of steroid estrogens and, if present, conjugated estrogens may add to the estrogen load released to soil and aquatic environments. Dairy shed effluent samples were collected from 18 farms for analysis of steroid estrogens by GC-MS, conjugated estrogens by LC-MS-MS, and estrogenic activity by E-screen in vitro bioassay. 17alpha-estradiol was found at highest concentrations (median 730 ng l(-1)), followed by estrone (100 ng l(-1)) and 17beta-estradiol (24 ng l(-1)). Conjugated estrogens (estrone-3-sulfate, 17alpha-estradiol-3-sulfate and 17beta-estradiol-3,17-disulfate) were measured in most samples (12-320 ng l(-1)). Median estrogenic activity was 46 ng l(-1) 17beta-estradiol equivalents. Conjugated estrogens contributed up to 22% of the total estrogen load from dairy farming, demonstrating their significance. Steroid estrogens dominated overall estrogenic activity measured in the samples. Significantly, 17alpha-estradiol contributed 25% of overall activity, despite potency 2% that of 17beta-estradiol, highlighting the importance in environmental risk assessments of this previously neglected compound.

Development of a harpacticoid copepod bioassay: selection of species and relative sensitivity to zinc, atrazine and phenanthrene.

Worldwide, estuaries are under increasing pressure from numerous contaminants. This study aimed to identify a suitable marine harpacticoid copepod species for toxicity testing of New Zealand estuaries. Multiple aspects were considered for species selection and included: a broad regional distribution, ease of culture, reproductive rate under laboratory conditions, sexual dimorphism, and sensitivity to contaminants. Five species were evaluated and two (Robertsonia propinqua and Quinquelaophonte sp.) were able to be cultured. The relative sensitivity of these copepods to three reference toxicants was assessed by determining the medial lethal values following a 96 h exposure (96 h LC(50)) to these toxicants in the aquatic phase. LC(50) values for zinc, phenanthrene, and atrazine respectively were 2.0, 0.89, and 7.58 mg/L in R. propinqua and 0.64, 0.75, and 20.8 mg/L in Quinquelaophonte sp. After evaluating all factors involved in choosing a bioassay species for New Zealand, Quinquelaophonte sp. was selected as the most suitable bioassay species.

Toxicity of estuarine sediments using a full life-cycle bioassay with the marine copepod Robertsonia propinqua.

Estuarine sediment contamination is a growing significant ecological issue in New Zealand. Methods of assessing toxicity and ecological impacts in a cost effective way are currently limited. Further to that is a need to develop bioassays that generate data quickly and cost effectively and have ecological relevance to the wider community. A chronic full life-cycle bioassay to assess the toxicity of New Zealand estuarine sediments using the marine harpacticoid copepod Robertsonia propinqua has been investigated. Sediment samples were collected from the Bay of Plenty region and included two polluted and one reference site. Sources of pollutants in the contaminated field sites originated from a variety of sources and generally include nutrients, pesticides and herbicides and the pollutants zinc, copper, lead and polycyclic aromatic hydrocarbons (PAHs). Conversely, the reference site was exposed to low levels of contaminants due to the relatively undeveloped catchment. Adult male and female copepods were exposed to field collected sediments for 24 days under flow-through conditions at 21 degrees C and 12h L:D cycles. Five endpoints were recorded: male and female survival, fecundity (number of gravid females per replicate at the end of the test), clutch size per female, number of eggs per sample and juvenile survival (number of nauplii and copepodites per replicate at the end of the test). Adult mortality was observed in all sediment samples but the number of males, gravid females, clutch size per female and number of eggs produced were not affected by either the contaminated or reference sediment samples. However, the contaminated sediments did reduce reproductive output (i.e. nauplii and copepodite production). Therefore, we conclude that reproductive endpoints provide a good measure of sediment-associated contaminant effects compared with adult R. propinqua survivorship. It may be that a change in focus from chemical thresholds without ecological relevance or lethal dose threshold methods, to more subtle but ecologically significant elements of faunal life, such as reproductive success, are a more sensitive and a long term ecologically informative method.

