3. Research (Part 3)

Part 3 of Canadian Environmental Protection Act, 1999 (CEPA 1999) allows the Minister to conduct research. Environment Canada and Health Canada scientists published numerous reports, papers, book chapters, articles and manuscripts on subjects related to the Act. CEPA 1999 directs the Minister to report on research in the annual report. Although it is not possible to describe all of these activities in this report, this chapter summarizes the types of research initiatives underway during the reporting periods, along with the key contributions of this research.

Air-related research under CEPA 1999 in 2006-2007and 2007-2008 included the following studies:

• Hexachlorobenzene in the Great Lakes -- Hexachlorobenzene, a persistent toxic substance, continues to have an impact on air quality in the Great Lakes environment years after it was banned for agricultural use in Canada (in the 1970s). Researchers therefore inventoried releases from major North American sources and used an atmospheric transport model to investigate how this fungicide finds its way into the Great Lakes Basin. The findings of the two-year study were summarized in an 85-page report entitled Emission Inventory and Multiple Pathways Modeling of HCB to the Great Lakes from North American Sources, submitted to Environment Canada and the U.S. Environmental Protection Agency in May 2008.
  
• Exchange and fate of persistent organic pollutants (POPs) and currently used pesticides in water, soil and air -- Researchers studied residues from currently used and banned pesticides in and above Hudson Bay waters, in Ontario precipitation and streams, and in the Great Lakes. In July 2007, Arctic researchers identified currently used pesticides (chlorpyirfos, dacthal, chlorothalonil and endosulfan), as well as legacy pesticides such as chlordane in Hudson Bay waters and in air samples collected at three different heights above the sea surface. In Ontario, the focus was on estimating the air/water exchange and fate of herbicides (mecoprop, dichlorprop and metolachlor). The lab work at the Canada Centre for Inland Waters was able to determine the rate at which pesticides, such as hexachlorocyclohexane, 1,1,1-trichoro-2,2-bis(4-chlorophenyl)ethane (DDT), chlordane and dieldrin, and brominated flame retardants are released from soil to air as residues age over time.
  
• Monitoring pesticides on Prince Edward Island -- In the second year of a three-year study, 106 air and soil samples were collected on Prince Edward Island at sites near a field where pesticides had been applied to a potato crop, and at residential and background sites. The overall preponderance of pesticides sprayed onto island potatoes includes carbofuran, chlorothalonil and methamidophos. Other currently used pesticides, such as endosulfan, trifluralin, chlorpyrifos, dacthan, and metribuzin, were also detected, although they were not applied to the investigated field, indicating spray drift and or long-range transport and deposition from other local/regional pesticide activities.
  
• Sampling and analytical techniques for measuring levels of perfluorinated chemicals, polybrominated ethers and other emerging chemicals -- A new type of sampling disc was successfully developed for volatile and ionic compounds. A hazardous air pollutants laboratory at Environment Canada was involved in the first international study investigating different sampling techniques for measuring perfluorinated chemicals. Air samples were collected collaboratively from a variety of international and Canadian outdoor sites, and these and archived indoor air and dust samples from Ottawa were analyzed. Scientists are assessing links between maternal thyroid hormone levels during pregnancy and exposure to these chemicals and analyzed target compounds in indoor and outdoor air samples, dryer lint and house dust.
  
• Activities of the Integrated Atmospheric Deposition Network -- The Canada-United States Integrated Atmospheric Deposition Network fulfills the requirement for surveillance and monitoring mandated by Annex 15 of the 1987 revision to the Canada-U.S. Great Lakes Water Quality Agreement. In addition to conducting routine sampling and analytical and administrative activities, the network developed a technical summary of its progress from 2002 to 2008.
  
• Global Atmospheric Passive Sampling network -- Results under the network help to fulfill Canada's obligations under the Stockholm Convention on Persistent Organic Pollutants. During the 2007 calendar year, the third year of activity under the network, Canadian research scientists contributed to, presented papers on and published results of air sampling at 55 worldwide sites, four of which were in Canada. In October 2007, Environment Canada launched a new website about the network.
  
• In support of the development of air quality objectives and guidelines, new mapping techniques were developed to interpolate air quality monitoring data across most of Canada. This information was combined with population data to determine population exposures at various levels of particulate matter (PM) and ozone.
  
• Critical load maps for acid deposition, which are used to assess progress toward mitigating acid rain in Canada (i.e. long-term environmental target for Canada), were updated with new information from western Canada and updated information for parts of eastern Canada.
  
• The Canadian Air and Precipitation Monitoring Network (CAPMoN), Canada's rural and background air and precipitation monitoring network, added new sites and analytical capacity in order to better define the air quality, human health and environmental impacts of domestic and international emissions.
  
• The Environment Canada-led federal-provincial National Air Pollution Surveillance (NAPS) network, added new sites and analytical capacity to better define the air quality and human health impacts of domestic emissions.
  
• A PM_2.5 inter-comparison network was formed to assess the performance and methods comparability of instruments for continuous monitoring of fine particulate matter (PM_2.5).
  
• A field survey was completed in northern Saskatchewan to estimate the chemical and biological sensitivity of lakes potentially affected by oil sands development.
  
• Chemical and biological data were collected to enhance assessments of the acidification and recovery of sensitive lakes in eastern Canada.
  
• Evaluations were made of the influence of nitrogen deposition, base cation depletion, release of stored sulphur and other alkalinity-generating mechanisms on the acidification status and recovery of selected eastern and western lake ecosystems.
  
• Improvements were made to Environment Canada's regional and global air quality models to improve their capacity to predict future levels of air pollution in Canada.
  
