L. Mark Hewitt

Altered reproduction in fish exposed to pulp and paper mill effluents: roles of individual compounds and mill operating conditions.

For the last 20 years, studies conducted in North America, Scandinavia, and New Zealand have shown that pulp and paper mill effluents affect fish reproduction. Despite the level of effort applied, few leads are available regarding the factors responsible. Effluents affect reproduction in multiple fish species, as evidenced by decreased gonad size, decreased circulating and gonadal production of reproductive steroids, altered expression of secondary sex characteristics, and decreased egg production. Several studies also have shown that effluent constituents are capable of accumulating in fish and binding to sex steroid receptors/ binding proteins. Studies aimed at isolating biologically active substances within the pulping and papermaking process have provided clues about their source, and work has progressed in identifying opportunities for in-mill treatment technologies. Following comparisons of manufacturing processes and fish responses before and after process changes, it can be concluded that effluent from all types of mill processes are capable of affecting fish reproduction and that any improvements could not be attributed to a specific process modification (because mills normally performed multiple modifications simultaneously). Improved reproductive performance in fish generally was associated with reduced use of molecular chlorine, improved condensate handling, and liquor spill control. Effluent biotreatment has been effective in reducing some effects, but biotreated effluents also have shown no difference or an exacerbation of effects. The role of biotreatment in relation to effects on fish reproduction remains unclear and needs to be resolved.

Effect-directed analysis supporting monitoring of aquatic environments — An in-depth overview

Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.

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

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.

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

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.

Accumulation of ligands for aryl hydrocarbon and sex steroid receptors in fish exposed to treated effluent from a bleached sulfite/groundwood pulp and paper mill

The accumulation of ligands for the aryl hydrocarbon receptor (AhR) and fish sex steroid receptors was investigated using two separate controlled fish exposures to final effluent from a bleached sulfite/groundwood mill in New Brunswick, Canada. In the first experiment, hepatic tissue extracts from exposed fish were fractionated according to lipophilicity. Fractions with different octanol-water (Kow) partition coefficients were tested for the presence of bioavailable chemicals that function as ligands for the AhR in H4IIE cells, rainbow trout hepatic estrogen receptors (ER), goldfish testicular androgen receptors (AR), and goldfish sex steroid binding protein (SSBP). Fish accumulated ligands for each receptor after 4-d exposure to effluent. Single fractions contained ligands for the AhR and the ER, while multiple fractions competed for the AR and SSBP. Fish also accumulated ligands for the AhR and SSBP from Saint John River dilution water, indicating upstream sources of bioactive substances. Semipermeable-membrane devices deployed concurrently with fish accumulated ligands from effluent for all receptors except the ER. In the second experiment, accumulated ligands were evaluated after exposure of fish to effluent for two different durations and following a depuration period. Hepatic mixed function oxygenase activity and whole-liver hormonal activity, measured as binding to SSBP, returned to background following 6 d depuration and were reduced but still significant after 12-d exposure to effluent. Whole-liver extract affinities for the AR were maintained after extended exposure and depuration, indicating the potential for AR ligands to bioaccumulate. The accumulation of AhR ligands and ligands for sex steroid receptors provides a mechanistic linkage to effects on growth, development, and performance of fish exposed to effluent from this and other mills.

Toxicity of naphthenic acid fraction components extracted from fresh and aged oil sands process-affected waters, and commercial naphthenic acid mixtures, to fathead minnow (Pimephales promelas) embryos.

