Mark E. McMaster

Pharmaceuticals and Personal Care Products in the Environment: What Are the Big Questions?

Background: Over the past 10–15 years, a substantial amount of work has been done by the scientific, regulatory, and business communities to elucidate the effects and risks of pharmaceuticals and personal care products (PPCPs) in the environment. Objective: This review was undertaken to identify key outstanding issues regarding the effects of PPCPs on human and ecological health in order to ensure that future resources will be focused on the most important areas. Data sources: To better understand and manage the risks of PPCPs in the environment, we used the “key question” approach to identify the principle issues that need to be addressed. Initially, questions were solicited from academic, government, and business communities around the world. A list of 101 questions was then discussed at an international expert workshop, and a top-20 list was developed. Following the workshop, workshop attendees ranked the 20 questions by importance. Data synthesis: The top 20 priority questions fell into seven categories: a) prioritization of substances for assessment, b) pathways of exposure, c) bioavailability and uptake, d) effects characterization, e) risk and relative risk, f ) antibiotic resistance, and g) risk management. Conclusions: A large body of information is now available on PPCPs in the environment. This exercise prioritized the most critical questions to aid in development of future research programs on the topic.

Intersex and reproductive impairment of wild fish exposed to multiple municipal wastewater discharges.

The Grand River watershed in Ontario, Canada, receives and assimilates the outflow of 29 Municipal Wastewater Effluent (MWWE) discharges which is a mixture of domestic and industrial wastes. The purpose of this study was to investigate the cumulative impact of multiple sewage discharges on populations of wild fish. In field studies, responses of fish populations and individual fish responses in terms of growth (condition factor), reproduction (in vitro sex steroid production, gonadosomatic indices, histology [cellular development and intersex]) were assessed upstream and downstream of two municipal discharges. Fish [Greenside Darters Etheostoma blennioides and Rainbow Darters E. caeruleum] collected downstream of two municipal wastewater plants had the potential to have greater growth (longer and heavier) when compared to reference fish collections regardless of sex. Fish were not assimilating additional anthropogenic resources into energy storage (increased condition, liver somatic index). Impacts on ovarian development appeared to be minor with no differences in growth, steroid production or cellular development. Sewage exposed male fish were experiencing impairment in the capacity to produce testosterone and 11-ketotestosterone in vitro, and in cellular development (GSI, intersex). Male darters of both species collected in the upstream agricultural region demonstrated no evidence of intersex whereas our urban reference sites had incidence of intersex of up to 20%. Rates of intersex were elevated downstream of both sewage discharges studied (33% and>60%, respectively). Lower rates of intersex at the intermediate sites, and then increases downstream of second sewage discharge suggests that fish populations have to potential to recover prior to exposure to the second sewage effluent. Pre-spawning darters demonstrated dramatically higher incidence of intersex in the spring at both urban reference sites (33% and 50%, respectively), and increased more so downstream of the near-field and far-field exposure sites (60% and 100%, respectively). These findings suggest that the compounds released in STP effluents have a tendency to act on the male reproductive system. These effects may become more pronounced as projected human population growth will require the aquatic environment to assimilate an increasing amount of sewage waste.

An overview of recent studies on the potential of pulp‐mill effluents to alter reproductive parameters in fish

In the early 1990s, many Canadian pulp and paper mills implemented process changes to comply with new regulations that came into effect in 1993. These regulations placed stricter guidelines on a number of parameters in effluent discharges, including limits on acute toxicity, on the discharges of suspended solids, and on biochemical oxygen demand. To meet these new regulations, many of the older Canadian pulp and paper mills had to install secondary treatment systems. The investment by the Canadian pulp and paper industry was in excess of

A Decade of Research on the Environmental Impacts of Pulp and Paper Mill Effluents in Canada: Sources and Characteristics of Bioactive Substances