• In support of the Canada-U.S. PM Annex discussions, modelling and monitoring work were assessed in order to quantify the impact of Canadian and U.S. emissions on the air quality of the other country.
  
• In support of the United Nations Economic Commission for Europe (UNECE) Gothenburg Protocol, scientific contributions were made to the Task Force on Hemispheric Transport of Air Pollution in order to better understand the scope and scale of regional and continental influences on air quality.
  
• Studies were undertaken with other government departments and industrial partners to investigate the impacts of renewable fuels (i.e. ethanol and biodiesel), engine hardware and aftertreatment devices (i.e. diesel particulate traps and selective catalytic control systems), and advanced combustion technologies on fuel efficiency and emissions. Within this research, additional studies were undertaken to investigate the human health impacts of transportation-related emissions, particularly the toxicity and mutagenicity of advanced fuels and related emissions.
  
• To support decision making, research was conducted to investigate and develop suitable emissions and fuel economy measurement methods and models for evaluating plug-in hybrid electric vehicles. The study provides some of the first available results of cold temperature laboratory testing of emissions, fuel economy and electrical energy consumption of a plug-in hybrid electric vehicle system as well as the performance and emissions of hybrids relevant to conventional utility trucks in a Canadian context.
  
• Emission research studies were completed under real-world test environments to quantify the emissions produced from various non-road vehicles and engines ranging from lawn, garden and construction equipment to ocean-going vessels and locomotives. The objective of this research was to gain a better understanding of the air emissions contributed from this sector under real-world conditions as well as the potential for mitigating these emissions through the introduction of clean fuels and/or emission control systems.
  
• Studying the significance of transportation-related sources to particulate matter levels in Canada -- Three projects (funded by the Program of Energy Research and Development) were undertaken to characterize ambient particulate matter (PM) and its precursors associated with transportation sources in Canada across a range of representative emissions, locations, and meteorological and topographic conditions. The research themes included the significance of on-road and non-road transportation-related emissions sources to PM levels in Canada; howPM was emitted and/or produced, transported, transformed and removed from the atmosphere; and the health and environmental (air quality) impacts of transportation-related PM in Canada. New science and technology capacities developed through this research include the ability to identify, within the particulate matter, organic matter and its sources, water-soluble organic compounds, and transportation-related organic acids.
  
• Development of analytical methodologies -- Two projects were initiated to support new regulations under CEPA 1999. The goal of the first project was to develop and implement an analytical method related to the 2-Butoxyethanol Regulations published in the Canada Gazette, Part II, on December 27, 2006 (Vol. 140, no.26, 2006, PDF 2,22 MB, go to page 82). The Regulations govern the concentration of 2-Butoxyethanol in products designed for indoor use (e.g. household cleaners, paints, paint stripers and solvents). The final report provides detailed information on sample preparation, the instrumental techniques for detection and quantification, and the quality assurance required to achieve reliable results.
  
  The second project was initiated to develop and implement a reference method to analyze chlorinated paraffins (CPs). CPs are subdivided into short-chain CPs (SCCPs, C_10-13), medium-chain CPs (MCCPs, C_14-17) and long-chain CPs (LCCPs, C>17). Over 200 CP formulations are used in industrial applications, such as flame retardants and plasticisers, and as additives in metal-working fluids, in sealants, paints and coatings. CPs in the environment result primarily from human activity. This project started in 2005 with the goal of developing a method to analyze CPs in various matrixes including soil, sediments and water effluents, and will be concluded in 2009.

Water-related research under CEPA 1999 in 2006-2007 included the following 14 studies:

• Occurrence and fate of the three most-prescribed fluoroquinoline antibiotics (ciprofloxacin, ofloxacin and norfloxacin) in the Canadian environment -- Researchers developed a method to measure the amount of these relatively new antibiotics in wastewater. Wastewater was collected from eight sewage treatment plants in southern Ontario. All three compounds were detected in all samples of sewage effluents, both treated and untreated. Development of analysis methods is ongoing. The research will make it possible to monitor the amounts of the three antibiotics being removed during treatment and improve treatment technologies.
  
• Effects of pharmaceuticals on aquatic microbial community structure, diversity and function -- Assessment of the effects on microbial communities of relevant concentrations of combinations of mixtures of anti-inflammatory drugs (e.g. diclofenac and acetaminophen) and other sewage effluent constituents (e.g. caffeine) indicated a variety of effects on protozoan and micrometazoan populations. In general, the results indicate that, of the three compounds tested, only diclofenac caused significant changes to community structure and function, although these effects can be influenced by nutrient availability and the presence of readily degraded co-contaminants.
  
• Effects of pharmaceuticals on attached (to sediments) and pelagic (free-living) microbial communities in a small stream -- A summer survey of pelagic microbial communities confirmed that primary production was depressed for at least 10 km downstream of Regina's sewage treatment plant in streams. Pharmaceuticals and personal care products were detected below the pharmaceutical plant, but were absent upstream. Two sets of experiments were conducted to test the effects of three antibiotics (clindamycin, erythromycin and trimethoprim) and nutrients on attached microbial communities. Preliminary results indicated differences in the biomass, primary production and metabolism of communities treated with the antibiotics compared to controls. Microcosm experiments showed negative effects only for erythromycin.
  
• Isomerisation and biological effects of chlortetracycline in wetlands -- Chlortetracycline is a broad-spectrum antimicrobial routinely added to feed in many intensive swine, poultry and cattle operations. Present in manure that is sometimes applied to pasture or cropland as a nutrient, chlortetracycline residues subsequently occur in various forms in aquatic ecosystems. Contamination of natural waters could accelerate the development of antimicrobial-resistant bacteria. Laboratory testing using water from a prairie wetland showed that chlortetracycline and its isomers generally break down quickly and have no harmful environmental effects. However, further testing of the effect of this antimicrobial on algal biomass at concentrations of more than 1000 µ/L is needed to assess impacts in streams near cultivated plots.
  
