[Book review] Aquatic Toxicology
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This chapter provides a listing of the increasing variety of organic moieties and heteroatom group for which Linear Solvation Energy Relationship (LSER) values are available, and the LSER variable estimation rules. The listings include values for typical nitrogen-, sulfur- and phosphorus-containing moieties, and general organosilicon and organotin groups. The contributions by an ion pair situation to the LSER values are also offered in Table 1, allowing estimation of parameters for salts and zwitterions. The guidelines permit quick estimation of values for the four primary LSER variables V i /100, π * , Β m , and α m by summing the contribtuions from its components. The use of guidelines and Table 1 significantly simplifies computation of values for the LSER variables for most possible organic comppounds in the environment, including the larger compounds of environmental and biological interest.
Available information on aquatic biota of the Willamette Basin was reviewed and summarized to describe current and historical conditions as part of the U.S. Geological Survey's National Water-Quality Assessment Program. Biological parameters emphasized include the status, distribution, and trends of aquatic biota, particularly algae, macroinvertebrates, and fish; the condition of aquatic and riparian habitat in which these biota reside; and the response of these biota to natural and human-associated impacts, including the level, type, and effect of contaminants. Considerable data are available on aquatic biota in the Willamette Basin, although the information is highly uneven relative to taxa and spatial scope. Extensive information exists for high-profile taxa, such as salmonid fishes, but less information is available for macroinvertebrates, and relatively little data exist for algae. Additionally, some areas such as the H.J. Andrews Experimental Forest and the main stem Willamette River have been extensively studied, whereas data are limited for many other areas. The basin supports a diverse aquatic macroinvertebrate fauna. Available data indicate a relatively high diversity of taxa and a high richness of Ephemeroptera, Plecoptera, Trichoptera (EPT) taxa in the upper reaches of the basin. In the lower main stem reaches, macroinvertebrate assemblages are dominated by pollution tolerant organisms and those adapted to low dissolved oxygen levels. Most of the limited data on algae are from sampling in the main stem Willamette River. Diatoms and blue-green algae are the dominant algal forms. Approximately 61 fish species occur in the basin, although nearly half are introduced. Species richness and distribution are highly correlated with elevation, stream gradient, and water temperature. High elevation, cold water, mountain streams are characterized by a few species of salmonids, sculpins, suckers, and whitefish. Low elevation, main stem reaches of major rivers and streams are dominated by warm water species, such as bass, catfish, and several species in the pan fish group. The only species of fish listed as threatened or endangered is the Oregon chub (Oregonichthys crameri). The effect of an expanding human presence in the Willamette Basin has substantially altered aquatic and riparian habitats, and the biota that use or reside in these habitats. Construction of dams, channelization and bank stabilization of rivers, species introductions, supplementations of fisheries through aquaculture, timber harvesting, agricultural activities, and urbanization have contributed to changes in aquatic habitats and biota from historical conditions. Aquatic toxicological investigations in the basin have focused primarily on fish. These studies have addressed chlorinated pesticides, polychlorinated biphenyls (PCBs), dioxins and furans, polycyclic aromatic hydrocarbons (PAHs), and trace elements in aquatic tissue, as well as fish health assessments, skeletal abnormalities, and aquatic toxicological responses. Several pesticides exceeded U.S. Environmental Protection Agency and State water-quality criteria for the protection of aquatic life. Elevated PCB, dioxin, and furan concentrations were associated with point sources, such as pulp and paper mills. Elevated concentrations of mercury in aquatic tissue were associated with several reservoirs. Fish health assessments and skeletal abnormality studies detected high levels of abnormalities in fish from the main stem Willamette River. Few investigations have examined aquatic toxicological responses, such as enzyme induction assays, growth assays, and biomarker studies.
