Acute toxicity of commonly used forestry herbicide mixtures to Ceriodaphnia dubia and Pimephales promelas.

Because many herbicides selectively control specific species or types of vegetation, they are often applied as mixtures to achieve better control over undesirable vegetation. When herbicides are applied in forest ecosystems, streams, ponds, and other bodies of water are typically protected by buffer zones in which no herbicide is applied. However, in some landscapes, small wetlands and streams are difficult to see and avoid, thus the potential acute toxicity of herbicide mixtures to aquatic organisms is of interest, yet it has not been well-studied. We examined the acute toxicity of 23 different herbicide mixtures to Ceriodaphnia dubia and fathead minnows (Pimephales promelas) at environmentally relevant concentrations, and, where possible, characterized mixture interactions using Marking's Additive Index. Maximum exposure concentrations were equivalent to applying the maximum allowable rate for each component directly to the surface of a 6-in. deep pond with no dissipation following application. Under the conditions of this study, herbicide formulations containing Accord Concentrate (glyphosate), Arsenal AC (imazapyr), Chopper (imazapyr), Escort (metsulfuron methyl), Oust XP (sulfometuron methyl), and Velpar L (hexazinone) were not associated with appreciable acute toxicity to fathead minnows or C. dubia when used alone or in mixtures with each other and various surfactants and adjuvants. Herbicide mixtures for which Additive Indexes could be calculated exhibited primarily antagonistic or simple additive toxicity. In the few cases where synergistic toxicity was observed, the degree of synergism was slight, never exceeding approximately twice the effect estimated based on additive toxicity. Based on the results of this study, neither acute toxicity nor enhanced acute aquatic toxicity due to synergistic mixture effects appears to be a significant concern for applications of the herbicide mixtures most commonly used in forestry.

Responses of fathead minnows (Pimephales promelas) during life-cycle exposures to pulp mill effluents at four long-term receiving water study sites.

We exposed fathead minnows (Pimephales promelas to 7 concentrations of effluents from pulp mills at 4 Long-Term Receiving Water Study (LTRWS) sites. The primary objective of these investigations was to determine the potential for toxicity, particularly on fish reproduction, of the pulp mill effluents using laboratory tests. These tests were performed as LTRWS fish community assessments were being completed, thus results of the laboratory fish reproduction tests could be compared to in-stream fish community measurements. In general, bioindicators measured during the life-cycle tests, including gonadosomatic index (GSI), hepatosomatic index, condition factor, numbers of tubercles on heads of males and females, and gonadal histology did not show consistent patterns or dose response and did not predict effects on egg production. Gonadosomatic indexes and tubercles also did not indicate estrogenic or androgenic responses to the effluents during the life-cycle tests. The most consistently sensitive test endpoint showing a dose response was the 25% inhibition concentration (IC25) for egg production. Based on this endpoint all 4 effluents had effects on fish reproduction from 8% by volume to 100% effluent. However, in-stream effects on fish reproduction would not be expected based on these 4 life-cycle tests for any of the LTRWS stream sites. The mean effluent concentration in Codorus Creek, Pennsylvania, USA was approximately 32%, and the IC25 for the life-cycle test was 100% effluent, providing a margin of safety of approximately 3 times. The margins of safety at the other sites are much greater: 34 times for Leaf River, Mississippi, USA (IC25 = 69%, 2% mean receiving water concentration), 36 times for the McKenzie River, Oregon, USA (IC25 = 18%, 0.5% mean receiving water concentration), and 40 times for the Willamette River, Oregon, USA (IC25 = 8%, 0.20% mean receiving water concentration). Effects on fish numbers, diversity, and community structure due to the effluent were also not found during the LTRWS, which is consistent with these laboratory results. These findings indicate that in this case, when laboratory results combined with in-stream effluent concentrations suggest in-stream effects on fish population are not expected, the laboratory results are consistent with the in-stream observations. However, inferences about situations where laboratory results predict in-stream effects cannot be made from these data.

A long-term, multitrophic level study to assess pulp and paper mill effluent effects on aquatic communities in four US receiving waters: lessons learned.