• Occurrence and fate of beta blockers in sewage treatment plants -- These drugs are used to treat high blood pressure, congestive heart failure and abnormal heart rhythms; relieve angina; and prevent heart attacks. Using a new liquid chromatography/mass spectrometry method, eight of the most commonly prescribed drugs (acebutolol, atenolol, bisopolol, metoprolol, nadolol, propranolol, labtalol and sotalol) were detected in 14 samples of primary and final effluents collected from seven sewage treatment plants located in southern Ontario. Rates of removal by primary and tertiary treatments ranged from 7% to 40%. Prior to this study, there were only a few reports of the occurrence of a handful of beta blockers in the Canadian environment. Their stability (failure to break down into substances that occur naturally) means that future work is required to assess their ultimate impacts on Canadian ecosystems.
  
• Field sampling protocol and solid-phase microextraction method for three antibiotics -- A field sampling protocol (optimum materials to use for sample collection, handling and storage) for three classes of antibiotics (fluoroquinolones, sulphonamides and tetracyclines) was developed that allowed the extracted samples to remain stable until their analysis. Using water samples collected from Woodland Beach, Ontario, according to the protocol, a solid-phase microextraction method was also developed for the three classes of antibiotics.
  
• Effects of environmentally relevant concentrations of certain antidepressants and antiepileptics (fluoxetine and carbamazepine) -- During this third year of research, scientists assessed the effects of these drugs on fish growth, health and reproduction of adult fathead minnow (Pimephales promelas) breeding groups exposed to environmentally relevant concentrations. The minnows exposed to fluoxetine had lowered egg production, but this could not be distinguished from the effects of the solvent (methanol) alone for the highest concentration. Fish exposed to lower concentrations of fluoxetine had decreased reproduction compared to control fish. Fish exposed to carbamazapine showed no effects on reproduction, but bacterial disease in fish in the temporary wetlab facilities caused several fish deaths that prevented definitive analyses of the data. Therefore, this part of the study will have to be repeated.
  
• Use and occurrence of perchlorate in Canadian natural waters -- The highest perchlorate occurrence in Canadian natural ground and surface waters was observed in samples taken in the vicinity of military sites, mining areas, sodium chlorate production facilities and fireworks displays. Concentrations in samples from these areas commonly exceeded interim drinking water guidelines. Elevated concentrations in Canada were also associated with fertilizer application, livestock farming, potash storage facilities, and pulp and paper operations. Perchlorate was detected in the majority of samples, but generally at fairly low concentrations. Concentrations greater than 0.3 µg/L were not found in any public water supplies. Perchlorate is used mainly in military applications, and more than half the 3174 samples were obtained by the Department of National Defence from five military sites across the country. Contamination from natural sources of perchlorate appears to be low in Canada.

• Effects of municipal wastewater effluents on resident fish populations -- Fathead minnows (Pimephales promelas) and brook stickleback (Cultaea inconstans) were collected on Wascana Creek upstream and downstream of the discharge from the Regina wastewater treatment facilities. The downstream male fatheads were significantly shorter and lighter than male fatheads collected upstream, but they did not differ in fish condition or gonadal development relative to body weight, despite having significantly reduced secondary sexual characteristics. Downstream female fatheads did not differ in any of the above characteristics compared to upstream females. Fatheads of both sexes had larger livers compared to controls collected upstream of the treatment plant. In March 2008, the stickleback had not yet been analyzed. In addition, greenside darters (Etheostoma blennioides) were collected on the Speed River upstream and downstream of the discharge from the Guelph wastewater treatment facility. Downstream male darters were shorter and lighter and had reduced gonadal development relative to length than upstream male darters, but did not differ in overall condition nor was there any significant difference in gonadal development relative to body weight. Darters of both sexes that were exposed to the effluents had smaller livers. There was 100% survival of darters caged for 21 days directly downstream of the discharge, which shows that the effluent is not acutely toxic to greenside darters.
  
• Exposure of goldfish to estradiol -- During this third year of the study, male goldfish (Carassius auratus) were exposed to estradiol and sampled after one and six days. Lab tests involving injections of estradiol showed that vitellogenin, a protein essential in the formation of egg yolk and expressed only in female fish (normally dormant in males), is an effective biomarker for the presence of exposure to estrogen. Vitellogenin was present at similar low levels in both control and treated fish after one day. The levels rose significantly in exposed fish after six days.

• Through the Environmental Effects Monitoring Program, a joint research initiative was established to investigate the cause of widespread effects on fish reproduction (small gonads) found near pulp and paper mills across Canada and associated with endocrine-disrupting substances. -- Monitoring the recovery of white suckers (Catostomus commersonii) after the February 2006 closure of a pulp mill at Terrace Bay in Ontario showed, in males collected in spring, improved gonad size and circulating steroid levels, but unimproved secondary sexual characteristics. Female white suckers failed to show recovery. In July 2006, tests began on effects of effluent from a new mill in La Tuque, Quebec. Testing proved the need to ship, receive and process samples of condensate of the mill's effluent as quickly as possible to reduce loss of compounds associated with steroid depressions. Smaller fish with enlarged livers were found both in fathead minnows (Pimephales promelas) used in the bioassays and wild fish captured downstream of the effluent outfall. Seven publications, four submitted or in-press articles, four peer-reviewed conference proceedings and eight conference presentations ensued from this project during 2006-2007.