Responses of Shortfin Eel (Anguilla Australis) Exposed In Situ to Pulp and Paper Effluent

The responses of shortfin eel (Anguilla australis) to discharges from two pulp and paper mills, municipal wastewater, and a geothermal power plant wastewater were examined. Eels were caged at 3 sites along the Tarawera River, North Island, New Zealand, to explore effects of a 3-wk exposure down a contamination gradient (Ref → D1 → D2). Most of the observed effects were seen in eels caged at the furthest downstream site (D2), below all the discharge areas. General hematology in eels was unaffected, as measures did not differ markedly at the two downstream sites compared with the reference site. At D2, eels were significantly lighter per unit length (reduced condition factor), although liver and spleen size (LSI and SSI) were unaffected. Significantly elevated circulating sex steroid concentrations (testosterone and estradiol) were measured in D2 eels and increasing sex steroid levels at both sites downstream of the reference site were observed. Significant ethoxyresorufin O-deethylase (EROD) activity induction was seen in D2 eels and bile chemistry showed significant accumulation of pyrene and retene equivalents. However, significantly greater concentrations of total resin acids were found in the bile of eels from the intermediate site (D1), between the two pulp and paper mills. The higher polycyclic aromatic hydrocarbon (PAH) equivalents found in the bile of D2 eels suggest that resin acid neutrals, particularly retene, are responsible for some of the effects observed in eels at the furthest downstream exposure site. Levels of pulp and paper mill extractives in sediment, including the PAH retene, support this conclusion.

Kia pono te mahi putaiao—doing science in the right spirit

Over the past two decades the challenges facing environmental and natural resource managers have become more complex. Natural resources are more contested and degraded, and as a result stakeholders are increasingly involved in their management. Single-issue management is often not proving effective. Policymakers, industry sectors, indigenous groups, communities and scientists alike have to recognise the interlinked nature of many apparent resource use problems. Successful outcomes are increasingly dependent on the coordinated actions of decision makers operating at many different levels and scales. Consequently, many viewpoints and sources of information have to be shared among the different stakeholders involved, and integrated to find solutions that will guide the way forward (Allen & Kilvington 2005; Berkes 2009 this issue; Robson et al. 2009 this issue). Multi-stakeholder research approaches that facilitate the wide involvement of people in problem solving and decision making with respect to issues and plans which impact on them are becoming widespread. Transdisciplinary research approaches such as sustainability science (Kates et al. 2001), post-normal science (Funtowitz & Ravetz 1993) and complexity science (Waldrop 1992) all call for more inclusive inquiry where local and other knowledge systems collaborate with science in research. The ownership of resulting knowledge production and ensuing management efforts are wider and can more adequately address issues of sustainability. The papers in this issue all highlight the importance of working with indigenous peoples on issues of environmental management. The use of indigenous knowledge in environmental research raises some particular issues for transdisciplinary approaches. We agree with Berkes (2009 this issue) that the science versus indigenous knowledge debate would be more usefully reframed as a dialogue and partnership. Moreover, if indigenous knowledge about the environment is viewed more broadly as a system through which indigenous peoples understand and engage in the world (Raffles 2002), then it encompasses much more than ecological knowledge. Work with traditional ecological knowledge therefore is necessarily framed within a wider cultural base (Berkes 2009 this issue). Because indigenous knowledge systems tend to view people, animals, plants and other elements of the universe as interconnected by a network of social relations and obligations (International Council for Science 2002),

Cumulative impacts assessment along a large river, using brown bullhead catfish (Ameiurus nebulosus) populations

The effects of point-source and diffuse discharges on resident populations of brown bullhead catfish (Ameiurus nebulosus (LeSueur, 1819)) in the Waikato River (New Zealand) were assessed at sites both upstream and downstream of point-source discharges. At each site, the population parameters, relative abundance, age structure, and individual indices, such as condition factor, organ (gonad, liver, and spleen) to somatic weight ratios, and number and size of follicles per female, were assessed. Physiological (blood), biochemical (hepatic ethoxyresorufin-O-deethylase [EROD] and plasma steroids), and other indicators (bile chemistry and liver metals) of exposure or response also were measured. No impacts on brown bullhead health were obvious at individual geothermal, municipal sewage, or thermal discharge sites or cumulatively along the river. Brown bullhead from the bleached kraft mill effluent site showed elevated levels of EROD, decreased numbers of red blood cells, increased numbers of white blood cells, and depressed levels of sex steroids. However, growth rates, condition factor, age structure, and gonadosomatic index suggest that discharges with significant heat or nutrients benefit catfish despite physiological impairment at one site. Consideration of brown bullhead population-level responses to discharges in a monitoring framework revealed three different population-level response patterns resulting from the point-source discharges.