Naphthenic acids (NAs) are constituents of oil sands process-affected water (OSPW). These compounds can be both toxic and persistent and thus are a primary concern for the ultimate remediation of tailings ponds in northern Albertas oil sands regions. Recent research has focused on the toxicity of NAs to the highly vulnerable early life-stages of fish. Here we examined fathead minnow embryonic survival, growth and deformities after exposure to extracted NA fraction components (NAFCs), from fresh and aged oil sands process-affected water (OSPW), as well as commercially available NA mixtures. Commercial NA mixtures were dominated by acyclic O2 species, while NAFCs from OSPW were dominated by bi- and tricyclic O2 species. Fathead minnow embryos less than 24h old were reared in tissue culture plates terminating at hatch. Both NAFC and commercial NA mixtures reduced hatch success, although NAFCs from OSPW were less toxic (EC50=5-12mg/L, nominal concentrations) than commercial NAs (2mg/L, nominal concentrations). The toxicities of NAFCs from aged and fresh OSPW were similar. Embryonic heart rates at 2 days post-fertilization (dpf) declined with increasing NAFC exposure, paralleling patterns of hatch success and rates of cardiovascular abnormalities (e.g., pericardial edemas) at hatch. Finfold deformities increased in exposures to commercial NA mixtures, not NAFCs. Thus, commercial NA mixtures are not appropriate surrogates for NAFC toxicity. Further work clarifying the mechanisms of action of NAFCs in OSPW, as well as comparisons with additional aged sources of OSPW, is merited.

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

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 Proposed Framework for Investigation of Cause for Environmental Effects Monitoring

Environmental Effects Monitoring (EEM) programs in Canada have been developed for the pulp and paper and metal mining industries, and require a cyclical evaluation of the receiving environment to determine whether effects exist when the facilities are in compliance with existing regulations. Identifying the cause of environmental effects is a specific, identified stage in this monitoring program, but as yet there has not been a synthesis of what is meant by “identification of cause”. We propose a multitiered guidance framework for the identification of the cause of environmental effects after they have been detected, confirmed, and their extent and magnitude documented. As part of point source confirmation, the framework includes levels to define whether there is an effect, whether it is related to the effluent discharge facility, and whether response patterns in the receiver are characteristic of a particular stressor type. The next tier involves investigating individual process wastes within the facility to determine the components that are contributing to effects caused by exposure to the final effluent. The last three tiers of the framework relate to characterizing the chemical classes involved in the effect and, ultimately, to identifying the specific chemicals associated with the responses. Although there is increasing knowledge of specific causes of environmental effects gained as one progresses through the levels of investigation, there is a concomitant increase in effort and costs required. Stakeholder input is critical in determining the depth of the investigation as well as how to proceed once the environmental effects information is available.

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.

Use of paired fathead minnow (Pimephales promelas) reproductive test. Part 1: Assessing biological effects of final bleached kraft pulp mill effluent using a mobile bioassay trailer system

Reproductive effects have been recorded in wild fish in waters receiving pulp mill effluent (PME) since the mid to late 1980s. Laboratory assays with fathead minnow (FHM; Pimephales promelas) have been developed to better understand fish responses to PME. However, observations from laboratory studies have been variable, making it difficult to establish cause/effect relationships. A lack of environmental relevance in these laboratory studies may have contributed to the variability observed. The objectives of the present study were, first, to determine the effects of bleached kraft PME (BKME) on FHM under environmentally realistic conditions (i.e., ambient water and effluent quality) and, second, to determine the suitability of pair-breeding FHM to better link BKME-induced changes in indicators at the biochemical, individual, and population levels. A mobile bioassay trailer was situated on-site at a bleached kraft mill for 60 d, allowing supply of both ambient water (Lake Superior, Canada) and final BKME. The reproductive output of FHM was initially assessed for 21 d to obtain baseline data pre-exposure. At the end of the pre-exposure period, selected breeding pairs were exposed to final BKME (100% v/v and 1% v/v) for 21 d. Results demonstrated a stimulatory response pattern at 1% BKME (e.g., increased egg production) compared to control. In the 100% treatment, spawning events were reduced and fewer eggs were produced during the first two weeks of exposure. Exposure to 100% (v/v) BKME also resulted in ovipositor development in males and development of male secondary sex characteristics in females. Obtaining pre-exposure data and use of pair-breeding FHM in this assay gave a sensitive indication of effluent effects and allowed accurate comparisons of endpoints to be made.