• New contaminants in the Arctic and Subarctic atmospheric and aquatic environments -- This study completed the analysis of samples for contaminants, including ice-cap and lake water, sediment cores, snowmelt, upper ocean waters, plankton and tissues from Arctic cod (Boreogadus saida). One article was published and two were in press at year-end. Four presentations were made at an annual meeting and a workshop. One major finding of ice-cap sampling was that perfluorooctane sulphonate (PFOS) was declining in concentrations consistent with the removal of certain types of PFOS-based industrial and consumer products, such as soil, oil, grease and water repellents used on textiles and packaging, from the market in 2001.

• In January 2007, under the Canadian Regulatory System for Biotechnology initiative, the New Substances Program led the organization and coordination of a two-day, government-wide conference held in Gatineau, Quebec. The conference provided a forum for researchers, policy analysts, program managers and policy makers to present results achieved during previous funding cycles, and to discuss existing and future challenges and directions. In addition to facilitating priority setting for research projects in support of the regulations, the New Substances Program used the success of the conference to establish an evidence-based decision-making process to call for and fund research project proposals that meet regulatory research needs consistent with priorities.
  
• Fate and effects of transgene DNA in aquatic environments -- Using the South Saskatchewan River as a model system, researchers developed baseline information on the composition and seasonal dynamics of microbial communities and the natural occurrence of the phosphotransferase gene in these river communities. This established a basis for monitoring certain effects of genetically modified micro-organisms in aquatic environments. Also, analysis of genes of bacteria found in rhizosphere soils (soil layer around roots) from multi-year field trials of three commercial corn lines suggested that different corn lines do influence microbial communities in the rhizosphere. Tests involving one commercially released genetically modified corn line concluded that evidence for potential transfer of the phosphotransferase gene to indigenous bacteria is lacking.
  
• Fate and effects of transgene DNA in aquatic environments -- Using the Richelieu River as a sink for transgenic DNA resulting from intense crop cultivation, the presence, fate and potential effects of transgenic corn and canola were examined in microbial flora and in wild and caged Elliptio complanata mussels. Recent studies at this site revealed that transgenic DNA from corn is found in local bacterial communities and accumulates in both wild and caged mussels. However, data on the bioactivity of these DNA fragments in bacterial and mussel tissues are lacking.
  
• Microbial biotechnology products subject to notification under the New Substances Notification Regulations (Organisms) were analyzed by DNA microarray and DNA fingerprinting techniques. These techniques were more effective than existing culture-based techniques at characterizing the composition of the microbial communities in these products, and determining their stability between batches.

• Occurrence of pathogenic and antibiotic-resistant Escherichia coli (E. coli) in aquatic ecosystems -- Researchers tested new techniques that will equip public health authorities to do more than detect and count the fecal-indicator bacterium E. coli in water. The more sophisticated techniques will allow researchers to identify disease-causing and antibiotic-resistant genes in the bacterium. Initial trials in Hamilton Harbour recreational waters identified the genes at proportions consistent with a potential concern for human health. The researchers then analysed the DNA fingerprints of E. coli isolates taken from different aquatic ecosystems in the Detroit and St. Clair river areas. Researchers then presented their results at a symposium and a workshop and published two articles in the journal Applied and Environmental Microbiology.
  
• Over 4500 water samples from four intensive agricultural watersheds across Canada were analyzed from July 2005 to December 2007. These analyses were conducted as part of research to develop a benchmark for ambient water quality impairment from waterborne pathogens under the National Agri-Environmental Standard Initiative (NAESI). Two provisional pathogen standards were derived based on annual mean concentrations of E. coli for sites on low- and high-order streams in agricultural watersheds. The standards were derived by accounting for the natural background occurrence of waterborne pathogens from sources such as wildlife at reference sites away from agricultural impacts in each watershed.

Water-related research under CEPA 1999 in 2007-2008 included the following 12 studies:

• Presence, fate and discharge of brominated flame retardants in municipal wastewaters and biosolids -- Debromination of eight polybrominated diphenyl ether congeners during sewage sludge digestion was monitored for 238 days under various conditions. The initial concentrations of five congeners in the sludge decreased by 18-50%. Breakdown is enhanced by higher temperatures. Debromination was not observed for the other three congeners.
  
• Emerging halogenated and siloxane contaminants in the Canadian aquatic environment -- Sampling studies were conducted and methods developed to assess the presence and concentration of halogenated and siloxane contaminants in water. Results indicated their presence and, in some environments, increased quantities over time.
  
• Methodology for detecting pharmaceuticals and personal care products in municipal wastewater and sludge samples -- Two methods were developed and validated for the detection of triclocarban in wastewater and sludge samples. Triclocarban is a preservative in many liquid and bar soaps, deodorants and cosmetics. Prior to this study, not much information was available about its toxicity or occurrence in the Canadian environment. It was detected in all water and effluent samples collected in southern Ontario. A third method was developed for the determination of the beta blockers commonly used in Canada. Atenolol, acebutolol, sotalol and metoprolol were found at the highest concentrations in sewage samples.
  
• Occurrence and fate of ciprofloxacin, ofloxacin, and norfloxacin in the Canadian environment -- A preliminary analytical method for measuring the concentrations of these fluoroquinolones (antibiotics prescribed to humans and other animals) was developed. The primary pathway into the environment is via human secretion in sewage.
  
• Recovery of antibiotics from groundwater and manure -- Sulfamethazine, chlortetracycline and tylosin are among the antibiotics administered routinely to cattle and swine to prevent disease and promote growth. A method was successfully developed to recover 70-90% of residues of these three veterinary antibiotics from groundwater under controlled conditions. The method was adapted to recover the same three compounds from control samples of raw and composted cattle manure, but the recovery rate was slightly lower, at 50-70%.