Lessons learned from the development, implementation, and initial 8 y of study findings from a long-term study to assess the effects of pulp and paper mill effluents on receiving waters are summarized as a conclusion to a series of articles (this issue) on study findings. The study, based on industry-defined information needs, was developed via a science-based experimental design into a long-term (>10 y) watershed-scale monitoring program that integrated in-stream population/ community assessment, laboratory chronic bioassays, and fathead minnow full life-cycle assays as well as water quality and effluent quality monitoring and habitat assessment in addressing the presence of effluent effects. The 4 study streams (Codorus Creek, PA; Leaf River, MS; and the McKenzie and Willamette rivers, OR) represented both bleached and unbleached kraft mill processes and effluent concentrations that ranged from near typical for the United States (0.4%) to very high (Codorus Creek= 32%). Following 8 y of monitoring, the weight of evidence suggests an absence of biological differences at stations downstream of the mill discharges for periphyton or macroinvertebrates and, with the exception of 1 of 9 large-bodied fish and 1 of 7 small-bodied fish community structure metrics for 1 river (McKenzie), an absence of differences for fish communities. Laboratory bioassay and fathead minnow full-life cycle tests supported a substantial "margin of safety" in that, depending on the effluent, adverse responses did not occur until effluent concentrations were from 2 times to more than 150 times in-stream concentrations. The incorporation of a watershed spatial scale illustrated that each sample site tended to be unique over the 28 to 50 km monitored segments with respect to habitat and that knowledge of these variables permitted accurate evaluations of effluent effects. Similarly, the multiyear study framework provided information regarding the natural seasonal and year-to-year variability in fish communities and consequently a better understanding of how potential effluent effects signals could be expressed within this variability. The study incorporated an adaptive management strategy that provided for study design and monitoring modifications over time as a way of benefiting from practical experience and knowledge gained through time and to optimize the use of study resources. Results from this initial 8 y of monitoring, to our knowledge, represent the longest-known population/community-level assessment of the in-stream effects of pulp and paper mill effluents. Beyond the lessons learned with respect to effluent effects are those related to the esign and conduct of long-term watershed-scale studies that may be of use to others in developing watershed assessment or management programs.

A long-term, multitrophic level study to assess pulp and paper mill effluent effects on aquatic communities in four US receiving waters: background and status.

An industry-funded, long-term, receiving water study was initiated in 1998/1999 to adress questions about the potential effects of pulp and paper mill effluent discharges on US receiving waters. Although the study continues, the knowledge gained to date provides an opportunity to reflect on the study development process, its progress, and its outcomes. As a backdrop to a series of articles in this special issue describing study results, this article describes the process by which study information objectives were identified as well as the process by which the experimental design was developed. A review of past literature and research identified gaps in long-term population/community data about effluent effects and that, consequently, emerged as a primary information objective. The selected streams for study included 1) Codorus Creek (Pennsylvania, USA), 2) Leaf River (Mississippi, USA), 3) McKenzie River (Oregon, USA), and 4) Willamette River (Oregon) represent a blend of mill process types, coldwater and warmwater stream types, and a range of effluent concentrations. Measurements included numbers of periphyton, macroinvertebrate, and fish communities; the assessment of water and effluent quality; laboratory bioassays; and fish full-life-cycle assays. Information objectives included addressing natural variability and, consequently, the study included long-term temporal (>10 y) and watershed-scale spatial frameworks. Regional-scale ecological risk assessments were performed for each site that aided in placing each site in an ecological and regulatory context. An adaptive-management process is described that allowed for modifications over time as a result of lessons learned as the study progressed. Results from the initial 7 to 8 y of monitoring, as described in the series of articles in this special issue, provide a unique data set with respect to addressing point-source pulp and paper mill effluent discharge concerns and may serve as a template for others to use in developing monitoring or management programs to assess or address water quality conditions or concerns.

A long-term, multitrophic level study to assess pulp and paper mill effluent effects on aquatic communities in four US receiving waters: characteristics of the study streams, sample sites, mills, and mill effluents.