5 billion, and the implementation of the new regulations and the process changes took several years. The new regulations were an extension of regulations designed in the early 1970s and were not designed specifically to address the reproductive responses recently reported in fish collected downstream of mills in Scandinavia and North America. This report describes a series of projects conducted between 1991 and 1996 to evaluate the effectiveness of the new regulations to address the issue of reproductive responses in fish associated with exposure to pulp-mill effluents. These studies have shown that the existing short-term bioassays do not adequately predict the potential of effluents to affect reproduction in wild fish. Laboratory testing using fathead minnows exposed over a full life cycle confirmed depression in sex steroid production, delay in sexual maturity, reduced egg production, and changes in secondary sex characteristics documented at some sites. Our studies demonstrated that both steroid hormone changes and induction of liver detoxification enzymes take place quickly. While short-term exposures can predict the potential of some effluents to impact steroid hormone production, there is no readily available assay that can be widely applied. In the absence of a usable and transferable laboratory bioassay, field collections were conducted at a number of sites. Generalizations are not possible at this time, but impacts have been seen at a variety of sites, and partial recovery has been documented at five sites in North America following various process and waste treatment changes. Data gaps and critical research areas are identified.

Biochemical and histopathological effects in pearl dace (Margariscus margarita) chronically exposed to a synthetic estrogen in a whole lake experiment.

This article is a review of research efforts over the last decade on the sources and characteristics of substances in Canadian pulp mill effluents associated with two responses in fish: (1) induction of detoxification enzymes and (2) reproductive effects. The initial uncertainty regarding the role of chlorine bleaching and dioxins in these responses was resolved by the mid 1990s, when it was determined that effects were not correlated with effluent adsorbable organic halogen (AOX) levels and that releases of dioxins had decreased substantially. In the mid 1990s researchers were able to partially attribute enzyme activity induction in fish to wood components, while other studies showed individual wood extractives had the potential to affect fish reproduction. A lack of correlation between threshold reproductive responses and effluent concentrations indicated additional unidentified compounds and mechanisms were involved. In the late 1990s, source identification approaches in concert with the development of mechanistically linked in vitro and in vivo bioassays showed multiple compounds are affecting production and signaling of sex steroids in fish. These substances are bioavailable and accumulated rapidly, consistent with the body of evidence that has shown a sustained exposure is required to produce both elevated enzyme activity and depressions in sex steroid levels. The patterns of these substances in effluents and fish tissues are not correlated with production type or effluent treatment. Collectively, these findings show that bioactive substances originate from wood and are derived from lignin and/or terpenoids, they are liberated during pulp digestion, and in kraft mills they are present in black liquor and chemical recovery condensates. Additional bioactive substances are also present in bleachery effluents containing residual lignin. The lack of a definitive identification of the responsible compounds has prevented an evaluation of the effectiveness of industry-wide process changes. Continued research into the identities, origins, and environmental fate of these substances and the efficacy of effluent treatment is required to determine their significance and relationship to the existing impacts of effluents from pulp and paper mills in Canadian aquatic ecosystems. Completion of this review would not have been possible without the helpful comments and scientific reviews provided by Drs. John Carey, Jim Maguire, Kelly Munkittrick, Glen Van Der Kraak, and Olöf Sandstrom.

Using reproductive endpoints in small forage fish species to evaluate the effects of athabasca oil sands activities