• Long-term effects of antidepressants in fathead minnows -- To correct a complete lack of data on the long-term effects of antidepressants or mixtures of anti-depressants in fish, fathead minnows (Pimephales promelas) were exposed for a life cycle to concentrations of venlafaxine (a selective serotonin uptake inhibitor) in municipal wastewater effluents. They were also exposed to a mixture of the top five selective serotonin uptake inhibitors at levels found in Ontario municipal wastewaters and at 10 times the actual levels. Larval fish growth was good and no significant effects on survival were seen. There were significant differences in egg production. Preliminary trials suggest an increase in aggression.
  
• Effects of pharmaceuticals and municipal wastewater effluents on aquatic microbial community structure, diversity and function -- In the final year of a survey that began in 2005-2006, 14 pharmaceutical products were found to always be present in Wascana Creek, Saskatchewan, independent of season or flow conditions. Research focused on the effects of selected anti-inflammatory drugs (ibuprofen, gemfibrozil, acetaminophen, salycylic acid, naproxen and diclofenac, and mixtures of these) typically found in municipal wastewater effluents, on aquatic microbial community structure, diversity and function. A wide range of results was obtained. Results from the microbial community from the eutrophic (excessive nutrients) Wascana Creek system differed from those from the oligotrophic (very low levels of nutrients) South Saskatchewan River. Results also varied according to the drug or drug mixture, and the organism or class of organisms tested. Primary bacterial production on Wascana Creek just below the effluent outlet of the Regina sewage treatment plant was three times less than that at an upstream site, and its ratio to upstream bacterial production was <1 to 1.26. Other detailed analyses are being conducted to identify cumulative effects and interactions.
  
• Effects of municipal wastewater effluents on resident fish populations -- Several fish from a variety of locations were collected during the third year of this study: fathead minnows (Pimephales promelas) and brook stickleback (Cultaea inconstans) from Wascana Creek, Saskatchewan, both upstream and downstream of the Regina sewage treatment plant; and feral greenside (Etheostoma blennioides) and rainbow (Etheostoma caeruleum) darters in the Grand River, Ontario, both upstream and downstream of the Kitchener and Waterloo wastewater plants. Both sexes of minnows were smaller downstream of the outlet and in poorer condition generally; the secondary sexual characteristics of exposed males were underdeveloped (this is the first time this has been demonstrated in wild fish populations in Canada); and exposed females had increased gonad size, higher testosterone levels and decreased estradiol steroid production capacity. Results for stickleback were inconclusive. In contrast, the downstream darters were longer and heavier, and in better condition than those collected upstream of the wastewater outlets. Assessments of the downstream fish communities showed improved abundance and diversity. These improvements are thought to be a response to nutrients in the wastewater plant effluent.
  
• Fate and effects of transgene DNA in microbial communities in aquatic environments and in soil, and its persistence in soil -- Scientists continued to compile baseline data on molecular biodiversity and function in aquatic ecosystems for use in monitoring the effects of genetically modified organisms on microbial biodiversity in these ecosystems. A related but separate investigation of the persistence of transgene DNA in rhizosphere soil samples suggested that transgene DNA degraded after the growing season. Investigations of the long-term effects of genetically modified crops showed that there is no significant difference in the abundance of AmoA genes between genetically modified and non genetically modified maize: AmoA genes are indicators of functioning nitrogen cycling.
  
• Persistence of Domestic Substances List (DSL) microbial strains in water microcosms -- The release of microbial strains listed on Environment Canada's DSL to environments raises concerns over ecological and public health impacts. Molecular techniques were developed and applied to investigate survival and persistence of DSL microbial strains in water microcosms. Scientists developed protocols using propidium monoazide to distinguish DNA carried by live and dead cells of microbial strains in real-time polymerase chain reaction. With the setup of water microcosms, preliminary results showed that introduced strains (e.g. Bacillus cells) could persist in water microcosms for periods ranging from a few weeks to over 100 days.

• Endocrine-disrupting substances in pulp and paper mill effluents -- Recovery of hormonally active compounds and sampling of suckers from a pulp mill's effluent receiver and from spawning wild fish took place in Terrace Bay, Ontario. Scientists did a detailed reproductive assessment and found that white sucker (Catostomus commersonii) males and females living in the effluent receiving water were significantly smaller and had smaller gonadal somatic indices.
  
• Microbial source tracking research was conducted to investigate the source of fecal pollution contaminating aquatic ecosystems and causing beach closures in the cities of Toronto, Hamilton and Ottawa. DNA fingerprinting and antibiotic resistance profiling techniques demonstrated the importance of E. coli contamination from gulls and geese at some beaches, while human gut DNA marker techniques demonstrated the importance of municipal sewage contamination at other beaches. These results are being used to target clean-up efforts and beach management actions in these cities.
  
• Occurrence of pathogenic and antibiotic-resistant E.coli in aquatic ecosystems -- Scientists who analyzed 1856 E. coli isolates from 20 sites in three intensively farmed agricultural watersheds found the highest frequency of antibiotic resistance in the pathogen in the Bras d'Henri watershed (swine farming) and lowest in the South Nation River watershed (dairy farming). Results in the Old Man River watershed in Alberta (beef cattle) were intermediate. The antibiotics were ampicillin, chloamphenicol and cephalothin.
  