Since their introduction to North America in the 1980s, research to develop effective control tools for invasive mussels ( Dreissena polymorpha and D. rostriformis bugensis ) has been ongoing across various research institutions using a range of testing methods. Inconsistencies in experimental methods and reporting present challenges for comparing data, repeating experiments, and applying results. The Invasive Mussel Collaborative established the Toxicity Testing Work Group (TTWG) in 2019 to identify “best practices” and guide development of a standard framework for dreissenid mussel toxicity testing protocols. We reviewed the literature related to laboratory-based dreissenid mussel toxicity tests and determined the degree to which standard guidelines have been used and their applicability to dreissenid mussel testing. We extracted detailed methodology from 99 studies from the peer-reviewed and gray literature and conducted a separate analysis for studies using presettlement and postsettlement mussels. We identified specific components of methods and approaches that could be refined or standardized for dreissenid mussels. These components included species identification, collection methods, size/age class distinction, maintenance practices, testing criteria, sample size, response measures, reporting parameters, exposure methods, and mortality criteria. We consulted experts in the field of aquatic toxicology and dreissenid mussel biology on our proposed. The final recommendations contained in the present review are based on published standard guidelines, methods reported in the published and gray literature, and the expertise of TTWG members and an external panel. In addition, our review identifies research needs for dreissenid mussel testing including improved methods for early–life stage testing, comparative data on life stages and between dreissenid mussel species, inclusion of a reference toxicant, and additional testing of nontarget species (i.e., other aquatic organisms). Environ Toxicol Chem 2023;42:1649–1666. © 2023 His Majesty the King in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.
Aquatic environments are being contaminated with a myriad of anthropogenic chemicals, a problem likely to continue due to both unintentional and intentional releases. To protect valuable natural resources, novel chemicals should be shown to be environmentally safe prior to use and potential release into the environment. Such proactive assessment is currently being applied to room-temperature ionic liquids (ILs). Because most ILs are water-soluble, their effects are likely to manifest in aquatic ecosystems. Information on the impacts of ILs on numerous aquatic organisms, focused primarily on acute LC50 and EC50 endpoints, is now available, and trends in toxicity are emerging. Cation structure tends to influence IL toxicity more so than anion structure, and within a cation class, the length of alkyl chain substituents is positively correlated with toxicity. While the effects of ILs on several aquatic organisms have been studied, the challenge for aquatic toxicology is now to predict the effects of ILs in complex natural environments that often include diverse mixtures of organisms, abiotic conditions, and additional stressors. To make robust predictions about ILs will require coupling of ecologically realistic laboratory and field experiments with standard toxicity bioassays and models. Such assessments would likely discourage the development of especially toxic ILs while shifting focus to those that are more environmentally benign. Understanding the broader ecological effects of emerging chemicals, incorporating that information into predictive models, and conveying the conclusions to those who develop, regulate, and use those chemicals, should help avoid future environmental degradation.
Introduction This circular provides an overview of selected activities that were conducted within the U.S. Geological Survey (USGS) Integrated Methods Development Project, an interdisciplinary project designed to develop new tools and conduct innovative research requiring integration of geologic, geophysical, geochemical, and remote-sensing expertise. The project was supported by the USGS Mineral Resources Program, and its products and acquired capabilities have broad applications to missions throughout the USGS and beyond. In addressing challenges associated with understanding the location, quantity, and quality of mineral resources, and in investigating the potential environmental consequences of resource development, a number of field and laboratory capabilities and interpretative methodologies evolved from the project that have applications to traditional resource studies as well as to studies related to ecosystem health, human health, disaster and hazard assessment, and planetary science. New or improved tools and research findings developed within the project have been applied to other projects and activities. Specifically, geophysical equipment and techniques have been applied to a variety of traditional and nontraditional mineral- and energy-resource studies, military applications, environmental investigations, and applied research activities that involve climate change, mapping techniques, and monitoring capabilities. Diverse applied geochemistry activities provide a process-level understanding of the mobility, chemical speciation, and bioavailability of elements, particularly metals and metalloids, in a variety of environmental settings. Imaging spectroscopy capabilities maintained and developed within the project have been applied to traditional resource studies as well as to studies related to ecosystem health, human health, disaster assessment, and planetary science. Brief descriptions of capabilities and laboratory facilities and summaries of some applications of project products and research findings are included in this circular. The work helped support the USGS mission to “provide reliable scientific information to describe and understand the Earth; minimize loss of life and property from natural disasters; manage water, biological, energy, and mineral resources; and enhance and protect our quality of