Bioactivity of POPs and their effects in mosquitofish in Sydney Olympic Park, Australia

The site of the 2000 Olympic Games (Sydney Olympic Park (SOP), Sydney, Australia) was contaminated by persistent organic pollutants (POPs) prior to remediation in the 1990s. This study investigates the bioactivity of POPs in the sediment and water of wetlands across SOP by in vitro 2,3,7,8-TCDD equivalence (TCDDeq) measurement (H4IIE cell line bioassay). Further, it examines whether disturbance of these sediments is likely to mobilise ligands for this receptor into the water column. Exposure to aryl hydrocarbon receptor (AhR) ligands was measured in vivo using hepatic cytochrome P4501A (CYP1A) induction (EROD) in the mosquitofish (Gambusia holbrooki). Aqueous TCDDeq ranged from 0.013 to 0.057 pM in SOP wetlands which was significantly (p<0.05) less that in urban reference sites. These concentrations were not correlated to physical or chemical characteristics of the wetlands. In the sediments, TCDDeq ranged from 0.0016 to 7.06 microg/kg and these were not significantly (p>or=0.05) different to that measured in urban reference sites. Simulated disturbance of small quantities of sediment in water samples significantly (p<0.05) increased the levels of TCDDeq measured in the water. Sediment TCDDeq was correlated to sediment SigmaPAH concentration in 2006 and sediment SigmaPCB, SigmaDDT concentrations and fine sediment grain size in 2005. While fish at one SOP wetland had hepatic EROD activity elevated above the estimated basal level for this species, these were at the lower end of the range measured in urban impacted, non-remediated wetlands. EROD activity was positively correlated with both the sediment SigmaPCB load and aqueous TCDDeq. Increased catchment size was correlated with increased EROD activity suggesting an even spread of POPs throughout the residential areas of the Sydney metropolitan area. The concentration of bioactive POPs in the wetlands of SOP is therefore low relative to urban reference sites demonstrating the ongoing success of the remediation program.

Distribution of inorganic and organic contaminants in sediments from Sydney Olympic Park and the surrounding Sydney metropolitan area.

Organic and inorganic contamination was assessed for sediments from wetlands and water bodies within the Sydney Olympic Park (SOP, remediated sites) and its surroundings (unremediated sites) and urban reference sites in the Sydney Basin. Among the seven elements analysed (As, Cd, Cr, Cu, Pb, Ni and Zn), Zn concentrations were the highest, followed by Pb, Cu and Cr in the sediments of SOP. Significantly higher concentrations (p < 0.05) of DDTs were found in sediments of the Homebush Bay and Parramatta River sites compared with the urban reference sites, mainly due to past manufacturing of DDT in the Homebush Bay area. However, no significant differences (p > 0.05) in concentrations were found for polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) as well as DDTs between sediments from SOP and the urban reference sites. Source indicators suggest that PAHs in the sediments originated from combustion processes. Two distinct groups of dioxin profiles were observed within SOP and its surroundings. Levels of dioxins were more than 100 pg WHO-TEQ/g dry weight of sediment at five sites adjacent to the SOP boundaries. Based on the findings of the chemical profiles of the contaminants, the remediated sites in SOP can be regarded as similar to the urban reference sites within the Sydney Basin, while the adjacent unremediated sites have higher concentrations, especially of dioxins, that could still affect organisms in the aquatic environment.

Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR)

Abstract A method to quantify induction of vitellogenin (Vtg) mRNA in adult male mosquitofish was developed. Male mosquitofish were exposed to 0, 1, 20 and 250 ng l−1 17β-oestradiol (E2) for 4 and 8 days in static exposures, and liver Vtg mRNA and 18S rRNA expression were quantified in duplex RT-PCR. Liver 18S rRNA expression was very consistent among individuals, and there was a highly significant increase in Vtg mRNA expression after exposure of mosquitofish for just 4 days at 250 ng l−1 E2. Lower doses did not induce Vtg mRNA expression even at 4 or 8 days. This method could be used as a rapid test to detect exposure of mosquitofish to oestrogenic chemicals. Further work is needed to determine if increased Vtg mRNA levels in male mosquitofish induce Vtg synthesis, and to determine the usefulness of the method in field sampling.