• Nanotechnology products in aquatic ecosystems -- Researchers developed a method to track nanotechnology products in municipal effluents and in biota. Lab tests revealed a wide range of sensitivity resulting from tests of the toxicity of nine metallic nano-powders and two organic nano-powders on several taxonomic groups, including decomposers, primary producers, and primary and secondary consumers. The researchers also tested the toxicity of four of the metallic nano-powders using natural St. Lawrence River sediments, with results indicating that adverse effects on biota will in part depend on the physical characteristics of the sediments, such as grain size. The third set of tests examined the toxic effects of a specific type of nano-particle on the freshwater mussel. Testing showed that the response of the organism varied based on the concentration of the contaminant, which allows researchers to establish biomarkers to distinguish the form (colloidal or molecular) in which the cadmium is present in the mussels.

Wildlife-related research under CEPA 1999 in 2006-2007 and 2007-2008 comprised the following studies:

• Continued monitoring of the long-term temporal and spatial trends of priority chemicals (e.g. halogenated substances on the Domestic Substances List, such as perfluoroalkyl compounds and brominated flame retardants) and regulated legacy compounds [e.g. DDT and polychlorinated biphenyls (PCBs)] in eggs of fish-eating bird bioindicator species (e.g. gulls, puffins and cormorants) and in other selected wildlife (e.g. polar bears Ursus maritimus, mink and otter) in the Arctic, Pacific (particularly in the Strait of Georgia) and Atlantic marine environments, and in the St. Lawrence River -- Great Lakes ecosystem, was conducted in order to obtain information on the presence, sources, environmental pathways and effects of these compounds on birds and other species and their food webs.
  
• An ongoing assessment of the impact of methylmercury, lake acidity and related stressors on the breeding success of the Common Loon (Gavia immer) and other wildlife in Eastern Canada continued through focused studies in Nova Scotia and in various parts of Quebec.
  
• The role of mercury and other organic chemical contaminants in the decline of the Ivory Gull (Pagophila eburnea) population in the Canadian Arctic was investigated. Some of the highest levels of mercury recorded in Arctic seabirds and steadily increasing concentrations (since 1976) of some brominated flame retardants have been found.
  
• Retrospective studies, using specimens that were archived at the National Wildlife Research Centre, of Northern Fulmars (Fulmarus glacialis) that were collected in 1975 and 1993 revealed the highest concentrations of some polychlorinated dibenzo-dioxins and -furans reported in tissues of Canadian Arctic biota.
  
• After remediation of a contaminated site in the Canadian Arctic, the toxicological impact of PCBs was studied in Black Guillemot (Cepphus grylle) chicks. It was discovered that PCB pollution near a military radar site at Saglek, Labrador, impaired the health of locally breeding seabirds and posed risks to the local marine ecosystem. PCBs disrupted the endocrine system, altered organ development and changed liver enzyme activity in exposed seabird chicks. In 2007, researchers visited the site to study the impacts of lower PCB exposures on seabird chicks and the effectiveness of PCB remediation at the site. A decline in PCB levels in guillemot chicks from 1999 to 2007 was associated with a decrease in the extent of adverse effects observed. However, some endocrine and neurochemical impacts still remain at current PCB concentrations.
  
• New methods based on analyses of gene expression, combined with cultures of neuronal and other tissue types from wild and domestic birds, were developed and applied to assess toxicity of commercial industrial contaminant mixtures and various individual congeners of polybrominated diphenyl and other priority substances.
  
• Tree Swallow (Tachycineta bicolor) colonies were established at reference and test sites, and test methods were selected and refined for wildlife sampling and standard toxicity testing. The methods established will be used for undertaking long-term studies, using selected wildlife indicators, to identify and assess the occurrence and ecological effects of chemicals found in sewage treatment plant effluents. In 2007 and 2008, differences between the effluents and reference sites were found in the reproductive success, growth and thyroid hormone concentrations of tree swallows.
  
• A laboratory was established for conducting analyses of fatty acids in birds and other wildlife. Ecological tracers, such as fatty acids, can be used in combination with other approaches, such as stable isotopes, in order to assess how contaminants, nutrients and pathogens are transferred to wildlife through food webs. This approach will lead to new insights into contaminant sources, and improve the ability to predict and assess the bioaccumulation potential of toxic substances and their associated risks.
  
• Investigations of the relationships between contaminant levels and parasite load in fish-eating birds (e.g. Double-crested Cormorants, Phalacrocorax auritus, from the Great Lakes) were conducted to improve understanding of how contaminants and parasites may be interacting to affect the health of wildlife.
  
• Studies of toxicity and effects at the molecular level of perfluoroalkyl compounds and other priority compounds in avian embryos and cells using egg injection and tissues of wild and domestic birds to establish quantitative structure-activity relationships for prediction of the toxic potential of these contaminants in birds were ongoing.
  
• A collaborative study with scientists from Denmark on the fate and effects of legacy and emerging organohalogen contaminants (e.g. PCBs and brominated flame retardants) in captive sled dogs was conducted. The dogs were a surrogate in a cause-effect study to assess how pollution affects the Arctic top mammalian predator, the polar bear. Results showed that, relative to control dogs fed a diet essentially free of organic pollutants, exposed dogs experienced deleterious effects on their immune system, as well as increased frequencies of kidney and liver lesions.
  
• Studies on East Greenland polar bears suggested that long-term exposure to legacy and emerging organohalogen contaminants (e.g. PCBs and brominated flame retardants) may be a factor in kidney lesion occurrence, although other factors, such as exposure to heavy metals and recurrent infections from micro-organisms, cannot be ruled out. This new information is important to the assessment of the health status of polar bear populations and humans relying on food resources that are contaminated with these substances.
  
• The composition of PCBs and organochlorine contaminants were shown to be similar in different major fat tissue deposits of polar bears from the Canadian Arctic. This study demonstrated that the part of the polar bear body where a fat sample is taken is not a serious confounding factor in the assessment of the trends of these legacy contaminants.
  