life.” Activities within the project include the following: Spanned scales from microscopic to planetary; Demonstrated broad applications across disciplines; Included life-cycle studies of mineral resources; Incorporated specialized areas of expertise in applied geochemistry including mineralogy, hydrogeology, analytical chemistry, aqueous geochemistry, biogeochemistry, microbiology, aquatic toxicology, and public health; and Incorporated specialized areas of expertise in geophysics including magnetics, gravity, radiometrics, electromagnetics, seismic, ground-penetrating radar, borehole radar, and imaging spectroscopy. This circular consists of eight sections that contain summaries of various activities under the project. The eight sections are listed below: Laboratory Facilities and Capabilities, which includes brief descriptions of the various types of laboratories and capabilities used for the project; Method and Software Development, which includes summaries of remote-sensing, geophysical, and mineralogical methods developed or enhanced by the project; Instrument Development, which includes descriptions of geophysical instruments developed under the project; Minerals, Energy, and Climate, which includes summaries of research that applies to mineral or energy resources, environmental processes and monitoring, and carbon sequestration by earth materials; Element Cycling, Toxicity, and Health, which includes summaries of several process-oriented geochemical and biogeochemical studies and health-related research activities; Hydrogeology and Water Quality, which includes descriptions of innovative geophysical, remote-sensing, and geochemical research pertaining to hydrogeology and water-quality applications; Hazards and Disaster Assessment, which includes summaries of research and method development that were applied to natural hazards, human-caused hazards, and disaster assessments; and Databases and Framework Studies, which includes descriptions of fundamental applications of geophysical studies and of the importance of archived data.
The Handbook of Ecotoxicology offers 34 chapters with contributions from over 50 selected international experts. The book is divided into four major sections: I. Quantifying and Measuring Ecotoxicological Effects, II. Contaminant Sources and Effects, III. Case Histories and Ecosystem Surveys, and IV. Methods for Making Estimates and Predictability in Ecotoxicology. Concepts and methodology are presented for many types of aquatic and terrestrial ecotoxicity test protocols for both controlled and field assessments. Chapters are offered on such diverse topics as sediment and soil ecotoxicity, landscape indicators, biomonitoring, and use of current bioindicators. The roles of deforestation and global warming, pathogens and disease in ecotoxicology, abiotic factors, urban runoff, predictive ecotoxicology, population modeling, and restoration ecology are discussed. This book was designed to serve as a reference book for students entering the fields of ecotoxicology, aquatic toxicology, terrestrial wildlife toxicology, and other environmental sciences. Many portions of this handbook will serve as a convenient reference text for established investigators, resource managers, and those involved in risk assessment and risk management within regulatory agencies and the private sector.
Recreational boating and personal watercraft use have the potential to adversely impact shallow water systems through contaminant release and physical disturbance of bottom sediments. These nearshore areas are often already degraded by surface runoff, municipal and industrial effluents, and other anthropogenic activities. For proper management, information is needed on the level of contamination and environmental quality of these systems. A number of field and laboratory procedures can be used to provide this much needed information. Contaminants, such as metals, pesticides, polychlorinated biphenyls and polycyclic aromatic hydrocarbons, entering aquatic environments generally attach to particulate matter that eventually settles and becomes incorporated into the bottom sediments. Because bottom sediments serve as a sink and as a source for contaminants, environmental assessments generally focus on this matrix. While contaminant residues in sediments and sediment pore waters can reflect environmental quality, characteristics of sediment (redox potential, sediment/pore-water chemistry, acid volatile sulfides, percent organic matter, and sediment particle size) influence their bioavailability and make interpretation of environmental significance difficult. Comparisons of contaminant concentrations in pore water (interstitial water) and sediment with water quality criteria and sediment quality guidelines, respectively, can provide insight into potential biological effects. Laboratory bioaccumulation studies and residue concentrations in resident or caged biota also yield information on potential biological impacts. The usefulness of these measurements may increase as data are developed relating in-situ concentrations, tissue residue levels, and biological responses. Exposure of test organisms in situ or to field-collected sediment and pore water are additional procedures that can be used to assess the biological effects of contaminants. A battery of tests using multi-species and/or various life stages with different sensitivities to contaminants may offer a more conservative assessment of toxicity than single species testing. Using a ?weight of evidence? approach, the Sediment Quality Trial produces a robust evaluation of habitat quality and includes a measure of contaminant concentrations in the sediment, an assessment of sediment/pore-water toxicity to laboratory animals, and an evaluation of in situ biological assemblages. Field and laboratory procedures are available that can be used to ascertain habitat quality, identify contaminants causing environmental degradation and delineate aquatic systems requiring mitigation of protective efforts. These studies provide the scientific data that are integral to developing an environmental risk assessment of contaminants from watercraft use in shallow water systems.