Potential effects of exposure to the synthetic estrogen 17alpha-ethynylestradiol (EE2) were examined in several species of fish from a lake experimentally treated with environmentally relevant concentrations of the contaminant. Ethynylestradiol was added to Lake 260, a small Precambrian shield lake at the Experimental Lakes Area in northwestern Ontario, Canada, from May to October of 2001, 2002, and 2003. Mean concentrations of EE2 in epilimnetic waters ranged between 4.5 and 8.1 ng/L during the three years, with overall means of 6.1 (+/- 2.8), 5.0 (+/- 1.8), and 4.8 (+/- 1.0) ng/L for the three years, respectively. Male and female pearl dace (Margariscus margarita) captured after EE2 additions began contained up to 4,000-fold higher concentrations of the egg yolk precursor vitellogenin than fish captured from the same lake before the EE2 additions or when compared to fish from reference lakes. Edema in the ovaries, inhibited development of testicular tissue, intersex, and histopathological kidney lesions were all evident in fish exposed to EE2. Some indications that EE2 exposure affected in vitro steroidogenic capacity of the ovaries and the testes existed, although results were not always consistent between years. Pearl dace abundance was similar in the lake treated with EE2 and the reference lake. A trend exists toward a reduced overall population of pearl dace from the treated and reference lakes, as do indications that young-of-the-year size classes are less abundant in the EE2-treated lake. Biochemical and histopathological impacts observed in fish exposed to EE2 in this study have not yet been linked to clear population level impacts in pearl dace. Monitoring of these populations is ongoing.

A Decade of Research on the Environmental Impacts of Pulp and Paper Mill Effluents in Canada: Field Studies and Mechanistic Research

The main objective of this study was to evaluate the influence of naturally occurring oil sands-related compounds (OSRC) on reproductive function in fish in order to assess the impacts of anthropogenic point-source inputs. The health of slimy sculpin (Cottus cognatus) and pearl dace (Semotilus margarita) collected from the Alberta Athabasca Oil Sands (Canada) watershed were examined. Two rivers were selected for study: the Steepbank and the Ells. These rivers originate outside the oil sands formation, where fish are unexposed (Ref), exposed to naturally occurring oil sands-related compounds (Nat), or exposed to naturally occurring compounds as well as adjacent to surface mining activity (Dev). Assessment endpoints included gonadosomatic indices (GSI), fecundity, and in vitro gonadal steroid production. In vitro gonadal incubations demonstrated lower levels of steroid production at sites along the Steepbank River within the oil sands deposit. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity, an indicator of exposure to OSRC, was elevated twofold at the site with natural compounds and up to 10-fold at the site adjacent to development compared to EROD activity in fish from the reference site. Fish collected in the Ells River had a threefold induction in EROD activity but no significant reduction in steroid production when compared to reference fish. No consistent alterations in gonadal development were seen in fish collected from sites within the oil sands deposit. This research in the Athabasca River basin provides baseline information of the health of fish populations within the oil sands deposit prior to further development in the area.

A decade of research on the environmental impacts of pulp and paper mill effluents in Canada: development and application of fish bioassays.

Studies conducted in Sweden in the early 1980s provided some of the first evidence that effluents from some pulp mills were capable of inducing toxic responses in fish at very low concentrations in the receiving environment. In response to these findings, studies were initated in Canada and impacts of primary treated bleached kraft mill effluent on reproductive function in fish were found. Reproductive impacts in fish were not limited to mills that used chlorine in the bleaching process and were also evident at some mills that employed secondary effluent treatment. In 1992, new federal regulations were passed under the Canadian Environmental Protection Act to control releases of dioxins and furans, and a new Pulp and Paper Effluent Regulation under the Fisheries Act set stricter limits for biological oxygen demand and total suspended solids. Very importantly, the new regulations included requirements for environmental effects monitoring (EEM) at all mill sites. This allowed the effectiveness of the control limits in protecting fish, fish habitat, and human use of fisheries resources to be assessed. At the same time, the Minister of the Environment launched an intensive government, industry, and university research program. Results from this research program along with feedback from the EEM program would then be used to define what additional control actions might be necessary. This article reviews the field studies and mechanistic research conducted in Canada following the implementation of the new federal regulations. Great progress has been made in this area, first demonstrating reproductive effects at various locations, then determining the mechanisms responsible for the reproductive effects at specific sites, followed by the demonstration of partial recovery in reproductive function following process and treatment changes in response to the new regulations. However, it is clear from the results of the first two cycles of the EEM program that mill effluents still affect the local receiving environments at a number of locations across Canada, and that continued research combining field studies, bioassay application, and chemical identification is required. Completion of this assessment would not have been possible without the helpful comments and scientific reviews provided by Drs. John Carey, Jim Maguire, Kelly Munkittrick, Glen Van Der Kraak, Jim Sherry, and Olöf Sandstrom.