• In collaboration with Norwegian researchers, a study was completed that examined priority organic pollutants (e.g. PCBs, organochlorine pesticides and brominated flame retardants) in Glaucous Gull (Larus hyperboreus) eggs and plasma samples of the laying females collected from the Norwegian Arctic. The study specifically examined the variability in the concentrations and composition of organohalogen pollutants associated with the laying sequence in this bird species. Organohalogen contamination in breeding Glaucous Gulls was also found to affect basal metabolism and circulating thyroid hormone levels. These results are important for health risk assessments in free-ranging Arctic seabird populations, including those in Canada.
  
• Exposure and effects of persistent halogenated compounds in nestling Bald Eagles (Haliaeetus leucocephalus) on the south coast of British Columbia were studied and compared with the results of a study of birds from California, in collaboration with U.S. colleagues. As a top predator, the eagle is a valuable ecosystem indicator. Eagles continue to be exposed to substantial concentrations of PCBs, organochlorine pesticides and brominated flame retardants, as well as halogenated metabolites of PCBs and brominated flame retardants.
  
• Non-intrusive methods using feces were developed to study river otter populations and contaminants in the Strait of Georgia region of the Pacific coast. Otters using the major harbours of Vancouver, Victoria and Esquimalt were most exposed to persistent contaminants, and otters from Victoria harbour had fecal concentrations exceeding reported reproductive effects criteria. Results have been communicated to federal contaminated sites managers, and follow-up studies are underway.
  
• Collaborative studies with U.S. and Mexican biologists were undertaken using satellite telemetry and measurements of contaminants in prey items to compare exposures of migrating Ospreys (Pandion haliaetus) to DDT and other contaminants at breeding versus wintering grounds. Greater exposure occurred at breeding sites, particularly on the lower Columbia River, compared to wintering grounds in Mexico.
  
• Research was initiated to investigate the effects of exposure during critical early development of birds, using the Zebra Finch (Taeniopygia guttata)and the starling as laboratory and field models. Their long-term fitness was also examined. A related field study showed that there were permanent neuro-anatomical effects on the song and courtship centres of the brain of adult American Robins (Turdus migratorius) that had been exposed to DDT-related compounds at the embryo and early nestling stages.
  
• A study of the Surf Scoter (Melanitta perspicillata), a sea duck species whose populations have been steadily declining across North America, was completed. The study examined the exposure to, and effects of, contaminants in populations wintering in the Strait of Georgia. Sea ducks were exposed to a wide range of persistent halogenated compounds and heavy metals. A number of biochemical, physiological and morphological characteristics were correlated with exposure, particularly to tributyltin, an anti-fouling compound.

• Health impacts of endocrine-disrupting substances -- A variety of both in vitro and in vivo animal and other studies investigated the health impacts of CEPA 1999 and Chemicals Management Plan priority substances with endocrine-disrupting properties. In particular, a significant effort was directed at understanding the long-term reproductive, neurodevelopmental and endocrine consequences of fetal and/or neonatal exposures to complex mixtures of common environmental contaminants present in human blood. A short report was published summarizing the effects of in utero and postnatal exposure to a mixture of methylmercury, organochlorine pesticides and polychlorinated biphenyl on the metabolism of estrogens and DNA methylation. Health Canada scientists continued to investigate the impacts of chemical exposure on endocrine physiology that is critical for normal development, and participated in activities sponsored by the Organisation for Economic Co-operation and Development to develop standardized regulatory testing protocols. Results from all of these studies have been or will be published.

• The Border Air Quality Strategy (2003-2007) was undertaken to evaluate the impacts of transboundary air pollution on human health in the border regions between Canada and the United States. As part of the Strategy, Canada and the United States conducted three joint air quality pilot projects, two of which involved Health Canada. Preliminary results indicated that these studies will provide valuable health-based evidence to support the development of strategies for improving air quality. The health evidence could also support future work on an international agreement on particulate matter.
  
• Data collected under the Strategy was provided to the Ontario Ministry of Transportation for inclusion in environmental impact assessments of any new border crossings. In addition, as part of the Windsor/Detroit research, Health Canada collaborated with the United States Environmental Protection Agency in the design and implementation of an extensive air pollution exposure study. Health Canada included approximately 100 Windsor residents in a personal, indoor and outdoor air pollution study to investigate the exposure of adults and asthmatic children to a variety of air pollutants, including fine particulate matter, coarse particulate matter, nitrogen dioxide, ozone and volatile organic compounds.
  
• As part of the Canada-U.S. Border Air Quality Strategy, the Georgia Basin/Puget Sound International Airshed Strategy investigated measures to reduce air emissions and address transboundary pollution in southwestern British Columbia and northwestern Washington State. In collaboration with the British Columbia Centre for Disease Control and academic partners, Health Canada conducted health and air quality studies in support of the objectives under the airshed pilot project.

• Health Canada conducted studies of the observed relationship between air pollutants and mortality and estimated the overall risk presented by the air pollution mixture in order to provide a health-based approach to presenting air quality information to the public. The resultant Air Quality Health Index, which will help Canadians understand how to protect their health from the negative effects of air pollution on a daily basis, was developed and introduced as a pilot program in areas of British Columbia and the city of Toronto in 2006 and 2007, respectively.

• In 2006-2007, Health Canada finalized the development and testing of the Air Quality Benefits Assessment Tool (AQBAT), a computer simulation tool designed to estimate the human health and welfare benefits or damages associated with changes in Canada's ambient air quality. This flexible tool can combine and link pollutants, health endpoints, geographic areas and scenario years. It contains historical and projected population counts and health endpoint occurrence rates, and allows for the input of pollutant concentration changes. A review version of the application was released for stakeholder access and was peer reviewed.