Aquatic organisms exposed to endocrine disrupting compounds (EDCs) at early life-stages may have reduced reproductive fitness via disruption of reproductive and non-reproductive behavioral and physiological pathways. Survival to reproductive age relies upon optimal non-reproductive trait expression, such as adequate predator avoidance responses, which may be impacted through EDC exposure. During a predator–prey confrontation, larval fish use an innate C-start escape behavior to rapidly move away from an approaching threat. We tested the hypotheses that (1) larval fathead minnows exposed to estro gens, a primary class of EDCs, singularly or in mixture, suffer a reduced ability to perform an innate C-start behavior when faced with a threat stimulus; (2) additive effects will cause greater reductions in C-start behavior; and (3) effects will differ among developmental stages. In this study, embryos (post-fertilization until hatching) were exposed for 5 days to environmentally relevant concentrations of estrone (E1), 17-estradiol (E2), and 17-ethinylestradiol (EE2) singularly and in mixture. Exposed embryos were allowed to hatch and grow in control well water until 12 days old. Similarly, post-hatch fathead minnows were exposed for 12 days to these compounds. High-speed (1000 frames/s) video recordings of escape behavior were collected and transferred to National Institutes of Health Image for frame-by-frame anal- ysis of latency period, escape velocity, and total escape response (combination of latency period and escape velocity). When tested 12 days post-hatch, only E1 adversely affected C-start performance of larvae exposed as embryos. Conversely, larvae exposed for 12 days post-hatch did not exhibit altered escape responses when exposed to E1, while adverse responses were seen in E2 and the estrogen mixture. Ethinylestradiol exposure did not elicit changes in escape behaviors at either developmental stage. The direct impact of reduced C-start performance on survival, and ultimately, reproductive fitness provides an avenue to assess the ecological relevance of exposure in an assay of relatively short duration.
Selenium contamination represents one of the few clear cases where environmental pollution has led to devastation of wildlife populations, most notably in agricultural drainage evaporation and power plant coal-fly ash receiving ponds. Complex biogeochemistry, in particular extensive biotransformations and foodchain transfer, governs Se ecotoxicology and toxicology, for which the mechanism(s) are still elusive. However, total waterborne Se concentration has been widely used as a criterion for regulating and mitigating Se risk in aquatic ecosystems, which does not account for Se biogeochemistry and its site-dependence. There is a need for more reliable indicator(s) that encompass Se ecotoxicity and/or toxicity. Selenomethionine warrants special attention since it simulates Se toxicosis of wildlife in laboratory feeding studies. While low in free selenomethionine, microphytes isolated from Se-laden agricultural evaporation ponds were abundant in proteinaceous selenomethionine. This prompted a more extensive survey of Se speciation in foodchain organisms including microphytes, macroinvertebrates, fish, and bird embryos residing mainly in the agricultural drainage systems of the San Joaquin Valley, California. Total Se in biomass, water-soluble fractions, and protein-rich fractions were measured along with GC-MS analysis of proteinaceous selenomethionine. In all foodchain organisms, water-soluble Se constituted the major fraction of total biomass Se, while proteinaceous Se was a substantial, if not dominant, fraction of the water-soluble Se. In turn, proteinaceous selenomethionine comprised an important fraction of proteinaceous Se. In terms of total biomass Se, an average 1400-fold of Se biomagnification from water to microphytes was observed while subsequent transfer from microphytes to macroinvertebrates exhibited an average of only 1.9-fold. The latter transfer was more consistent and greater in extent for proteinaceous Se and proteinaceous selenomethionine, which is consistent with their importance in foodchain transfer. Proteinaceous Se in the omnivorous carp (Cyprinus carpio) liver also demonstrated a relation to ovarian lesions, while deformed stilt (Himantopus mexicanus) embryo was more abundant in proteinaceous selenomethionine than were normal embryos. Although limited in the number of organisms surveyed, these findings provide an impetus for further field and laboratory feeding studies to substantiate the hypothesis that proteinaceous selenomethionine underlies Se ecotoxicity, which may in turn prove to be a reliable indicator of Se risk in aquatic ecosystems. Copyright ?? 2002 Elsevier Science B.V.