Watershed characteristics, study streams, sample sites, mills, and mill effluents are provided for 4 streams included in a long-term study to assess potential effects of pulp and paper mill effluents on US receiving waters. The study streams are Codorus Creek (Pennsylvania, USA), Leaf River (Mississippi, USA) and McKenzie and Willamette rivers (Oregon, USA) and were chosen to represent a blend of mill process types, effluent concentrations, and coldwater/warmwater stream systems. The described effluent quality, water quality, and habitat data sets encompass the initial 7 to 8 y of a study anticipated to continue >10 y and provide a backdrop to a series of articles describing periphyton, macroinvertebrate, and fish community properties in these same streams. The mean in-stream waste concentration (IWC) for these 4 effluent discharges was 32.4%, 2.0%, 0.5%, and 0.2% v/v for Codorus Creek and Leaf, McKenzie, and Willamette rivers, respectively, as compared with a median of 0.4% for US mills. Effluent quality measurements included Selenastrum capricornutum, Ceriodaphnia dubia, and Pimephales promelas chronic bioassays as sanctioned by the US Environmental Protection Agency for estimating effluent effects on receiving-water aquatic communities. Based on mean bioassay inhibition concentration for a 25% effect and on mean IWC, a margin of safety against adverse biological effects of 2, 25, 137, and 150 times was indicated for Codorus Creek and Leaf, McKenzie, and Willamette rivers, respectively. Habitat and water quality assessment was carried out over a gradient of sample sites above and below the effluent discharge to determine nonmill-related conditions that might interfere with interpretation of effluent effects. Noneffluent related localized differences in conditions for some parameters, including current velocity (McKenzie River), and surface incident photosynthetically active radiation (Codorus Creek and Willamette River) occurred at the sample stations immediately upstream or downstream of the effluent discharge. In addition, broader watershed differences were evident on Codorus Creek, where a relatively rich riparian corridor and stream structure occurred upstream in contrast to areas of canopy and stream-structure loss in the downstream urban area. The mill effluent discharges contributed to increases in receiving-water color and conductivity, although upstream tributaries contributed additional conductivity to Codorus Creek and color to the Leaf River. The McKenzie River provided the only example of a nutrient increase immediately downstream of a mill discharge. This increase in total nitrogen (0.11 vs 0.16 mg/L) could not, however, be differentiated with respect to whether it was of mill effluent or tributary stream origin. Tributary streams were potentially important total nitrogen contributors on Codorus Creek and the Willamette River. As an integrated study, the effluent quality and physical/chemical watershed descriptions provided here represent 1 component of the broader study addressing potential point-source effluent effects within the context of the larger watershed and a multiyear timescale. The absence of effluent-related in-stream chemical/physical responses, other than increases in conductivity and color, and a considerable bioassay-based margin of safety, provides for a working hypothesis that there will be no effluent-related biological population/community responses from these 4 mill discharges. This hypothesis, as it relates to periphyton, macroinvertebrate, and fish communities, will be addressed in other articles in this series.

Bluegill (Lepomis macrochirus) vitellogenin: purification and enzyme-linked immunosorbent assay for detection of endocrine disruption by papermill effluent.

Vitellogenin (VTG) is a highly specific marker of exposure to environmental estrogens and has been used extensively in field and laboratory studies of estrogenic endocrine disruption in fishes. The purpose of this study was to develop and validate a sensitive, competitive, enzyme-linked immunosorbent assay (ELISA) specific for bluegill (Lepomis macrochirus) vitellogenin. Bluegill VTG was purified by anion exchange chromatography on DEAE-agarose. The polypeptide had an apparent mass of 170 kDa and was specifically recognized by the rabbit antiserum raised against bluegill female-specific plasma protein. Plasma samples from vitellogenic females diluted in parallel with the purified VTG standard curve in the ELISA. The detection limit of the assay was 29 ng/ml and the working range extended to 2700 ng/ml. Recovery of purified VTG was 85.8+/-9.5%, intra-assay variation was 6.4% and interassay variation was 12.3%. We used this ELISA to analyze the seasonal cycle of vitellogenesis in female bluegill and to evaluate potential disruption of this process by exposure to bleached kraft mill effluent (BKME). Captive female bluegill stocked in outdoor experimental streams in New Bern, NC had the lowest levels of VTG, estradiol-17beta (E2), and testosterone (T) and the smallest oocyte diameters in January, but these variables increased in March and remained elevated through August, suggesting an extended spawning season. Plasma VTG, E2, T and oocyte diameter were unaffected by exposure to BKME concentrations as high as 30%. Development of the VTG ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to potential endocrine disrupting compounds.