• Health Canada research on priority substances under the Chemicals Management Plan emphasized the health impacts of mixtures, endocrine-disrupting substances and metals. The Department increased its capacity to investigate hazards posed by thyroid hormone or androgen-disrupting substances.
  
• Health Canada scientists carried out research on the identification and measurement of certain chemicals in indoor environments. The flame retardant dechlorane plus was measured in indoor dust. Another previously unreported flame retardant, hexachlorocyclopentadienyl-dibromocyclooctane, was detected in both indoor air and indoor dust. These results were published in peer-reviewed science journals.
  
• Research on airborne aldehyde species (formaldehyde, acetaldehydes, medium-chain aldehydes, alpha-beta unsaturated aldehydes) was performed and is ongoing. A paper describing the structure-activity relationship of alpha-beta unsaturated aldehydes was accepted for publication.

• An investigation in rodents to identify the critical period of development sensitive to endocrine disruption was undertaken and is ongoing. Effects of low-dose exposure during the in utero and/or the postnatal periods to mixtures of contaminants, such as PCBs, polychlorinated dibenzofurans, polychlorinated dibenzo-p-dioxin, pesticides and methylmercury, were investigated (and are ongoing) by measuring numerous endpoints (systemic, neuro-endocrine, epigenetic) indicative of a susceptibility to develop cancers, learning disabilities, and psychological and metabolic problems. Preliminary endocrine results have suggested that individuals can tolerate exposure to mixtures with no consequences unless they are subjected to early postnatal stress.

• Work on di(2-ethylhexyl) phthalate (DEHP), perflurooctane sulphonate (PFOS) and perfluorooctanoic acid (PFOA) with peroxisome proliferator properties was undertaken and is ongoing. Comparative studies of human and animal responses were initiated and are nearing completion. Results were presented at two international conferences. A final report will be prepared to support the new substances assessment and control activities.

• To identify new biomarkers of exposure and health effects and explain the molecular mechanisms of toxicity, Health Canada research scientists used genomics and proteomics methodologies to support regulatory activity. This research work has led to discoveries of relevant biomarkers of exposure, susceptibility and health outcomes of exposure to a toxicant or toxicants, including endocrine disruptors. There are five prominent research themes:
  • Critical examination of assumptions used in regulatory toxicology and risk assessment -- Gene expression profiles from laboratory experiments were investigated to examine cross-species assumptions in toxicology.
  • Functional genomics and biological validation of novel health effects -- The research involved the verification of the identified "prospective" biomarkers of thyroid hormone deficiency. This resulted in the definition of a more limited set of genes as candidate biomarkers that do not exhibit a species-specific response. This research has also examined carcinogens that disrupt cellular signal transduction networks for growth and other hormones.
  • Methodology development and assessment -- Technical validation exercises were undertaken that included inter-laboratory validation of DNA microarray data, which is crucial for international harmonization. These research activities have generated innovative approaches to the optimization of microarray design, experimental use and data normalization.
  • Discovery of biomarkers -- Characterization of oxidative stress biomarkers was developed to screen model particles and metal compounds with different physical and chemical properties for relative potencies in in vitro cell culture experiments based on peptide/protein changes. Diverse instrumental analysis methods are employed to discover biomarkers and the pathways of biological responses to toxic environmental contaminants.
  • Assessment of micro-organisms and their by-products -- This has resulted in the updating of information critical for definitive micro-organism identification through the use of advanced methodologies and the investigation of the toxicity and immunologic effects of certain selective genera, such as Bacillus, Enterobacter, Escherichia and Pseudomas, which are considered sources of new or existing biotechnology applications and include opportunistic and pathogenic relatives.

• Health Canada research scientists conducted a small-scale study of Canadian drinking water treatment plants in which residual aluminum was reported at levels higher than those recommended in the Guidelines for Canadian Drinking Water Quality (greater than 100 µg/L). While the seasonal monitoring of the other main parameters (turbidity and dissolved organic carbon) had not shown problems, this monitoring of aluminum at various treatment stages provided evidence that residual aluminum levels peak in winter and summer. The variations occur because it is difficult to adapt water treatment to the large seasonal temperature range in Canada.

• In 2007-2008, the Air Quality Benefits Assessment Tool was used to run various scenarios to assess the impact of changes in levels of greenhouse gases, emissions from ethanol-blended gasoline and specific Clean Air Regulatory Agenda scenarios.

• Data were gathered on the persistence of 6 out of 10 high-priority Domestic Substances List (DSL) micro-organisms in soil microcosms. Genetic fingerprints were developed for the micro-organisms, and then the micro-organisms were inoculated into soil microcosms and incubated for up to 180 days. Genetic material from the microcosms was then retrieved and the persistence of each strain determined. One DSL strain persisted for the entire incubation period, while three strains persisted for 126 days and the remaining two for 62 and 5 days, respectively. Data from this study will be used to support possible new regulatory data requirements under amendments to the New Substances Notification Regulations (Organisms).
  
• Data were gathered to assess the pathogenicity and/or toxicity to plants and soil invertebrates of the high-priority Domestic Substances List micro-organisms. Test methodology consisted of procedures recommended in Environment Canada's Guidance Document for Testing the Pathogenicity and Toxicity of New Microbial Substances to Aquatic and Terrestrial Organisms (EPS 1/RM/44, 2004). Tests evaluated the effect of single and continuous inoculation events, with confirmation of the presence of the micro-organisms via the retrieval of genetic material from soil samples throughout the test. Data from this study will be used to support possible new regulatory data requirements under amendments to the New Substances Notification Regulations (Organisms).