The invasion of the Laurentian Great Lakes of North America by sea lampreys ( Petromyzon marinus ) in the early 20th century contributed to the depletion of commercial, recreational and culturally important fish populations, devastating the economies of communities that relied on the fishery. Sea lamprey populations were subsequently controlled using an aggressive integrated pest-management program which employed barriers and traps to prevent sea lamprey from migrating to their spawning grounds and the use of the piscicides (lampricides) 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide to eliminate larval sea lampreys from their nursery streams. Although sea lampreys have not been eradicated from the Great Lakes, populations have been suppressed to less than 10% of their peak numbers in the mid-1900s. The ongoing use of lampricides provides the foundation for sea lamprey control in the Great Lakes, one of the most successful invasive species control programs in the world. Yet, significant gaps remain in our understanding of how lampricides are taken-up and handled by sea lampreys, how lampricides exert their toxic effects, and how they adversely affect non-target invertebrate and vertebrates species. In this review we examine what has been learned about the uptake, handling and elimination, and the mode of TFM and niclosamide toxicity in lampreys and in non-target animals, particularly in the last 10 years. It is now clear that the mode of TFM toxicity is the same in non-target fishes and lampreys, in which TFM interferes with oxidative phosphorylation by the mitochondria leading to decreased ATP production. Vulnerability to TFM is related to abiotic factors such as water pH and alkalinity, which we propose changes the relative amounts of the bioavailable un-ionized form of TFM in the gill microenvironment. Niclosamide, which is also a molluscicide used to control snails in areas prone to schistosomiasis infections of humans, also likely works by uncoupling oxidative phosphorylation, but less is known about other aspects of its toxicology. The effects of TFM include reductions in energy stores, particularly glycogen and high energy phosphagens. However, non-target fishes readily recover from sub-lethal TFM exposure as demonstrated by the rapid restoration of energy stores and clearance of TFM. Although both TFM and niclosamide are non-persistent in the environment and critical for sea lamprey control, increasing public and institutional concerns about pesticides in the environment makes it imperative to explore other means of sea lamprey control. Accordingly, we also address possible “next-generation” strategies of sea lamprey control including genetic tools such as RNA interference and CRISPR-Cas9 to impair critical physiological processes (e.g. reproduction, digestion, metamorphosis) in lamprey, and the use of green chemistry to develop more environmentally benign chemical methods of sea lamprey control.
Current toxicological methods often miss contaminant effects, particularly when immune suppression is involved. The failure to recognize and evaluate indirect and sublethal effects severely limits the applicability of those methods at the population level. In this study, the Vitality model is used to evaluate the population level effects of a contaminant exerting only indirect, sublethal effects at the individual level. Juvenile rainbow trout ( Oncorhynchus mykiss ) were injected with 2.5 or 10.0 mg/kg doses of the model CYP1A inducer, β-naphthoflavone (BNF) as a pre-stressor, then exposed to a challenge dose of 10 2 or 10 4 pfu/fish of infectious hematopoietic necrosis virus (IHNV), an important viral pathogen of salmonids in North America. At the end of the 28-d challenge, the mortality data were processed according to the Vitality model which indicated that the correlation between the average rate of vitality loss and the pre-stressor dose was strong: R 2 = 0.9944. Average time to death and cumulative mortality were dependent on the BNF dose, while no significant difference between the two viral dosages was shown, implying that the history of the organism at the time of stressor exposure is an important factor in determining the virulence or toxicity of the stressor. The conceptual framework of this model permits a smoother transfer of results to a more complex stratum, namely the population level, which allows the immunosuppressive results generated by doses of a CYP1A inducer that more accurately represent the effects elicited by environmentally-relevant contaminant concentrations to be extrapolated to target populations. The indirect effects of other environmental contaminants with similar biotransformation pathways, such as polycyclic aromatic hydrocarbons (PAH), could be assessed and quantified with this model and the results applied to a more complex biological hierarchy.