Occurrence and degree of intersex (testis–ova) in darters (Etheostoma SPP.) across an urban gradient in the Grand River, Ontario, Canada

Laboratory tests have been used to assess the regulatory and research questions related to the effects of pulp mill effluents on aquatic biota. Acute, short-term laboratory tests have clearly shown the improvement in final effluent quality following installation of secondary treatment at Canadian pulp mills. In an effort to predict and investigate impacts on wild fish, laboratory bioassays were developed to examine sublethal endpoints: induction of hepatic mixed function oxygenase activity and reduction of sex steroid concentrations. These laboratory assays have been used to assess whole effluents, specific chemicals, and components of pulp mill processes, and to discriminate between historical and present-day effluent discharges. These tests have shown that induction of mixed-function oxygenase activity and reduction of sex steroid concentrations are produced by effluents from a variety of mill types, with and without chlorine bleaching, in hardwood and softwood pulping facilities, and before and after effluent treatment. These short-term bioassays have enabled reductions in sex steroid concentrations to be linked to mill process streams, and have provided information on effective waste stream treatment. Longer term, life-cycle fish bioassays have shown that chronic exposure to pulp mill effluents commonly results in growth enhancement, liver enlargement, and decreases in gonad size, secondary sex characteristics, and fecundity. These long-term laboratory exposures are able to mimic the most commonly observed alterations of wild fish exposed to pulp mill effluents: increases in condition factors, increases in liver-somatic indices, and decreases in gonadosomatic indices. This pattern of response is a combination of nutrient enrichment with metabolic disruption. The most sensitive and biologically meaningful endpoint is decreased reproduction in fish life-cycle exposures. As the laboratory tests move forward into the next decade, attention will focus on the reproductive endpoints and on the possibility of shortening the fish bioassays while still maintaining sensitivity and biological relevance.

A framework for assessing cumulative effects in watersheds: An introduction to Canadian case studies

The variability and extent of the intersex condition (oocytes in testes, or testis-ova) was documented in fish along an urban gradient in the Grand River, Ontario, Canada, that included major wastewater treatment plant outfalls. A method for rapid enumeration of testis-ova was developed and applied that increased the capacity to quantify intersex prevalence and severity. Male rainbow darters (Etheostoma caeruleum) sampled downstream of the first major wastewater outfall (Waterloo) had a significant increase, relative to 4 upstream reference sites, in the mean proportion of fish with at least 1 testis-oocyte per lobe of testes (9-20% proportion with ≤ 1 testis-oocyte/lobe vs 32-53% and >1.4 testis-oocyte/lobe). A much higher mean incidence of intersex proportion and degree was observed immediately downstream of the second wastewater outfall (Kitchener; 73-100% and 8-70 testis-oocyte/lobe); but only 6.3 km downstream of the Kitchener outfall, the occurrence of intersex dropped to those of the reference sites. In contrast, downstream of a tertiary treated wastewater outfall on a small tributary, intersex was similar to reference sites. Estrogenicity, measured using a yeast estrogen screen, followed a similar pattern, increasing from 0.81 ± 0.02 ng/L 17b-estradiol equivalents (EEq) (Guelph), to 4.32 ± 0.07 ng/L (Waterloo), and 16.99 ± 0.40 ng/L (Kitchener). Female rainbow darter downstream of the Kitchener outfall showed significant decreases in gonadosomatic index and liver somatic index, and increases in condition factor (k) relative to corresponding reference sites. The prevalence of intersex and alterations in somatic indices suggest that exposure to municipal wastewater effluent discharges can impact endocrine function, energy use, and energy storage in wild fish.