A 16-wk feeding study was conducted to evaluate the chronic toxicity of graded levels (0, 1.0, 2.5.5, 10 and 15 mg/kg of chemically pure dietary T-2 toxin (4,15-diacetoxy-8-(3-methylbutyryloxy)-12,13-epoxy-Δ 9 -tricothecen-3-ol) in 1-g rainbow trout, Salmo gairdneri , held in 9°C single-passage well water. Levels of T-2 toxin > 2.5 mg/kg depressed growth, efficiency of feed use, hematocrit, blood hemoglobin concentration and feed acceptance, and caused a transitory edema in a dose-dependent manner. Growth of trout fed a semipurified diet containing the toxin was described by the function: Y = 0.265 + 142.075 e (0.029 X 1 − 1.554x 2 3.7 ), where Y = gain as percentage starting weight per wk; X 1 is time in wk and 0 ⩽ X 1 ⩽16; and X 2 is T-2 content of diet in mg/kgand 0⩽ X 2 ⩽15. Exposure of fish to T-2 toxin did not affect activity of intestinal lumen chymoirypsin or trypsin, nitrogen digestibility or metabolizabte energy. Feeding of 15 mg/kg T-2 toxin to adult trout caused hemorrhaging in the intestines and regurgitation of subsequently intubated feed regardless of T-2 loxin content.
The toxicity of monochlorobenzene (CB) was investigated in rainbow trout following acute intraperitoneal (i.p.) administration and chronic exposure via the water in a continuously flowing system for 15 or 30 days. In the acute study overt toxicity and hepatotoxicity were monitored over a 96-h time period. Variables measured to assess toxicity included weight changes, liver weight to body weight ratios, behavioral changes, alanine aminotransferase activity (GPT), sulfobromophthalein (BSP) retention, total plasma protein concentration and liver histopathology. In the chronic study the same measures of toxicity were followed as well as food consumption and alkaline phosphatase (AP) activity. Upon acute i.p. exposure the toxicant (9.8 mmol/kg) caused behavioral changes in the fish which were consistent with the known anesthetic properties of CB in mammals. Elevations in BSP retention and GPT activity, and histopathology indicated that CB was hepatotoxic in fish. The LC 50 of CB in trout exposed via the water for 96 h was 4.7 mg/l. Chronic exposure of trout to 2 or 3 mg/l CB resulted in similar behavioral changes as seen in the acute study. Liver toxicity was evident from elevations in GPT activity. BSP retention and AP activity appeared to be affected by the nutritional status of the trout as much as by the CB treatment. After 30 days of exposure to 3 mg/l CB, trout appeared to have developed some tolerance to the toxic effects.
Yearling bluegills ( Lepomis macrochirus ) were exposed to sublethal concentrations of [ 14 C]rotenone (5.2 μg/l) for 30 days in a continuous flow exposure system and then transferred to clean, flowing water for an additional 21-day depuration period. Rates of uptake and elimination and profile of the rotenoid metabolites in head, viscera, and carcass components were evaluated by 14 C counting and by high performance liquid chromatography. Total [ 14 C]rotenone derived activity was relatively uniform in all body components within 3 days after initial exposure and remained constant during the ensuing 27 days of exposure. Initial uptake rate coefficients were highest in viscera (K u = 80· h -1 ) and were nearly identical for head (K u = 14 · h) and carcass (K u = 10 · h -1 ). Analyses of tissue extracts by high performance liquid chromatography confirmed the presence of at least six biotransformation products of rotenone. More than 60% of the activity extracted from viscera was present as a single peak which represented a compound that was extremely soluble in water. Rotenone composed only 0.3% of the extractable activity in viscera taken from fish exposed to rotenone for 30 days; however, rotenone accounted for 15.4% of extractable activity in the head and 20.1% in the carcass components. Rotenolone and 6 ' ,7 ' -dihydro-6 ' -,7 ' --dihydroxyrotenolone were tentatively identified as oxidation products in all tissue extracts. Elimination of 14 C activity from all body components was biphasic; both phases followed first-order kinetics. The rate of elimination was nearly equal for all body components during the initial phase but was most rapid from viscera during the second phase of elimination. Bioconcentration factors for the head, viscera, and carcass were 165, 3,550, and 125, respectively, when calculated on the basis of total 14 C activity but only 25.4, 11, and 26 when calculated as the concentration of parent material.